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'Российская биоэнергетика:
от молекул к клетке'
21 - 23 февраля
2005 г.
Факультет биоинженерии и биоинформатики и
Институт физико-химической биологии им. А.Н. Белозерского Московского государственного университета,
Москва
School
of Bioengineering
and Bioinformatics and
A.N.
Belozersky Institute of Physico-Chemical Biology,
Moscow
State University,
Moscow
Оргкомитет конференции:
Акад. А.А. Богданов, председатель; В.А. Драчев, Т.В. Выгодина, отв. секретарь
(тел 939 55 49, E-mailvygodina@belozersky.msu.ru),
Д.Б. Зоров, М.С. Мунтян, В.Б. Сапрунова, проф. В. Швядас,
Organizing
Committee:
Prof. A.A.Bogdanov, Chairmen; V.A. Drachev, T.V.Vygodina, Executive Secretary
(tel 939 55 49, E-mail vygodina@belozersky.msu.ru); D.B. Zorov, M.S Muntyan,
V.B. Saprunova, Prof. W.Shvyadas
Конференция организована при финансовой поддержке Российского Фонда Фундаментальных Исследований 05-04-58028
По
данным на 16.02.05
Corrected
- February 16, 2005
Программа
21
февраля (Биологический факультет МГУ, ауд. М1)
9-30
Регистрация участников
10-00Открытиеконференции
Section
1.Общие
проблемы биоэнергетики
Basic
problems of bioenergetics
Председатель А.А.Богданов
A.A.
Bogdanov, Chairman
10-00 Л.Л. Киселев
Геном митохондрий: норма и патология.
Институт молекулярной биологии РАН, Москва
Lev
L. Kisselev
Mitochondrial
genome, favour and harm
Engelhardt
Institute of Molecular Biology, Russian Academy of Sciences,
Moscow,
Russia
10.30В.А. Шувалов
Первичные превращения энергии при фотосинтезе.
Институт фундаментальных проблем биологии РАН, Пущино, Моск. обл.
Vladimir
A. Shuvalov
The
primary light energy conversion at photosynthesis.
Institute
of Basic Problems of Biology, Russian Academy of Sciences, Pushchino, Moscow
Region, 142290 Russia
11-00 Е.В.
Кунин
Происхождение
и эволюция апоптоза эукариот
E.V.
Koonin
Origin
and evolution of eukaryotic apoptosis: the bacterial connections
National
Center for Biotechnology Information
National Library of Medicine, National Institutes of Health
Bethesda, MD 20894, USA
11-30 Р.
Меджитов
Пути передачи сигнала от тол-подобных рецепторов
Ruslan
Medzhitov
Toll-like
receptor signaling pathways
Section
of Immunobiology, Howard Hughes Medical Institute, Yale University School
of Medicine, New Haven, CT 06510, USA.
12-00 - Перерыв на кофе
12.30Л.
Гринюс
От протонов к генам
L.
Grinius
From
Protons to Genes
Fermentas,
Inc.,Vilnius, Lithuania.
13-00 К.
Чумаков
Эволюция вирусов и безопасность антивирусных вакцин.
Konstantin
Chumakov
Evolution
of viruses and safety of viral vaccines
Rockville,
MD 20852 USA.
13.30М.Ю.
Гальперин
Градиенты Na+ иK+ : геномные перспективы
M.Y.
Galperin
Na+
and K+ gradients in genomics perspective
National
Library of Medicine, National Institute of Health
Bethesda
MD 20894 USA
14-00К. Льюис
Биоэнергетика, множественная лекарственная устойчивость и судьба клетки
Kim
Lewis
Bioenergetics,
multidrug resistance, and cell fate
Department
of Biology, Northeastern University, Boston, MA
14.30Л.Е.
Бакеева
Ультраструктура митохондрий
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
Lora
E.Bakeeva
Ultrastructure
of mitochondria
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
15-00 - 17-00Обед
17-00-18-00 В.П.
Скулачев
Публичная
лекция:
Старение
как программа-атавизм, подлежащая отмене.
Public
Lecture: Aging
as a program-atavism which should be switched off
22
февраля (Лабораторный корпус Б, ауд.
221)
Section
2.Активные
формы кислорода
ReactiveOxygenSpecies
Председатель: В.П.Скулачев.
V.P.
Skulachev, Chairman
10-00В.С.
Кунц
Генерация АФК дыхательной цепью митохондрий - последствия при нейродегенеративных заболеваниях
Wolfram
S. Kunz
ROS
generation by mitochondrial respiratory chain - implication for neurodegenerative
diseases
University
Bonn Medical Center, Bonn, Germany
10-30Ю.А.
Лабас
Генерация активных форм кислорода наружными поверхностями водных многоклеточных животных и некоторые механизмы их защиты от окислительного стресса
Институт биохимии им.А.Н. Баха, Москва, Россия
Yulij
A. Labas
Generation
of reactive oxygen species by the external surface of water multicellular
animals and some protective mechanisms against oxidative stress
Bach
Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071,
Russia
11-00Д.Б.
Зоров
Друзья и враги. Активные формы кислорода и азота
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
Dmitry
B. Zorov
Friends
and Foes. Reactive Oxygen and Nitrogen Species
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
11-30
- 12-30 Постерная сессия (размер стенда 85 х 110 см)
Poster
section (85 x 110 cm)
12-30 - Перерыв на кофе
Section
3.Транспортные
системы и рецепторы
Transport
systems and receptors
Председатель:Р. Меджитов
R. Medzhitov, Chairman
12-50Л.Б.
Марголис
Взаимодействие ВИЧ с другими патогенами в лимфатической ткани человека ex
vivo
L.
Margolis
HIV
cross-talk with other pathogens in human lymphoid tissue ex vivo
National
Institute of Child Health and Human Development, National Institutes of
Health, Bethesda MD 20892, USA
13-20Р.А.
Звягильская
Системы транспорта фосфата и энергетический статус клеток дрожжей Yarrowialipolytica, выращенных в щелочных условиях
Институт биохимии им.А.Н. Баха, Москва, Россия
R.A.
Zvyagilskaya
Phosphate
transport systems and energy status of yeast Yarrowia lipolytica cells
cultivated under alcaline conditions
Bach
Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071,
Russia
13-50А.М.
Дижур
На
перектестке зрения и слепоты. Роль цГМФ и кальция в фоторецепции
и наследственной дистрофии сетчатки
A.
M. Dizhoor
At
the crossroads of vision and blindness. Cyclic GMP and calcium
in photoreceptors
Pennsylvania
College of Optometry, 8360 Old York Road, Elkins Park, PA 19027, USA
14-20 - 15-30 Обед
Продолжение сессии 3
15-30С.И.
Бибиков
Аэротаксис
S.I.
Bibikov
Aerotaxis
EMD
BioSciences, San Diego, CA, USA
16-00С.
Зайцев
К преодолению недостаточности секреции инсулина
S.
Zaitsev
Targeting
defective insulin secretion
Department
of Molecular Medicine, Karolinska Institute,
Karolinska
Hospital, Stockholm, Sweden
Section
4.Старение
и избирательная гибель клеток
Aging
and selective cell death
Председатель: Е. Кунин
E. Koonin, Chairman
16-30Б.
Животовский
Функция каспазы 2 в апоптозе, опосредованная митохондриями
B.
Zhivotovsky
Caspase-2
function in mitochondria-mediated apoptosis
Department
of Toxicology, Institute of Environmental Medicine
Karolinska Institutet, Box 210, SE-171 77 STOCKHOLM, Sweden
17-00М.
Циглер
Новая жизнь столетнего: сигнальные функции НАД
M.
Ziegler
The
new life of a centenarian: Signaling functions of NAD
Department
of Molecular Biology, University of Bergen
Thorm?hlens gt. 55 5020 Bergen, Norway
17-30 Перерыв на кофе
18-00А.Е.
Донцов
Роль старческого пигмента липофусцина и А2Е в механизмах
фотоповреждения сетчатки
Институт биохимической физики им. Эммануэля РАН, Москва, Россия
A.
E. Dontsov
Retinal
photodamage: role of 'age pigment' lipofuscin and A2E
Emanuel
Institute of Biochemical Physics, Russian Academy of Sciences,
Moscow,
Russia
18-30Б.В. Черняк
Биоэнергетика митохондрии и гибель клеток
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
B.V.
Chernyak
Mitochondrial
bioenergetics and cell death
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
19-00К.Г.
Лямзаев
Избирательное уничтожение митохондрий в клетке (митоптоз), индуцированное митохондриальными ингибиторами
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
K.G.
Lyamzaev
The
selective elimination of cell mitochindria (mitoptosis), initiated by mitochondrial
inhibitors
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
19.30Д.А.
Кнорре
Программируемая клеточная смерть дрожжей, вызванная феромоном
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
D.A.
Knorre
Programmed
death of yeast caused by a pheromone.
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
23
февраля (Лабораторный корпус Б, ауд. 221)
Section
5.Свободноеокисление
'Free'
respiration
Председатель: Н.Б. Гусев
N.B.
Gusev, Chairman
10-00 А.В. Богачев
Роль ферментов несопряженного
окисления в реализации дыхательной
защиты у азотофиксирующей бактерии Azotobacter vinelandii и в процессе
теплопродукции у термогенного растения Arum orientale
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
A.V.
Bogachev
Role
of noncoupled oxidation enzymes in respiratory protection of nitric bacterium
Azotobacter vinelandii and in heart production of thermogenic plant Arum
orientale
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
10-30В.Н.
Попов
Разобщенное и несопряженное дыхание в растительных митохондриях: биохимические механизмы и физиологическая роль
Кафедра биохимии растений и физиологии Воронежского государственного
Университета, Россия
V. N. Popov
Uncoupled
and non-coupled respiration in plant mitochondria: biochemical mechanisms
and physiological role
Dept.
of plant biochemistry and physiology, Voronezh State University, Russia
Section
6.Энергопреобразующие
ферменты и цитохромные
комплексы
Председатель: Л.С. Ягужинский
L.S.
Yagizhinsky, Chairman
11-00Н.Б.
Гусев
Молекулярные механизмы регуляции мышечного сокращения
Кафедра биохимии, Биологический факультет МГУ, Москва, Россия
Nikolaj
B. Gusev
Molecular
mechanisms of muscle contraction regulation
Department
of Biochemistry, Biology Faculty, Moscow State University, Russia
11-30А.Д.
Виноградов
Работают ли энергопреобразующие ферменты как обратимые молекулярные машины ?
Кафедра биохимии, Биологический факультет МГУ, Москва, Россия
A.
D. Vinogradov
Do
energy-transducing enzymes operate as the reversible molecular machines?
Department
of Biochemistry, Biology Faculty, Moscow State University, Russia
12-00А.Ю.
Семенов
Электрогенные реакции в фотосинтетических реакционных центрах
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
A.Y.
Semenov
Electrogenic
reactions in photosynthetic reaction centers.
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
12-30 Перерыв на кофе
Председатель: А.А. Константинов
A.A.
Konstantinov, Chairman
13-00М.
Верховский
Почему оксидаза должна работать быстро
Michael
Verkhovsky
Why
oxidase should operated fast
Helsinki
Bioenergetics Group, Institute of Biotechnology, University of Helsinki,
PB 65 (Viikinkaari 1), FIN-00014, Helsinki, Finland
13-30Д.А.
Блох
Транслокация протона в процессе восстановительной фазы каталитического
цикла цитохром с-оксидазы
D.A.
Bloch
Proton
translocation during the reductive phase in the catalytic
cycle of cytochrome c oxidase
Helsinki
Bioenergetics Group, Institute of Biotechnology, University of Helsinki,
PB 65 (Viikinkaari 1), FIN-00014, Helsinki, Finland
14-00 -15-30 Обед
15-30В.Б.
Борисов
Изучение механизма работы бактериального комплекса цитохромов bd
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
V.B.
Borisov
The
study of functioning mechanism of bacterial cytochrome bd
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
16-00Ю.А.
Каменский
Перенос электронов в мембранах хромаффинных гранул. Структурно-функциональное исследование цитохрома b561
Y.A.
Kamensky
Electron
Transfer Across the Chromaffin Granule Membrane. Structure/Function Studies
of Cytochrome b561
Dept.
of Biochemistry and Cell Biology, Rice University, 6100 S. Main, Houston
16-30М.С.
Мунтян
Транспорт ионов, сопряженный с работой терминальных оксидаз в экстремально- алкалофильных бактериях
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
Maria
S. Muntyan
Ion
transport coupled to terminal oxidases functioning in the extremely alkaliphilic
bacteria
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
Section
7.Биоэнергетика
мембранных систем
Bioenergetics
of membrane systems
.
Председатель: А.Д. Виноградов
A.D.
Vinogradov, Chairman
17-00 Л.С. Ягужинский
О двух механизмах сопряжения дыхания и фосфорилирования в митохондриях
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
L.S.
Yaguzhinsky
About
two mechanisms of respiration and phosphorylation coupling in mitochondria
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
17-30Е.И.
Милейковская
Кардиолипин в энергопреобразующих мембранах
E.I.
Mileykovskaya
Cardiolipin
in energy transducing membranes
Department
of Biochemistry & Molecular Biology,University of Texas-Houston
Medical
School, Houston
18-00А.Я. Мулкиджанян
Динамика переноса протонов на границе мембрана/вода и механизм преобразования энергии
Armen
Y. Mulkidjanian
Proton
transfer dynamics at the membrane/water interface and mechanism of biological
energy conversion
Max-Planck
Institute of Biophysics, Frankfurt, Germany; Department of Biophysics
18-30М.Ю.
Высоких
Образование переходного комплекса фермента с субстратом дестабилизирует октамер митохондриальной креатинкиназы в условиях окислительного стресса
Институт физико-химической биологии им.А.Н. Белозерского, МГУ, Москва
M.Y.
Vyssokikh
Transition
State Analog Complex (TSAC) formation affected oligomer structure of mitochondria
creatine kinase under conditions of oxidative stress.
Belozersky
Institute of Phys-Chem. Biology, Moscow State University, Russia
19-00 Д.О.
Левицкий
Является ли место связывания
кальция с высоким сродством в натрий/кальций обменнике калмодулинподобным
модулятором кальция?
D.
O. Levitsky
A
high affinity calcium-binding domain in sodium/calcium exchanger: a
calmodulin-like calcium modulator?
University
of Nantes, Nantes, France
19-30Заключительный
вечер в 536 ауд. Корпуса
'А'
Closing
party (room 536, Bldg.'A')
Abstract
of participants
67-kDa
melittin-like protein from mouse kidney seems to belong to new family of
proteins
N.V.
Dolgova, A.M.
Rubtsov, O.D. Lopina
Bee
venom peptide melittin is known to be an inhibitor of P-type ATPases including
H,K-ATPase from gastric mucosa, sarcoplasmic reticulum (SR) Ca-ATPase,
and Na,K-ATPase. It was shown earlier that the inhibition of Na,K- and
SR Ca-ATPase by melittin is not due to the melittin interaction with lipid
bilayer but is connected with direct interaction of melittin with catalytic
subunits of the enzymes. These imply that melittin can simulate some intracellular
protein(s) that interact(s) with catalytic subunits of P-type ATPases.
It was shown that protein with molecular mass of about 67 kDa interacting
with antibodies against melittin exists in gastric mucosa cells. The aim
of this research was to find and to study 'melittin-like' protein in mammalian
kidney tissue.
Immunoprecipitation
of Na,K-ATPase by antibodies against its a-subunit
revealed that protein with molecular mass about 67 +
3 kDa was among the proteins co-precipitated with Na,K-ATPase from Triton
X-100 solubilized homogenate of mouse kidney. The protein was stained by
antimelittin antibodies. The 67 kDa-protein was purified from homogenate
using immunoaffinity chromatography and its N-terminal sequence was determined.
That is the following: h p p k r v r s r l n g.
We
did not find any protein with this N-terminal sequence among the proteins
presented in available protein database. However we found 7 protein sequences
that contain this sequence inside the plausible sequences on a distance
of 553 amino acids from C-terminus. From these seven sequences only Alfy
(Autophagy-linked FYVE protein) is known at present time as a protein.
Other sequences were obtained by translation of cDNA or genomes from different
sources. Analysis of sequences using RPS-BLAST
2.2.9 program showed that all plausible proteins (except 67-kDa 'melittin-like'protein
and 3-d isoform of protein 3) should contain domains Beach, WD40 и
FYVE. Two latter proteins should have only domains WD40 and FYVE.
Domains
WD40 are presented in many eukariotic proteins participating as adaptor
and regulatory proteins in signal transduction and cytoskeleton assembling.
Using Blat program we found significant homology between WD40 domain and
melittin. Domain FYVE is known to interact specifically with PIP-3 lipid
that is mainly presented in endosomal membranes. We suggest that 67-kDa
protein is a member of protein family containing WD40 and FYVE domains.
Is partially supported by grant ? 01-0224 from INTAS.
Antioxidant
Mito Q protects the cells from apoptosis, induced by H2O2
L.V.
Domnina, O.Yu.
Ivanova, I.V. Skulachev and Ju.M. Vasiliev.
A.N.
Belozersky Phys.-Chem. Biology Institute, Moscow State University, Moscow.
E-mail:
domnina@belozersky.msu.ru, domnina@mbio.genebee.msu.su
The
effect of oxidative stress and antioxidants on cytoskeleton and adhesive
structures is poorly understood. We investigated the effect of the antioxidant
MitoQ (Mitochondric-tergetted antioxidant) [Smith R.A. et al, Methods Enzimol.
2004; 382: 45-67], which in very small concentrations protects cells from
death, induced by H2O2.
We
studied the effect of antioxidant MitoQ on the next cells:HeLa
(epithelial human cells), IAR-2 (rat epithelial cells), on human fibroblasts,
Rat-1 (rat fibroblasts), of L line (mouse fibroblasts). The changes of
cell shape, cytoskeleton, adhesive structures and mitochondrial system
were investigated.The method of morphometry [Dunn and Brown, 1986] was
used to analyze cell shape changes. The confocal microscope in combination
(revealing?) with immunofluorescence were used in studies of cytoskeleton
(bundles of actin microfilaments and the microtubules system). The vinculin
and paxillin staining was used to reveal focal adhesive structures. Mitochondria
in living cells were stained with tetramethilrhodamin. The difference of
pothencial was removed by damage of the small mithohondria area by laser
beam (wave length 488 nm). It was shown that after the antioxidant treatment
the area of the spread cells increased and the cells shifted from fibroblasts
to ephithelioid shape. Simultaneously essential increase and thickening
of the actin microfilament bundles (stress-fibers) took place as well as
increase of the amount of connected myosin. Areas and lengths of focal
contacts substantially increased. In the wounding assay the cultured fibroblasts
after antioxidant treatment also equared epithelioid behavior and
moved into the wound as a single sheet. In experiments with laser beam
pointlike damage in HeLa cells the formation of the mithochondrial network
under the influence of MitoQ and SQ was shown, in contrast to control cells
where the single mithochondrias were present. The possible mechanism of
the observed alteration will be discussed.
This
work was supported by grants from the Ludwig Institute for Cancer Research
(PO 863) and the Russian Foundation for Basic Research (02-04-48792, 00-04-48090).
Unltrastructural
changes in plant cells accompanying programmed cell death
E.V.
Dzyubinskaya1,
L.E.Bakeeva2, V.D. Samuilov1
1.Department
of Biology, M. V Lomonosov Moscow State University, Moscow, 119992, Russia,
2A.N. Belozersky Phys.-Chem. Biology Institute, Moscow State
University, Moscow, Russia.
Fax
(095)939-38-07, E-mail: alena@8.cellimm.bio.msu.ru
The
data on the plant cell ultrastructure in apoptosis are presented. The treatment
of the pea leaf epidermal peels by cyanide resulted in destruction of nuclei
of guard cells visible by a light microscope. The process was accelerated
by illumination. According to electron microscopy, the ultrastructure of
guard cells undergoes appreciable СN--induced
changes in time. After 1 h of incubation of the isolated epidermis with СN-
chromatin condensation and margination was already revealed in guard cells.
Further condensation of chromatin, the swelling of endoplasmatic reticulum
together with formation of large tanks covered with ribosomes, the changes
in the structure of dictyosomes, and a negligible swelling of mitochondria
were revealed after 3 h of the epidermis incubation with СN-.
After 6 h of incubation with СN-,
the bulk volume of the guard cells was filled with vacuoles, the cytoplasm
occupied a thin marginal zone, while the nucleus, similar to in the control
experiment, was placed in the center of the cell, but took the shape of
a polyhedral frame and became stretched in narrow cytoplasmic bands as
self-contained structure, despite the loss of integrity of its membrane.
In the open fields of the nucleus devoid of the membrane, mitochondria
and chloroplasts came into direct contact with the chromatin. The chloroplasts,
similarly to the cellular nucleus, lost outer membrane continuity but did
not swell, preserving the stroma and the integrity of the thylakoid system.
A dramatic surge in volume of vacuoles and a decline in cytoplasm volume
were observed.Plant cells contain
no lysosomes. The enzymes causing cell degradation are located in vacuoles.
Therefore, the significant increase in the volume of vacuoles and the presence
ofmembrane structures in them would
suggest an initial apoptosis at this stage of our test. The significant
increase in the volume of vacuoles and the presence of membrane structures
in them would suggest an initial apoptosis. The antioxidant
di-tret-butyl-4-hydroxytolueneprevented the
ultrastructural changes of the cells observed after 6 h of incubation with СN-.
The data obtained show that the СN--induced
death of guard cells is realized through the mechanism of apoptosis.
This
work was supported by grant from the Russian Foundation for Basic Research
(04-04-48121).
Mitochondrial
metabolism and neuronal activity cross-talk
L.A.
Erokhova, G.V.
Maksimov
M.V.
Lomonosov Moscow State University, Biological Faculty, Biophysics Dep.,
Cell BiophysicsLab., 119992, Leninskie Gori, 1, build. 12.
Tel.
939-19-66; E-mail: erokhova@rambler.ru, maxim@biophys.msu.ru
Increased
neuronal activity in the CNS is associated with accumulation of Na+,
Ca2+and
Cl-within
nerve cells and activity-dependent release of K+into
the extracellular space. In order to maintain neuronal excitability, ion
transport processes at plasma membranes (Na+/K+-ATPase,
Na+/Ca2+-exchange,
etc.) and at endoplasmic/ sarcoplasmic reticulum (Ca2+-ATPase)
must be activated. These processes ultimately depend on sufficient availability
of ATP. Approximately 95% of the ATP consumption in the CNS is thought
to be generated by oxidative metabolism within mitochondria. The
reduced cofactors, NAD(P)H and FADH2, are necessary to establish
membrane potential (DY)
across the inner mitochondrial membrane. Central to the homeostatic role
of mitochondria is the maintenance of a DY.
The
changes of spontaneous activity and mitochondrial DY
of identified neurons during sensory signal propagation were studied. Rz-neurons
from segmental ganglia of a leech, Hirudo medicinalis, were chosen
as an object of investigation. Explored preparations were the skin patches
connected by lateral nerves to the exposed ganglia dissected from the front,
posterior or middle-body regions. It was observed that skin thermal stimulation
results in a reversal increase in action potential (AP) generation frequency
of Rz-neurons from any ganglia. The AP frequency increase in Rz-neurons
is correlated with mitochondrial DY
and NAD(P)H decline, as revealed by fluorescence measurements. All the
parameters restore to their initial values with termination of stimuli.
Similar events were shown under direct acetylcholine application to ganglion.
The
results suggest that mitochondrial oxidative metabolism is tuned to physiological
activity in neurons.
The
electric field action on the index of refraction of water bilayers
S.S.
Golubev
A.N.
Belozersky Institute of Physico-Chemical Biology
,
Moscow State University, Moscow, 119899. Fax (095) 939-0338;
The
new effect of electric field on the index of refraction of water bylayers
in membranes system was shown in this work. Energization of inner mitochondrial
membrane leads to increase of optical height of mitochondria. In the present
work we carried out some experiments on artifical model - phospholipids
multilayers. The measurement of the path-length difference dependence from
the gradient of electric field was carried out by using phase microscope.The
constant of electrooptical effect of one membrane (embodied in multilayer
structure) was 5.5?10-9
m/V. Similar values was obtained in experiments with electrooptical effect
on the mitochondrial membrane. The numerical values of this constant was
from 2.4?10-9
m/V to 1.2?10-8
m/V. Bases on similarity of this values, we conclude the phase-shift on
mitochondrial membranes and black membrane to have the same physical nature.
After the weak-bounded water was removed from multilayers, the constant
of electrooptical effect was decreases many times. This experiments shows,
that the phase-length changes (index of refraction) in the electric field
to be linked with existence of the weak-bounded water layer. The obtained
results opens new aspects of the influence of the membrane electric potential
on functioning of the biological systems.
The
Net Charge transport by Neurospora Rhodopsin Photocycle
O.
Gopta,
L. Khitrina, I. Kalaidzidis, L. Brown, Y. Kalaidzidis
A.N.Belozersky
Institute of Physico-Chemical Biology, MSU, Moscow, 119992 Russia. E-mail:yannis@computer.org;
ikalaidz@mpi-cbg.de
The
Neurospora Rhodopsin (NR) is a type-I rhodopsin from Neurospora
crassa (Beiszke at al, 1999). The absorbsion maximum of light-adapted
NR (530 mn) is blue shifted relative to bacteriorhodopsin (BR) from Halobacterium
salinarium. The photocycle of NR was studied in (Brown at al, 2001,
Furutani at al, 2004). It was shown that NR releases proton under illumination
and forms blue-shifted intermediate similar to M intermediate of BR and
then after conversion into red-shifted intermediate (similar to O intermediate
of BR) relaxes to ground state. The kinetical analysis reveals 5 exponents
in the photocycle (0.12, 0.9, 30, 700, 5000 ms). The exponent's values
are vary twofold in the pH range from 5 to 8 (Brown at al, 2001).
In
the present work the photoabsorption photocycle of NR's membranes washed
in 0.5%DM was measured at 26 wavelengths in the range 380-660 nm (pH=7.4,
t=22њC,
150mM NaCl). The measurements was done by the logarithm sweep in the range
from 1 ms
to 40 sec. The apparent differential maximums of main intermediates were
found at 390 and 590 nm. An exponential analysis shows that 5 exponents
mentioned above are not enough to describe the photocycle. Global fitting
gave maximum probability to 8 exponents (0.004, 0.18, 0.75,6.1,
75.5, 1100, 2100, 9300 ms). The decay ofO
and M intermediates includes 3 exponents in second's range (1.1, 2.1 and
9.3 sec) instead of two (0.7 and 5 sec). The exponent 2.1 sec is positive
for both intermediates but 1.1 and 9.3 sec have different sign for M and
O intermediates. Different sign of amplitudes allows reliably extract this
closely spaced exponents. The same sign change allows split exponents 6
and 75 ms. New additional fast exponent (5ms)
could be artifact of laser excitation.
The
NR containing proteoliposomes were adsorbed on the teflon film and charge
transfer kinetic were measured by the direct electrical method (Drachev
at al, 1978). The teflon film with high resistance (discharge constant
28 sec) allows to get kinetics up to second range. The net charge transport
during NR photocycle was shown. Surprisingly the shape of NR electrical
response is qualitatively different from that in the BR. In the middle
cycle time (0.1 - 500 ms) the voltage has sign opposite to the final one.
The exponential analysis reveals components in line with absorption kinetics
data. The net-charge transfer components are (3, 40, 130, 745, 2750 ms)
This
work was partly supported by the RFBR (grant No. 03-04-49153) and ISTC
(grant No. 866).
Location
and chemical reactivity of the slow active-inactive transition-sensitive
SH-group in the mitochondrial NADH: ubiquinone oxidoreductase (Complex
I)
I.
S. Gostimskaya,
A. D. Vinogradov
Department
of Biochemistry, School of Biology, Moscow State University, Moscow 119992,
Russian Federation.
The
membrane permeant (N-ethylmaleimide, NEM) and membrane non-permeant (5,5?-dithiobis-(2-nitrobenzoate),
DTNB) SH-reagents were employed as the specific inhibitors of the mitochondrial
NADH:ubiqinone oxidoreductase (Complex I) in intact mitochondria and inside-out
submitochondrial particles (SMP) to locate the SH-group involved in slow
interconversion between the active (A) and de-activated (D) forms of the
enzyme. Both NEM and DTNB rapidly reacted with the D-form in SMP. DTNB
prevented the irreversible inhibition of the enzyme (D-form) by NEM thus
indicating that both reagents interact with the same SH-group. In intact
mitochondria NEM blocked the D-form whereas the enzyme was not susceptible
to inhibition by DTNB. The channel-forming antibiotic, alamethicin [1],
induced the sensitivity of the D-form to DTNB in intact mitochondria. The
reactivity of the A-D transition-sensitive SH-group in SMP towards dianionic
DTNB and neutral 2,2?-dithiobis-(5-nitropyridine),
DTNP were compared. The pseudo-first order rate constant for the enzyme
inhibition by DTNP was 14-times higher than that by DTNB if the efficiency
of either reagent in thiol-disulfide exchange reaction was normalized to
their reactivity towards low molecular mass neutral dithiothreitol. The
pH-profile of the pseudo-first order inhibition rate constant for DTNB
showed abnormally high apparent pKa of the enzyme SH-group (>10).
The D-form is insensitive to prolonged incubation (75 min) in the presence
of 10 mM oxidised glutathione. Taken together the results suggest that
the transition sensitive-SH-group is located in the mitochondrial matrix-protruding
part of Complex I within negatively charged local environment. Supported
by the Russian Foundation for Fundamental Research (grant 02-04-48679 and
03-04-48202), Program Leading Schools in Science (grant 596.2003.4) and
NIH Fogarty International Research grant 1R03TW006041.
References:
1.
I.S. Gostimskaya, V.G. Grivennikova, T.V. Zharova, L.E. Bakeeva, and A.D.
Vinogradov, Analytical Biochemistry 313 (2003), 46-52.
Stoichiometry
of cytochrome b559 in PSII
O.P.
Kaminskaya1, J.
Kern2, V.A. Shuvalov1,
G. Renger2
1Institute
of Basic
Biological Problems,
Russian Academy of Sciences, Pushchino, Moscow Region 142292, Russia, 2Max-Volmer-Institute
for Biophysical Chemistry and Biochemistry, Technical University Berlin,
Strasse des 17. Juni 135, D-10623 Berlin, Germany.
Cyt
b559 is a heme protein closely associated with reaction center of PS II
which properties and function are not fully understood. The number of Cyt
b559 copies bound to a native PSII complex is a matter of controversial
discussion. Here we show that the controversy in this subject mostly arises
from a substantial underestimation in the value of 'reduced minus oxidized
' difference extinction coefficient De
of Cyt b559. We report for the first time De
of Cyt b559 determined on the basis of the amplitude of the full redox
titration curve in the wide range from -250 up to +450 mV allowing to monitor
redox transitions of all Cyt b559 redox forms. The value of the a-bandDe
of Cyt b559 was found to be 25.1 +
0.5 mM-1cm-1 in PS II core complexes from the thermophilic
cyanobacterium Thermosynechococcus elongatus which sample represents
current mostly purified functional PS II preparation. On the basis of corrected
value of the De
and assuming close similarity in biochemical properties of cyanobacterial
and higher plant Cyts b559 the Cyt b559:PSII stoichiometry is inferred
to be 1:1 in both thermophilic cyanobacteria and plants.
Effect
of Membrane Cell Excitation on the Thylakoid pH Gradient and Photosynthetic
Electron Transport in Chara Cells
N.A.
Kamzolkina,
A.A. Bulychev
Department
of Biophysics, Faculty of Biology, Moscow State University.
It
was shown earlier that a propagation of an action potential (AP) in Chara
corallina leads to transient suppression of spatially periodic pH profiles
along the illuminated cell. The suppression was manifested as a large pH
decrease in the alkaline zones and a slight pH increase in the acid zones.
This data indicates an interference of membrane excitation in the mechanisms
responsible for pH banding patterns in Chara
cells. In illuminated
Chara cells the pH pattern correlates fairly well with the pattern
of photosystem II (PSII) photochemistry. In the view of this correlation
we examined the effect of AP generation on the maximum yield of chlorophyll
fluorescence (Fm').
In
the alkaline cell region the AP propagation caused a fast substantial decrease
of the Fm' fluorescence followed by its gradual recovery. The quantum efficiency
of PSII photochemistry (?F/Fm')
showed a similar pattern. In the acid cell region we did not observe substantial
changes in Fm' and?F/Fm'
after cell excitation. The decrease in ?F/Fm'
suggests that the linear electron transport is retarded after AP. Besides,
the AP-induced depression of the Fm' fluorescence in the alkaline cell
regions is indicative of nonphotochemical quenching associated with the
pH gradient at the thylakoid membrane that facilitates thermal dissipation
of the light energy absorbed.
The
mechanistic basis of AP-induced increase of the ?pH
is not known yet but this may result from the increase in cytosolic Ca2+
([Ca2+]c) during excitation that contributes to many
processes in plant cells, including changes in ion conductance of thylakoid
membrane. The increase in [Ca2+]c should elevate
the Ca2+ level in the chloroplast stroma, thereby leading to
inhibition of the Calvin cycle and to over-energization of the thylakoid
membrane (the increase in?pH
and nonphotochemical quenching). The subsequent recovery of Fm' could be
due to the operation of the Ca2+/H+ antiporter of
the thylakoid membrane, which should reduce the stromal Ca2+
level and ?pH.
Apparently, the electrical excitation of the plasma membrane in the alkaline
cell region initiates a pathway that can modulate membrane events at the
thylakoid membrane.
ROS
and telomere shrinkage. Are they really responsible for natural aging?
A.V.
Khalyavkin
Institute
of Biochemical Physics of RAS, Moscow, Russia.
E-mail:
ab3711@mail.sitek.net
We
didn't know yet why the
control and co-ordination of vital processes normally deteriorates with
age, and senescent individual becomes more vulnerable. Recently it was
found out, that both free-radical theory and telomere shrinkage concept
are not suitable for an explanation the origin of natural non-pathological
aging. Author of telomere shrinkage hypothesis, AM
Olovnikov, has rejected it as explanation of an aging origin and
has put forward a new one (Biochemistry (Moscow) 2003; 86: 2-33; Adv Gerontol.
2003; 12: 28-45). On the other
hand, the most commonly used free-radical theory of aging also failed to
explain a non-pathological senescence. WC
Orr and RS Sohal, who werelong-standing supporters
of the free-radical concept of aging, reconsidered the role of the reactive
oxygen species (ROS) in
development of natural aging (Exp. Gerontol. 2003; 38:
227-230). Summarizing
data of SOD + catalase influence on longevity of fruit flies they came
to conclusion that oxidative stress does not cause normal aging. At the
same time the role of signaling in aging received more attention (Cell
2001; 105: 165-168).
Moreover, recent studies have implicated ROS as the natural second messengers
along with cAMP, Ca2+, and phospholipid metabolites contributing
to signal transduction from membrane receptors of extracellular signaling
ligands to intracellular systems involved in gene transcription control
(Biochemistry (Moscow) 2002; 67: 339-352; Physiol. Rev. 2002; 42: 47-95).
The rate of ROS generation in cells is under strict control of several
hormones, cytokines, and growth factors. Therefore uncontrolled antioxidant
therapy will also inhibit essential cellular functions and can be dangerous.
We know that each organism react as a whole on a set of environmental influences
by means of certain changes in control systems at the physiological levels
as well as via some shifts in signal transduction pathways at the cellular
level. According
to our concept natural aging is originated from inevitable shift incertain
parameters of physiological control systems under influence of environmental
conditions, which are not able to fully support organism's 'optimal' existence
in the self-maintenance mode (Adv. Gerontol. 1998; 2: 43-48; Ibid. 2001;
7: 46-49). Therefore,
outer cues can evoke or retard and even reverse the aging manifestations
via launching or abolishment of the built-in self-destruction program.
Substitution
of isoleucine L177 by histidine affects the pigment composition and properties
of the reaction center of the purple bacterium Rhodobacter sphaeroides
R.
A. Khatypov,
L.G. Vasilyeva, T.Y. Fufina, T. I. Bolgarina, I.B.Klenina, A.Ya. Shkuropatov,
V. A. Shuvalov
Institute
of Basic Biological Problems, Pushchino-city, Institutskaya str., 2, 142290.
Using
method of site-directed mutagenesis we obtained the mutant of the purple
bacterium Rhodobacter sphaeroides with
Iso-His substitution in the position 177 in the L- subunit of the photosynthetic
reaction center (RC). The mutant strain forms stable photochemically active
RC complexes. Spectral and photochemical properties of the mutant RC have
changed significantly in the absorption bands corresponding to the primary
donor and the monomer bacteriochlorophyll (BChl) absorption. It was shown,
that the pigment content in the mutant RC I(L177)H has changed significantly.
Pigment extraction and analysis revealed that the mutant RC contains only
three BChl molecules comparing to four BChl molecules in the wild type
RC. EPR linewidth
of the photoinduced signal of the primary donor P870 was different in the
wild type and I(L177)H mutant chromatophorepreparations
(1.05 mT and 1.3 mT at T=10 K, respectively). This is an indication of
different spin delocalization in the primary donor, for the mutant being
typical of a monomeric oxidized BChl. Considering
the fact that the properties of both isolated and membrane-associated mutant
RCs were similar, we conclude that missing BChl molecule from the mutant
RC was the result of the introduced mutation but not of the protein purification
procedure. The new mutant with the altered RC pigment composition is still
able to perform light-induced reactions forming the state with the separated
charges P+QA-, appears to be an interesting
object to study the mechanisms of the first steps of the primary electron
transfer in photosynthesis.
Authors
gratefully acknowledge the support by the Russian Basic Research Foundation
and RAS grants.
"To
be, or not to be" in terms of cytogerontology
A.N.
Khokhlov
Evolutionary
Cytogerontology Sector, School of Biology, Moscow State University.
E-mail: khokhlov@genebee.msu.su
The
famous Hamlet's question in some translations reads as "Life or death",
or "To live, or not to live". In terms of cytogerontology (investigations
of aging mechanisms on cultured cells) it could be formulated as "Alive
or dead are those cells we study in our flasks and wells". We have already
repeatedly mentioned in our publications that it is not so trivial question
as it seems to be from the first sight. With the help of standard aging
definition as mortality rate increase with time we should certainly draw
a conclusion about necessity of getting of survival curves for cultured
cells in cytogerontologicalexperiments.
However, the regular approach used for getting of survival curves for cohorts
of experimental animals and humans is absolutely not suitable in the case.
The first problem - the cells divide. This leads to the lack of cell corpse.
Animals also reproduce but we always can distinguish between parents and
descendants. For the cells it is almost impossible. Basically, we can suppress
cells' proliferation (and we really do it in our experiments in terms of
"stationary phase aging" model) and then follow their dying out. However,
in the situation we cannot say that the cells exist at normal conditions
and, that is the most important, - it is very difficult to correctly register
the cell death moment because it lasts for the time period comparable with
the time of all the experiment, i.e. there is almost no way to get "the
right" point on the survival curve. Besides, most of the current cell viability
tests (trypan blue, neutral red, MTT and XTT, naphthalene black, 57Cr,
etc.) give absolutely different results for the same cell population studied.
A number of the problem studies in our lab, unfortunately, just confirmed
the conclusion. We have tried to evaluate the live cells' ratio in "stationary
phase aging" cultures by several staining methods with the following standard
microscopic analysis of the preparations, or with help of computer analysis
of their digital images. Significant dispersion of the data obtained by
different methods was revealed and, besides, we were not able to completely
get rid of researcher's subjectivism contribution to evaluation of the
results. Say, in the most popular trypan blue test appeared to be no accurate
criterion of staining density allowing us to relate the cell to dead or
live. Paradoxically, the live cells' ratio evaluation just "by eye", without
any staining, allowed us to get the results being within the limits of
data dispersion mentioned. At present we try to understand how much effective
for the problem solving could be various computer programs of automatic
image analysis.
Ca2+-binding
site in mammalian cytochrome c oxidase: kinetic studies and bioinformatic
analysis.
A.
Kirichenko1,
T.V. Vygodina1, N. Tretiakova2, A. Guiffre3
and A. Konstantinov1
1A.N.
Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Russia, 2Department of Bioengineering and Bioinformatics, Moscow
State University, Russia, 3 CNR Center of Molecular Biology,
University of Rome, Italy.
3-dimentional
structure reveals Ca-binding site near outside surface of subunit I both
in mitochondrial and bacterial cytochrome c oxidase (COX). In mammalian
enzyme Ca2+ binds reversibly bringing about a small red shift
of the heme a absorption spectrum whereas bacterial COX contains
tightly bound cation. Ca2+ -induced spectral changes can serve
as convenient indicator of cation interaction with mammalian COX. Spectrophotometric
studies using stopped-flow techniques has been carried out on fully oxidized
and mixed-valence bovine COX to characterize kinetic parameters of Ca2+
interaction. The reaction of Ca2+ binding
with bovine COX is second-order with a rate constant kon of
ca. 4 x 103 M-1s-1 for oxidised and 6.5x103
M-1s-1
for half-reduced forms. Dissociation of Ca2+ is characterized
by koff (4-6)x10-2 s-1 and (9-10)x10-2s-1
for oxidized and half-reduced forms respectively. Data were confirmed by
fluorescence spectroscopy monitoring Ca2+ dissociationfrom
COX with fluorescent calcium chelator Quin-2. Ca2+ dissociation
rate shows a strong temperature dependence of 19.6 kcal/mol. The apparent
Kd obtained as the koff/kon ratio (ca.15чM)
is much higher than the equilibrium value of 1чM indicating that formation
of the Ca2+-adduct involves several steps including initial
low affinity binding detected spectroscopically followed by an exergonic
isomerization to a final tight complex.
Ca-binding
shaer is composed by two patterns P1 and P2. P1 has key E-residue in the
middle and D-claster (D51/52 in bovine numbering) at the end and is localized
in the loop connecting ?-helixes
I-II. P2 is typically RRYSD with S441 in bovine and inserted in the loop
between helixes XI-XII. Bioinformatic analysis of Ca-binding site based
on sequences P1 and P2 was carried out on 135 eukaryotic COX and revealed
that it is widely spread and highly conserved in evolution. According to
published data D51 in P1 and S441 in P2 appear in evolution simultaneously
not early than Echinodermata but we found it also in some Chidaria
(Metridium senile), Mollusca (Albinaria coerulea), Annelida and Arthropoda
(was found in Insecta and Arachnida). In agreement with
earlier observations Ca site was not found in Plants and Fungi. It would
be also noted that E/D replacement is quite frequent in P1 while in P2
D/E change never occurs. Supported by RFBR grant 03-04-48203
Programmed
death of pea guard cells
D.B.
Kiselevsky
Department
of Biology, Lomonosov Moscow State University, Moscow, Russia.
Е-mail:
kis@8.cellimm.bio.msu.ru
Programmed
death (PCD) of pea guard cells was investigated. CN--Induced
death of guard cells in epidermal peels isolated from pea leaves was detected
by disappearance of nuclei. Fragmentation of the nucleus and their sensitivity
to pharmacological agents caused us to regard the death of pea guard cells
as apoptosis. Light stimulated CN--induced death. PCD of pea
guard cells depended on reactive oxygen species (ROS). PCD was inhibited
by antioxidants and prevented by anaerobiosis. The effect of various electron
acceptors and inhibitors of photosynthesis and respiration indicated that
PCD of pea guard cells was apparently regulated by the redox state of plastoquinone
in the o site of the chloroplast cytochrome b6f
complex.
In contrast to plastoquinone, the reduced ubiquinone of mitochondria inhibited
the PCD and, therefore presumably acted as antioxidant. Staurosporine,
an inhibitor of protein kinases, and non-specific protease inhibitors N-ethylmaleimide,
iodoacetamide and phenylmethylsulfonyl fluoride prevented the CN--induced
PCD. De-energization of guard cells by 2-deoxyglucose, an inhibitor of
glycolysis, or CCCP, a disconnector of oxidative and photosynthetic phosphorylation,
induced the destruction of nuclei and cell death. The effect was inhibited
by light. The combined action of 2-deoxyglucose and CCCP did not induce
a significant destruction of guard cell nuclei. Probably, an extensive
de-energization prevented apoptosis and stimulated necrosis. Cycloheximide,
an inhibitor of protein synthesis in the cytoplasm, increased the CN--induced
destruction of nuclei. Lincomycin, an inhibitor of protein synthesis in
mitochondria and chloroplasts, suppressed PCD. This data indicate that
the PCD of guard cells depends on the combined action of both proteins
synthesized in the cytoplasm and in mitochondria and chloroplasts. Possibly,
proteins synthesized in cytoplasm protect the nucleus against degradation
induced by cyanide, and proteins synthesized in the organelles promote
the destruction of nuclei. The data obtained with illumination, inhibitors
of photosynthetic electron transfer chain, electron acceptors and lincomycin
support the hypothesisthat
chloroplasts are involved in plant PCD. The effect of ROS-generating compounds
and traps specific to different ROS indicate that superoxide anion-radical
(О2-ћ), Н2О2
and hydroxyl radical (ОНћ),
but not singlet oxygen (1O2), stimulated the destruction
of nuclei. Quinacrine, an inhibitor of NADPH oxidase of plasma membrane,
prevented the CN--induced destruction of nuclei. This data supports
the idea that NADPH oxidase, similar to chloroplasts and mitochondria,
is involved in ROS-generation in guard cells that accompanies PCD induced
by CN-.
Proton
transfer at the quinone oxidizing site of the cytochrome bc1
complex of Rhodobacter
capsulatus
S.
S. Klishin 1and A.
Y. Mulkidjanian1,2,3
1A.N.BelozerskyInstitute
of Physico-Chemical Biology, Moscow University, 119992, Moscow, Russia,
2Dept. of Biophysics, University of Osnabr?ck, Osnabr?ck, Germany,3Max
Planck Institute of Biophysics, Frankfurt, Germany.
We
studied the flash-triggered operation of cytochrome bc1
complex (bc1) in the vesicular preparations of
the inner cellular membranes (chromatophores), which were isolated from
phototrophically grown cells of Rhodobacter capsulatus. As
shown earlier [2], Zn2+, an acknowledged inhibitor of the mitochondrial
bc1 [5, 3, 1], retarded the heme bh
oxidation and made its flash-induced redox changes visible. We have found
that the electrogenic reaction in bc1
slowed down in the presence of Zn2+ but bc1
remained fully functional. Under all conditions tested, the reduction of
heme bh
remained much faster than the voltage generation and the reduction of cytochrome
c1. The latter two reactions followed the oxidation of
heme bh.
The ions of Zn2+ also retarded the proton release from center P.
We suggest a mechanistic scheme that helps to rationalize how the
proton release by bc1, the voltage generation, and the
cytochrome c1 re-reduction by ubiquinol can be coupled
with each other and with the oxidation of heme bh.
According to this scheme, the turnover of bc1 proceeds
in two steps. During the first faster step, the quinol oxidation
in center P leads to the coupled
electron and proton transfer to the FeS cluster yielding a semiquinone
in center P. The next electron
goes from the semiquinone first to heme bl
and then to heme bh.This
transfer of a negative charge across the membrane seems to be essentially
electrically silenced as no compatibly fast Dy
generation was observed. The
reduced and protonated FeS domain remains docked to cytochrome
b. During
the next step, the oxidation of heme bh
in center N enables, in agreement with the earlier suggestion of
the allosteric coupling between centers N and P [4],
the undocking of the FeS domain and its movement towards cytochrome
c1.Based on analogy with other
enzymes, we suggest that the histidine-rich Zn2+-binding
patch of cytochrome b serves as a proton exit from center P.
This exit opens with the movement of the FeS domain and
proton(s) leave center P.
This proton release, as well as the reversion of the reactions that silenced
the ET towards heme bh
upon the previous turnover step, account together for the electrogenic
reaction.
2.
Klishin SS, Junge W, Mulkidjanian AY (2002) Biochim. Biophys. Acta
1553, 177-182.
3.Link
TA, von Jagow G (1995)J. Biol
Chem. 270, 25001-25006.
4.Mulkidjanian
AY, Junge W (1995) in Photosynthesis: From Light to Biosphere. (Edited
by P Mathis), pp. 547-550. Kluwer,
Dordrecht, The Netherlands.
5.Skulachev
VP, Chistyakov VV, Jasaitis AA, Smirnova EG (1967) Biochem. Biophys.
Res. Commun. 261-6.
Protein
phosphorylation as a way of the regulation of Ca-ATPase activity in skeletal
muscle sarcoplasmic reticulum of hibernator, the groun squirrel Spermophilus
undulatus
A.
S. Kondrashev-Lugovskii,
O. D. Lopina, A. M. Rubtsov
Department
of Biochemistry, School of Biology, Lomonosov Moscow State University,
119992 Moscow, Russia.
Hibernation
(winter torpor state) is a way of adaptation of many small mammals for
severe external conditions. This process is accompanied by numerous changes
in animal metabolism directed to minimization of energy consumption, in
particular, by significant changes in skeletal muscle metabolism. Sarcoplasmic
reticulum Ca-ATPase is a key enzyme providing intracellular exchange of
Ca2+ ions. We have found that the activity of Ca-ATPase is 2-fold
decreased during hibernation despite of not very significant change of
the content of this enzyme in sarcoplasmic reticulum membranes. It was
shown earlier that the incubation of homogenates of ground squirrel skeletal
muscles in the medium activating endogenous protein kinases results in
activation of Ca-ATPase in the case of hibernating animals but does not
change the enzyme activity in homogenates of summer active ones. The same
results were obtained with the use of isolated preparations of sarcoplasmic
reticulum. The data of radioautographic studies shows that sarcoplasmic
reticulum vesicles isolated from skeletal muscles of both active and hibernating
ground squirrels possess relatively high endogenous protein kinase activity;
the level of phosphate incorporation into sarcoplasmic reticulum proteins
was about 200 pmol/mg of protein in both cases. However, the Ca-ATPase
protein was not a target for endogenous protein kinases. Solubilization
of sarcoplasmic reticulum vesicles by nonionic detergent, Triton X-100,
significantly increases the level of phosphate incorporation into the proteins
in preparations from summer active and winter hibernating animals (3-fold
and 5-fold, respectively). Nonspecific inhibitor of protein kinases, heparin,
which does not penetrate across sarcoplasmic reticulum membrane, practically
does not affect the protein phosphorylation in native sarcoplasmic reticulum
vesicles but totally suppress phosphorylation in preparations treated by
Triton X-100. The high sensitivity of endogenous protein kinase activity
to heparin and effect of Triton X-100 on protein phosphorylation allow
to suggest that this activity in sarcoplasmic reticulum belongs mainly
to kasein kinase which is located in sarcoplasmic reticulum lumen. We suggest
also that the activity of Ca-ATPase is regulated by reversible phosphorylation
of non-identified protein(s) located in sarcoplasmic reticulum lumen. Supported
in part by RFBR Grant 03-04-48661.
Periplasmic
superoxide radical formation in E. coli: respiratory chain as a major source
S.
S. Korshunov and
J. A. Imlay
Department
of Microbiology, University of Illinois at Urbana- Champaign, 601 S. Goodwin
Ave, CLSL, Urbana, Illinois, 61801.
Superoxide
dismutases (SODs) are ubiquitous in aerobic organisms.Cytosolic
SODs protect vulnerable enzymes from internal sources of O2-.E.
coli and some other gram-negative bacteria also express periplasmic SODs
in stationary phase. The functions of those enzymes are not yet clear.
A popular hypothesis is that they protect periplasmic targets from O2-
that is made by external sources in the environment.However,
in the present study we show that both log-phase and stationary-phase E.
coli generate O2- that can reduce extracellular cytochrome
c.Overproduction of periplasmic
SOD blocked O2- detection, indicating that the O2-
was generated in that compartment. In log phase the rate of periplasmic
O2- production was about 0.04% of total oxygen consumption,
which is similar to the rate of cytosolic O2- production.
The rate of O2- production increased linearly with
oxygen concentration, showing that the O2- was formed
by an adventitious chemical reaction. The respiratory chain was the primary
source, as mutants lacking NADH dehydrogenases made little O2-
in either growth phase. In log-phase cells, little O2-
was produced by menA mutants, even though menaquinone is a minor
component of the quinone pool. General overreduction of quinones led to
increased O2- formation.It
seems likely either that these reduced low-potential quinones react directly
with oxygen, or that they do so when complexed to respiratory enzymes.However,
none of the major complexes of the aerobic respiratory chain was required
for periplasmic O2- production.Stationary-phase
cells produced periplasmic O2- at rates about half
that of log-phase cells. While stationary-phase O2-
formation required a functional respiratory chain, the mechanism was different
than log-phase production, since menaquinone was not essential.We
conclude that periplasmic SODs probably defend the cell against O2-
that is released from the periplasmic face of the respiratory chain.
Formation
of redox-dependent complex at participation of ADP/ATP- and aspartate/glutamate
antiporters during the uncouplingmof oxidative phosphorilathion by fatty
acid in liver mitochondria of old rats
O.V.
Kozhina, V.N. Samartsev
Mari
State University, pl. Lenina 1, 424001, Yoshkar-Ola, Russia
E-mail: laiken@marsu.ru
Protonophoric
uncoupling action of palmitate and recoupling effects of carboxyatractylate
and aspartate were studied on liver mitochondria of 22-28-month-old rats
with body mass of 400-500 g (old rats). It has been established that mitochondria
of old rats, by contrast of young rats, are characterized by lower rate
of respiration in state 4 and smaller uncoupling palmitate activity. Recoupling
effects of carboxyatractylate and aspartate are very small at addition
of these substances after palmitate, but when these substances are added
in other sequence - carboxyatractylate after aspartate or aspartate after
carboxyatractylate - their recoupling effects are significantly increased.
These data are considered as evidence that in old rats mitochondria ADP/ATP-
and aspartate/glutamate antiporters are involved in uncoupling function
as single uncoupling complex with the common fatty acids pool. The fatty
acid molecules are able to move: from ADP/ATP antiporter to aspartate/glutamate
antiporter on carboxyatractylate addition, and in the opposite direction
on aspartate addition. In the presence of pyruvate or ?-hydroxybutyrate,
which are able to reduce mitochondrial pyridine nucleotides, and also in
the presence thiourea - one of the reducers of thiol groups of mitochondrial
proteins, the complex does not occur. Possible mechanisms of formation
of uncoupling complex at participation of ADP/ATP- and aspartate/glutamate
antiporters and its destruction by reducing of protein thiols at ageing
of animals are discussed.
This
work was supported by the Interuniversity Scientific Program 'Universities
of Russia' (UR.07.01.013).
The
effects of the Pro L209 mutations in reaction center of Rb. sphaeroides
M.
A. Kozlova1,2,
D. A. Cherepanov1,2, L. Baciou3, P. Sebban3
& A. Y. Mulkidjanian1,2
1
A.N.Belozersky Institute of Physico-Chemical Biology, Moscow University,
119899, Moscow, Russia, 2 Division of Biophysics, Department
of Biology/Chemistry, University of Osnabr?ck, Osnabr?ck, Germany, 3
Laboratoire de Chimie Physique, Bat. 350, University Paris-IX, Orsay, France.
The
rate of the electron transfer in protein can be limited by charge redistribution
and/or conformational changes [1]. We studied the effect of the Pro L209
replacement by Tyr or Glu in the Rb. sphaeroides reaction center
(RC) [2,3] on protein relaxation accompanying the flash-induced reduction
of the secondary quinone QB.
Electrogenic
proton uptake was monitored electrometrically in the membrane vesicles
(chromatophores) upon semiquinone and quinol formation (in response to
the first and second flash, respectively). The mutant RCs had lower activation
energies of proton uptake slow kinetic component both after the first and
second flash (~30 kJ/mol) than wild type RCs (~50 kJ/mol). Together with
observed increase in accessibility for inhibitors in the mutant quinone-binding
sites, this evidence pointed to less tight quinone binding in mutant RCs.
Based
on steered molecular dynamics simulations of the QB rotational
motion inside the pocket, activation energy was calculated for WT (60 kJ/mol)
and L209PE mutant (40 kJ/mol) RCs.
Both
computational and experimental results indicate that high activation energy
of the electron/proton transfer to QB
might be due to the conformational changes in the protein adjacent to QB
site, as suggested in [1, 4-6], and Pro L209 essentially contributes to
the tightness of the quinone-binding pocket.
2. Kuglstatter
A, Ermler U, Michel H, Baciou L, Fritzsch G. Biochemistry2001
40, 4253-4260
3. Tandori,
J., Maroti, P., Alexov, E., Sebban, P., Baciou, L. Proc. Natl.
Acad. Sci. USA
2002, 99 6702-6706.
4. O.A.
Gopta, D.A. Bloch, D.A. Cherepanov, A.Y. Mulkidjanian, FEBS Lett.
1997, 412, 490-494
5.
Mulkidjanian, A. Y. FEBS Lett.1999, 463, 199-204.
6.
Cherepanov, D. A., Bibikov, S. I., Bibikova, M. V., Bloch, D. A., Drachev,
L. A., Gopta, O. A., Oesterhelt, D., Semenov, A. Y., and Mulkidjanian,
A. Y. Biochim. Biophys. Acta 2000, 1459, 10-34.
Proton
translocation catalyzed by Paracoccus denitrificans NADH:quinone
oxidoreductase (NDH-1)
M.
A. Kulikova,
A. V. Ushakova, and V. G. Grivennikova
Department
of Biochemistry, School of Biology, Moscow State University, Moscow 119992,
Russia.
The
stoichiometry quotient n ()
for vectorial translocation of protons coupled with NADH:ubiquinone oxidoreduction
is a key parameter for any possible model of energy transduction mechanism
catalyzed by Complex I. The value of 4 has been determined for both rotenone-sensitive
and rotenone insensitive NADH:ubiquinone oxidoreduction in tightly coupled
bovine heart submitochondrial particles [1]. Prokaryotic homologue of the
mammalian Complex I (46 different subunits) is composed of only 14 polypeptides.
The trivial proposal can be envisaged that extra 32 subunits of the eukaryotic
enzyme have evolved to increase thermodynamic efficiency of the energy
accumulating machinery. To check this possibility and to get closer insight
into coupling mechanism the experimental procedure similar to that previously
used for the mitochondrial system was applied to determine n for
the simpler prokaryotic NDH-1 dehydrogenase operating in tightly coupled
inside-out vesicles derived from Paracoccus denitrificans.
We
found that 'non-permeable' pH-indicator Phenol Red shows abnormal spectral
response when used with Paracoccus denitrificans vesicles under
standard experimental conditions in NADH-induced pH-pulse experiments previously
employed for submitochondrial particles. The abnormal response was energy-dependent
and most likely due to either redox activity of the dye or its imperfect
non-permeability through the bacterial plasma membrane. The experimental
conditions that allow to eliminate side reaction of Phenol Red were found.
The stoichiometry of
3,8 + 0,4 for proton-translocating NADH:quinone oxidoreductase reaction
was determined. Supported by the Russian Foundation for Fundamental Research
(grant 02-04-48679 and 03-04-48202), Program Leading Schools in Science
(grant 596.2003.4) and NIH Fogarty International Research grant 1R03TW006041.
References:
1.
A.S. Galkin, V.G. Grivennikova, A.D. Vinogradov, FEBS Let. 451 (1999),
157-161.
Сytochrome
c oxidase inhibition by zinc ions
S.S.
Kuznetsova, N.V. Azarkina, T.V. Vygodina, S.A. Siletsky, A.A. Konstantinov.
A.N.
Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Moscow, Russia.
Zn2+
ions inhibition of cytochrome c oxidase (COX) activity has been
studied on mitochondrial and bacterial COX both in solubilized and liposome-reconstituted
forms. We find out that the effect of zinc on the solubilized COX develops
in two time-scales. Initial rapid interaction of zinc with the site exposed
to inner aqueous phase (corresponding to mitochondrial matrix.) is fully
reversed by EDTA and results in a partial inhibition of the enzyme activity
(50-90%, depending on preparation) with an effective Ki ofca.
10-5 M. Presumably, zinc blocks the entrance of the D-proton
channel. Rapid phase is followed by slow (tens minutes-hours) zinc binding
to COX which results in almost complete inhibition of enzyme and is only
partly reversible. The slow phase is characterized by increased affinity
to the inhibitor. The rate of inhibition in the slow phase is proportional
to Zn2+ concentration. At less than 10-5 M Zn2+
binding to COX converts to slow. According to published data outside Zn2+
inhibit COX activity in proteoliposomes (COV) only in the coupled state.
In fact effect of Zn2+ is not observed in an uncoupled COV but
re-appears when proteoliposomes are supplied with alamethicin that makes
the membrane permeable to low-molecular substances and allows Zn2+
to go inside. Outside Zn2+ does not change steady-state ??-generation
by COV measured as safranine consumption but inhibits proton pumping especially
effective in mammalian COX.. At the same time we observed slow inhibitory
effect of outside zinc in turning over uncoupled liposomes similar to that
described in earlier observations. Possible explanation comes when we found
out that in the presence of weak reductants outside zinc demonstrated fast
and reversible inhibition of cytochrome c oxidation by the uncoupled
COV. Presence of weak reductants also restored Zn2+ inhibitory
effect on ??generation
by COV. Inhibitory effect is highly enhanced if Zn2+ and weak
reductans are preincubated with COV whereas incubation with zinc alone
does not change COV's enzymatic activity. This inhibition is only partly
reversed by chelators. Thus cytochrome oxidase has at least two Zn2+
binding sites. One is located inside near D-channel and is responsible
for main Zn2+ effects on solubilized COX. The second is somewhere
outside and becomes accessible only in turning over COV or in the presence
of weak reductants. Detailed spectrophotometric measurements nevertheless
do not reveal reduction of COX heme centers or formation of oxygen intermediates
under proteoliposome treatment with weak reductants so we presumed the
effect to be associated with reduction of the 'invisible' CuB..
Independently it was shown by Siletsky in our group that outside Zn2+inhibit
specifically the slow electrogenic phase of proton transfer coupled to
F => Ox transition corresponding to transfer of the 4-th electronin
the COX catalytic cycle. Supported by RFBR grant 03-04-48203.
Effects
of palmitate on the mitochondrial NADH:ubiquinone oxidoreductase (complex
I)
M.
V. Loskovich,
V. G. Grivennikova, and A. D. Vinogradov
Department
of Biochemistry, School of Biology, Moscow State University, Moscow 119992,
Russia.
Palmitate
rapidly, specifically and reversibly inhibits the uncoupled NADH oxidase
activity catalysed by active Complex I in inside-out bovine heart submitochondrial
particles (IC50 extrapolated to zero enzyme concentration is
equal to 9 mM
at 25њC,
pH 8.0) and in permealized mitochondria. The NADH:hexaammineruthenium reductase
activity of Complex I is insensitive to palmitate. Partial (about 50%)
inhibition of the NADH:external quinone reductase activity is seen at saturating
palmitate concentration and the residual activity is fully sensitive to
piericidin. The uncoupled succinate oxidase activity is considerably less
sensitive to palmitate. Only slight stimulation of tightly coupled respiration
with NADH as the substrate is seen at optimal palmitate concentrations
whereas complete relief of the respiratory control is observed with succinate
as the substrate. Palmitate strongly prevents the turnover-induced activation
of the de-activated Complex I (IC50 extrapolated to zero enzyme
concentration is equal to 3 mM
at 25њC,
pH 8.0). The mode of action of palmitate on the NADH oxidase is qualitatively
temperature-dependent. Rapid and reversible inhibition of the Complex I
catalytic activity and its de-active to active state transition are seen
at 25њC,
whereas the slow progressive inactivation of the NADH oxidase proceeds
at 37њC.
Palmitate drastically increases the rate of the temperature-dependent de-activation
of Complex I in the absence of NADH. The time-dependent inactivation of
the NADH oxidase is prevented but only partially reversed by bovine serum
albumin. Taken together these data suggest that free fatty acids act as
specific Complex I directed inhibitors; at physiologically relevant temperature
(37њC)
their inhibitory effects on mitochondrial NADH oxidation is due to perturbation
of the pseudo-reversible active - de-active Complex I transition [1]. Supported
by the Russian Foundation for Fundamental Research (grant 02-04-48679 and
grant 03-04-48202), Program Leading Schools in Science (grant 596.2003.4),
NIH Fogarty International Research (grant 1R03TW006041).
References:
1.
Loskovich, M. V., Grivennikova, V. G., Cecchini, G. and Vinogradov, A.
D. (2005) Biochem. J. 386, in the press.
Kinetic
model of mitochondrial adeninenucleotide translocase
E.
Metelkin, O.
Demin
Moscow
State University, A.N. Belozersky Institute of Physico-Chemical Biology,
Moscow, 119992, Russia.
Tel.
(903)793-1645, Fax: (095)939-3181, e-mail: emetelkin@yandex.ru
Adenine
nucleotide translocase (ANT) is one of the main protein components of mitochondria.
It catalyzes ATP/ADP exchange across inner mitochondrial membrane and can
limit velocity of oxidative phosphorylation. Resuming available data about
the carrier monomer structure and dependences of exchange velocity on membrane
potential difference the kinetic model was developed by us which was based
on dimeric structure assumption. The model describes the dependences of
exchange velocity on nucleotide concentrations, magnesium concentrations,
pH and potential difference value. Model verification shows a good
agreement between the model results and exchange velocity dependences (1)
in liposomal particles. Using the experimental data (1) the kinetic constants
were fitted which were contained in velocity equation. Using estimated
parameters and model equations theoretical dependences on potential difference
and pH was plotted.
References:
1.Kramer
R., Klingenberg M., Biochemistry, 1982, v. 21, pp. 1082-1089.
Permeability
transition independent depletion of NAD(P)H in heart mitochondria induced
by Ca2+ overload
V.
Milda?ien?1,2,
R. Banien?1, J. Grigien?1, R. ??kiene2,
A. Dapk?nas2
1Inst.
for Biomed. Res., Kaunas University of Medicine, Eiveniu 4, 50009, 2Vytautas
Magnus University, Vileikos 8, 44404 Kaunas, Lithuania
The
redox state of mitochondrial pyridine nucleotides is important for the
function of mitochondria and for their structural integrity. The opening
of the permeability transition pore (PTP) under condition of calcium overload
is regulated by the redox state of pyridine nucleotides. We have found
that Ca2+ overload causes very strong inhibition of pyruvate+
malate oxidation that is explained by draining off the substrate of the
respiratory chain, NADH. At the same time the amount of NADPH in mitochondria
is also substantially decreased. The rotenone insensitive decline in NAD(P)H
occurs only under condition when Ca2+ is accumulated in mitochondria
and it does not depend on the origin the substrate (succinate+rotenone,
endogenous substrates or NAD-dependent exogenous substrates). By measuring
NAD(P)H fluorescence we have determined the kinetic dependence of Ca2+
induced NAD(P)H oxidation in heart mitochondria respiring with pyruvate+
malate on PTP inhibitors ATP and cyclosporin A (CsA). In line with the
suggestion (DiLisa et. al., 2001) that loss of mitochondrial pyridine nucleotides
is explained by their leak through PTP, we obtained that decrease in NAD(P)H
concentration occurs 2.5 times faster in the absence of ATP and CsA. However,
even in the presence of ATP and CsA, an appreciable rate of NAD(P)H oxidation
was registered that comprised about 1/3 of the rate when the PTP is open.
The rate of mitochondrial respiration with all NAD-dependent substrates
is substantially inhibited by Ca2+ overload even under condition
when PTP remains closed. Therefore we suggest that Ca2+ overload
activates yet unidentified rotenone insensitive NAD(P)H-oxidazing system
in mitochondrial matrix that competes for NADH with the respiratory chain
and therefore may lead to: 1) inhibition of mitochondrial respiration;
2) reduction of free radical generation by the respiratory chain; 3) generation
of NAD+ and NADP+ for the stress response pathways;
4) and finally - to the opening PTP that is followed by very fast release
of NAD+ and NADP+ to cytoplasm.
Plasoquinone
participation in oxygen reduction in thylakoids and intramembrane formation
of hydrogen peroxide
M.M.
Mubarakshina, S.A.
Khorobrykh, B.N. Ivanov
Institute
of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow
region, 142290. E-mail: ivabor@issp.serpukhov.su
Photosynthetic
electron transport chain is one of the main places of reactive oxygen species
generation in plants. To characterize
in detail such a generation the rates of oxygen consumption in the light
in a whole photosynthetic electron transport chain (PETC) as well asin
a short chain containing only Photosystem I (PSI) in the presence of diuron
and donor pair ascorbate/ N,N,N',N' -tetramethyl-p-phenylenediamine
(TMPD) were measured in isolatedpea
thylakoids at рН
5.0, рН 6.5
and рН 7.8.
At рН 5.0
these rates were saturated at light intensity close to 500
mmols quanta m-2
s-1; at higher pHs they were not saturated up to 900 mmols
quanta m-2 s-1. The difference between the rates
of oxygen reduction in the whole chain and in the PSI-chain was used for
an estimation of contribution in this reduction of other, besides PSI,
segments of PETC. At high light intensities this contribution achieved
a constant value 50 % at рН
5.0, and was close to 70 % at рН
6.5 and рН
7.8. The data are explained as the result of plastoquinone pool participation
in two processes of oxygen reduction in PETC, namely, in a reduction of
molecules О2
by plastosemiquinone as well as in a reduction of superoxide radicals generated
in the first process and in PSI, by plastohydroquinone. This hypothesis
implies that hydrogen peroxide formation can occur inside of thylakoid
membranes. In the presence of cyt. C,which
traps superoxide radicals outside thylakoid membrane, we have really found
the hydrogen peroxide formation in the light. The study of effects of both
lumen pH and lumen volume on the electron flow to hydrogen peroxide showed
that its formation unlikely occurred in lumen. The possibility of hydrogen
peroxide generation inside the thylakoid membrane is the fact of importance
since such molecules can leave the membrane and even the chloroplast, avoiding
the scavenging systems.
Investigation
of the mitochondrial ultrastructure by the small angle neutron scattering
method (SANS)
T.N.
Murugova,
V.I. Gordeliy, A.I. Kuklin, A. Nuernberg, L.S. Yaguzhinsky
Frank
Laboratory of Neutron Physics, Joint Institute for Nuclear Research,Dubna,Russia,
A.N.
Belozersky Institute of Physico-Chemical Biology
,
Moscow State University, Moscow.
In
present work the small angle neutron scattering (SANS) was applied for
investigation of mitochondrial membranes structural changes induced by
osmotic swelling. The aim of the work is finding out structural reconstructions,
which relate to mitochondrial functional changes. SANS experiments with
intact rat liver mitochondria were carried out on the YuMO spectrometer
(Joint Institute for Nuclear Research,
reactor IBR-2, Dubna, Russia) [2,3]. Treatment of SANS data has
been realized by program package SAS [4]. Mitochondrial swelling
was induced in two ways: placing mitochondria into hypotonic media and
opening nonspecific pore in mitochondrial membranes. Incubation media was
based on variety ratio mass of H2O and D2O
to vary scattering density of media. This allows to observe neutron scattering
on protein and lipidic components of mitochondrial membrane separately.
Scattering
curves for mitochondria in isotonic media (when mitochondrial matrix is
in native state) and in hypotonic media (when matrix is swelled) differ
each from other. In case of D2O- hypotonic media scattering
curve has structural maximum(Fig.
1b). In case of isotonic media the maximum is absent (Fig. 1а).
Scattering curves for mitochondria swelled by opening nonspecific pore
resembles to curves for mitochondria in hypotonic media. So effect of swelling
do not depends on means of swelling.
Isolated
from overall scattering intensity structural factor presents two peaks.
The peaks positions are near 0.042 ?-1 and 0.079 ?-1;
that corresponds to 150 ? and 80 ? in direct space. We connect this two
correlative parameters with distance between membranes forming mitochondrial
crista and distance between Otter and inner membrane correspondingly. Considering
thickness of mitochondrial inner membrane to be 75 ?, we
obtained the
crista thickness
to be equal 225 ?, and the thickness of interface of inner and otter mitochondrial
membrane to be equal 155 ?. At that the thickness of gap between cristae
membrane proved to be equal 75 ? and between otter and inner membrane -
5 ?.
Electron
microscopy report cristae thickness under swelling to be 236 ? and the
thickness of interface of inner and otter mitochondrial membrane to be
133 ? [1]. Thus data of SANS come to an agreement with data of electron
microscopy. This confirms our supposition about cause of appearance of
the peak. With help of contrast variation method it was shown, that the
appearance of the maximum is determinedby
scattering of neutrons on lipidic component of the mitochondrial membrane.
Hence
SANS allows to register qualitative structural changes in mitochondrion.
As a result of swelling crista collapsed itself as well as inner membrane
is closely adjacent to otter membrane, so the gap between cristae membrane
has got equal 65 ? and between otter and inner membrane - about 5 ?,. This
effect doesn't depend on way of inducing swelling of the organelle: hypotonic
media or nonspecific pore. Results obtained are correspond to Manella's
'closed cristae' model of inner mitochondrial membrane [5].
References:
1. I.
P. Krasinskaya, I.S. Litvinov, S.D. Zaharov, L. E. Bakeeva, L. S. Yaguzhinsky.
Two qualitatively different structural-and-functional states of mitochondria,
Biochim. Т.54?9.
1989. 1556-1561.
2.
Kuklin A.I., Islamov A. Kh., V.I. Gordeliy "Two Detectors System for Small
Angle Neutron Scattering Instrument", Submitted to J. Appl. Crystal.
3. http://nfdb.jinr.ru:800/cocoon/hipns/ibr-2.instr?instr_id=905.
4. http://www.jinr.ru/~tsap/Koi/jinrlib/sas/index.html
5. Mannella
CA, Marko M, Buttle K. Reconsidering mitochondrial structure: new views
of an old organelle. Trends Biochem Sci. 1997 Feb;22(2):37-8.
The
role of oxidation of glyceraldehyde-3-phosphate dehydrogenase in the induction
of apoptosis and in the formation of amyloid structures
I.N.Naletova1,2, I.
N. Shalova1, L. Saso3, E.V. Schmalhausen2,
V.I. Muronetz1,2
1School
of Bioengineering and Bioinformatics, Lomonosov Moscow State University,
2Belozersky Institute of Physico-Chemical Biology, Moscow
State University, Moscow 119992, Russian Federation, E-mail: naletova@belozersky.msu.ru,
3Department of Pharmacology of Natural Substances and
General Physiology, University of Rome La Sapienza, Piazzale Aldo Moro
5, 00185 Rome, Italy.
The
goal of the work was to study the role of oxidation of glyceraldehyde-3-phosphatedehydrogenase
(GAPDH) in the induction of apoptosis and inthe
formation of amyloid structures. On the first step, it was shown that the
denatured GAPDH oxidized by hydrogen peroxide does not affect the spontaneous
renaturation of GAPDH and other investigated enzymes, but inhibit the chaperon-depending
step of the renaturation. Presumably, the oxidized forms of the denatured
GAPDH that are unable to acquire the native conformation bind irreversibly
to the chaperonin and prevent its functioning. A decrease in the concentration
of active chaperones can result in the accumulation of denatures proteins
and their aggregation. This observation is the first confirmation of the
main idea of the work concerning the role of oxidative stress in the formation
of the amyloid structures. Immunofluorecsent spectroscopy revealed the
translocation of the non-native forms of GADPH from the cytoplasm to the
nucleus in the murine L-fibroblasts under the oxidative stress caused by
the addition of hydrogen peroxide. In the transgenic mice with the Alzheimer's
disease, 25-30% decrease in the specific activity of GAPDH was observed
in different parts of brain. The content of GAPDH determined with the specific
antibodies did not change, this supporting the idea concerning the accumulation
of the non-native (presumably oxidized) forms of GAPDH in the development
of this disease.
The
work was supported by the Russian Foundation of Basic Research (grant02-04-48076
and the grant ? 04-04-81038
for collaborative studies with Byelorussia), INTAS (03-51-4813) and NATO
(LST.CLG. 979533).
Reactive
oxygen species and their sources in programmed death of pea guard cells
A.V.
Nesov, Yu.V.
Kusnetsova, A.V. Shestak, D.B. Kiselevsky
Department
of Biology, Lomonosov Moscow State University, Moscow.
Е-mail:
kis@8.cellimm.bio.msu.ru
CN-
was used as an inducer of programmed death (PCD) of guard cells in epidermal
peels isolated from pea leaves. PCD was detected by light microscopy of
the destruction of cell nuclei. It was shown that CN--induced
death of pea guard cells was stimulated by light and depended on reactive
oxygen species (ROS). Methyl viologen and menadione generate superoxide
anion-radical (О2-ћ).
These compounds are reduced by photosynthetic and respiratory electron
transfer chains and spontaneously oxidized by oxygen. Methyl viologen and
menadione did not induce the disappearance of nuclei in guard cells per
se and not stimulated but prevented CN--induced destruction
of nuclei. Presumably, this effect is caused by methyl viologen and menadione
that operate as electron acceptors in the Hill reaction. Nitroblue tetrazolium,
a trap for О2-ћ,
inhibited CN--induced PCD. Н2O2
per se did not induce destruction of nuclei at concentrations up to 10
mM in the light and 50 mM in the dark but significantly stimulated CN--induced
degradation of nuclei. Mannitol and ethanol, acting as quenchers of hydroxyl
radical (ОНћ),
prevented PCD caused by CN-. NaN3, an inhibitor of
heme-contaning enzymes like cyanide, possesses quencher properties with
respect to singlet oxygen (1O2). NaN3
induced degradation of nuclei and stimulated CN--induced destruction
of nuclei. Bengal rose generates 1O2 in green light.
This compound insignificantly induced destruction of nuclei in the dark,
but not in the light, had no influence on CN--induced destruction
of nuclei without the illumination, and inhibited CN--induced
PCD in the light. Probably, the effect of Bengal rose involved 1O2-stimulated
degradation of protein D1 in photosystem II. Histidine was tested as an
antioxidant specific to 1O2. This reagent at various
concentrations had no influence on PCD caused by CN-. The results
indicate that О2-ћ, Н2О2
and ОНћ,
but not 1O2, stimulated destruction of nuclei. The
main sources of ROS in plant cells are enzymes performing redox reactions
involving components of chloroplast and mitochondria electron transfer
chains. The NADPH oxidase of the plasma membrane plays a significant role
in plant diseases. This multi-enzyme complex generates ROS protecting plant
organisms from the invasion of pathogens. Quinacrine, an inhibitor of the
NADPH oxidase, prevented CN--induced destruction of nuclei.
This supports the idea that NADPH oxidase, as well as chloroplasts and
mitochondria, participates in ROS-generation in guard cells during CN--induced
PCD. Fluorometric and oxymetric dates were obtained that demonstrate ROS-generation
in epidermal peels isolated from pea leaves at the primary stages of PCD.
The
regulation of cation transport at the level of plastoqinone reduction by
photosystem II of chloroplasts
V.
K. Opanasenko
Institute
of Basic Problems ofBiology, RAS,
Pushchino
E-mail: opanasenko@hotbox.ru
The
interaction of DCCD with ionic channels of various cell organelle is studied
more than 30 years. It has been detail investigated on the plant thylakoid
membranes inthe laboratories ofW.
Junge (1992-1994) and P. Horton (1994-1998). The authors used the chloroplasts
after 10 mines dark incubation with a reagent at the ratio DCCD/Chl=2.
The choice of such a processing was explained by the fact that it led to
the occurrence of effects of cyclic protons transfer in the complex PS
II observing only at the pulse illumination of chloroplasts. The covalent
linkage of DCCD with two polypeptide of LHCII - СР26
and СР29
has been shown, and carboxylic groups modified by DCCD in these proteins
have been determined.
Our
studies have shown the occurrence of the interaction between DCCD and a
membrane observing at the stationary illumination. We have found two new
functional actions of DCCD caused by the linkage of this reagent with proteins
of PSII complex. The first effect (A) is the stimulation of leaking protons,
K+and NH4+from
a lumen at the presence of penetrating amines or nigericine, and the second
one (B) is the inhibition of this leak by the increase of DCCD concentration.
The interaction ofDCCD with carboxylic
groups of the membrane proteins concerningto
effects А
and B did not lead to the covalent binding of DCCD isotope.
The
groups participating in the effect A are modified by DCCD irreversibly,
but then they interact with amino groups or OH-groups of alcohol with the
losing of a label of DCCD. The groups concerning to effect B are modified
convertible. They gradually lose an isotope interacting with water during
the dark incubation after processing chloroplasts by DCCD. The data show
that at the uncoupling of processes of energy transformation the amines
induce the occurrence in PS II complex a channel conducting ions from a
lumen to a stroma. The channel consists of two DCCD-sensitive parts, hydrophilic
(A) and hydrophobic one (B). The part A connects the lumen with Q(B)-site,
and the part B connects Q(B)-site with the stroma. Preincubation of chloroplasts
with DCCD or DCMU leads to the fixing of an open state of the channel.
In this case the amine concentration which is necessary for the induction
of uncoupling is sharply reduced. Hence, DCCD irreversibly modifies one
or several carboxylic groups in the "gate" of the A-part of the channel.
The modification of the groups in the part B is reversible and it is necessary
to add new portion of DCCD to observe the inhibition of the protons flow
through the B-part of the channel.
Functional
characteristics of Ca-release channels (ryanodine receptors) in skeletal
muscle sarcoplasmic reticulum of the ground squirrel Spermophilus
undulatus
A.T.
Pashovkin,
O.D. Lopina, A.M. Rubtsov
Department
of Biochemistry, School of Biology, Lomonosov Moscow State University,
119992 Moscow, Russia.
Heavy
fraction of sarcoplasmic reticulum vesicles containing functional Ca-release
channels (ryanodine receptors) was isolated from skeletal muscles of typical
hibernator, the ground squirrel Spermophilus undulatus. It
was shown that the content of Ca-release channel protein (apparent molecular
mass 550 kDa) is 3-fold decreased during winter time as well as the content
of its regulatory Ca-binding proteins: 63 kDa calsequestrin (4-fold decreased),
130 kDa sarcalumenin (1.5-fold decreased), and 165 kDa histidine-rich Ca-binding
protein (1.5-fold decreased). At the same time the content of 55 kDa Ca-binding
protein calreticulin is increased 2.5-fold as well as the content of non
identified 44 kDa (2-fold increased) and 30 kDa (1.5-fold increased) proteins.
The level of phosphate incorporated into sarcoplasmic reticulum proteins
by endogenous protein kinases was significantly higher in preparations
of winter hibernating ground squirrels (220 pmol/mg of protein) in comparison
to preparations of summer active animals (140 pmol/mg of protein). In preparations
of winter hibernated ground squirrels 16 individual proteins with molecular
masses ranging from 16 to 165 kDa were the targets of endogenous protein
kinases. In preparations of summer active animals the radioactive phosphate
was found only in 10 individual protein bands. The relative level of phosphorylaton
of proteins calculated with correction on the content of individual protein
was significantly different in sarcoplasmic reticulum of winter hibernating
and summer active ground squirrels. For example, in preparations of winter
hibernating animals the level of phosphorylation of 130 kDa sarcalumenin
was almost 20-fold higher, 44 kDa protein was 10 fold higher, and 30 kDa
protein was 5-fold higher, but the level of phosphorylation of 18 kDa protein
was 4-fold lower in comparison with sarcoplasmic reticulum preparations
of summer active animals. Under conditions of passive loading of sarcoplasmic
reticulum vesicles by radioactive calcium the preparations of winter hibernated
ground squirrels demonstrate significantly lower Ca-capacity (about 10
and 30 nmol Ca2+/mg of protein, respectively) and the rate of
Ca-release (about 30 and 100 nmol Ca2+/min per mg of protein,
respectively) in comparison with summer animals. It was shown also that
in contrast to Ca-release channels of rabbit skeletal muscle sarcoplasmic
reticulum the Ca-release channels of skeletal muscles of the ground squirrels
are weakly inhibited by high Ca2+ concentrations, Mg2+
ions, and ruthenium red and are almost insensitive to well-known activators
of ryanodine receptors, AMP and caffeine. Supported in part by RFBR Grant
03-04-48661.
Kinetic
model of Bovine Liver Fructose-1,6-bisphosphatase
K.V.
Peskov1,
O.V. Demin2
1Institute
of Theoretical andExperimental
Biophysics RAS (142290, Moscow region Pushchno, Institutscaja
str. 3, Russia,) kirill_peskov@front.ru,
2A.N.Belozersky
Institute of Physico-Chemical Biology (Build.
"A", Moscow State University, Moscow,119899, Russia.) demin@genebee.msu.su
Fructose-1,6-bisphosphatase
catalyses degradation offructose-1,6-bisphosphate
to fructose-6-phosphate and inorganic phosphate and it is one of the central
enzymes of gliconeogenesis pathway. This enzyme has complex oligomeric
structure. The kinetic mechanism of monomer subunit is Random Bi Bi, where
a magnesium ion serves as initiator of the reaction [5]. There are at least
three ways of metabolic regulatios of the fructose-1,6-bisphosphatase:
inhibition with effectors (fructose-1,6-bisphosphate and AMP),substrate
inhibition with both substrates of enzyme and inhibition with products
of the reaction. By this time there is no clear understanding of the mechanism
of substrate inhibition of this enzyme [1].Another
properties of fructose-1,6-bisphosphatase isn't clear-cut too [2-5]. In
our work we made an attempt to develop kinetic model of the fructose-1,6-bisphosphatase
and investigate mechanism of substrate inhibition.
Methods
we use to develop kinetic model have been described in [6]. In our work
we estimated unknown model parameters using experimental data published
in [1-3, 5] and DbSolve package. To understand how fructose-1,6-bisphosphatase
is inhibited by its substrates. We developed a number of kinetic models
corresponding to different possible mechanisms. Each of these models has
been tested against available experimental data [1]. We found that three
aspects of substrate inhibition of fructose-1,6-bisphosphatase were necessary
to describe this experimental data:
1.Mg2+
ions can bind to fructose-1,6-bisphophate with formation of chelate complex.
2.Chelate
complex competes with fructose-1,6-bisphophate for catalytic site of the
fructose-1,6-bisphosphatase.
3.This
substrate probably has another allosteric binding site on the enzyme. Such
assumption was discussed in [4]. Allosteric inhibition is necessary to
describe the dependence of rate of the reaction from concentration of fructose-1,6-bisphophate
under conditions more than 250 чM.
Thus,
using methods of kinetic modeling, we could find mechanism of substrate
inhibition of the fructose-1,6-bisphosphatase. Also, we estimated 34 from
44 model parameters using available experimental data (32 kinetic curves).
Based on these results, we could construct kinetic model, which adequately
describes known experimental facts and data available from literature.
References:
1.
Marcus CJ at al.// JBC (1973) 248(24), 8567-73.
2.
Casazza JP at al.// JBC (1979) 254 (11), 4661-65.
3.
Ekdahl KN, Ekman P// JBC (1985) 260(26), 14173-79.
4.
Nimmo HG, Tipton KF,// Eur J Biochem (1975) 58, 575-85.
5.
Liu F, Fromm HJ // JBC (1990) 265 (13), 7401-7406.
6.
Demin OV et al. Kinetic Modeling as a Modern Technology to Explore and
Modify Living Cells.// Modeling in Molecular biology. 2004. Springer. Pp
59-105
Participation
of mitochondria in oxidative and nitrosative stress in rat kidney cells
after ischemia/reperfusion
E.Y.
Plotnikov,
M.Y. Vyssokikh, D.B. Zorov
A.N.Belozersky
Institute, Biological Faculty, Moscow State University, Moscow, Russia.
Organ
transplantation, laparotomic operations, thrombosis and low blood pressure
can provoke kidney tissue damage mediated by ischemia/reperfusion transition.
The latter can result in both oxidative and nitrosative stress in the cells
of the organ. In order to gain an insight into the cause of the damage,
we have undergone the study of the functional and structural changes of
mitochondria in vitro and in situ in kidney cells after ischemia/reperfusion.
In order to evaluate the mitochondrial transmenbrane potential, production
of reactive oxygen species (ROS) and nitric oxide (NO) we used specific
fluorescent probes: tetramethylrhodamine, 2,7-dichlorofluoresceine and
2,5-diaminofluoresceine, respectively. After staining the living kidney
slices were analyzed using laser scanning confocal microscopy, and a fluorescence
intensity in cells was measured. We were able to detect an activity of
NO-synthase (NOS) in tubule cells mitochondria, which was significantly
elevated after ischemia/reperfusion. Under these conditions, mitochondrial
membrane potential was dramatically reduced accompanied by mitochondrial
swelling. Reactive oxygen species production in ischemia-exposed kidney
was doubled as compared to control values and was also associated with
mitochondria. The treatment with NOS inhibitor and especially with mitochondrial
permeability transition inhibitor, cyclosporin A partially abolished Dy
collapse. Production of ROS and NO increased as early as 10 min of reperfusion,
and after 180 min an obvious apoptotic changes (i.e. Bax translocation
to mitochondria and cytochrome c release from mitochondria) was apparent
in kidney tubule cells.
Chemiosmotic
logic as a tool to consider self-organization in a multicellular system
T.V.
Potapova
Belozersky
Institute of Physico-Chemical Biology, Moscow State University
Ionic
transport through the plasma membrane both in procaryotic and eucaryotic
cells is a necessary link in maintaining ion-osmotic homeostasis and transmission
of information, energy provision of cell metabolism, provision of cells
with substrates and removal of active life products. In steady state conditions,
all the ionic fluxes are interequilibrated and are in conformity with the
energetic status of the cell.
Permeable
intercellular junctions (PJ) are present in most multicellular systems
(organs, tissues, developing embryos, cell cultures, multicellular microorganisms)
permitting adjacent cells to "socialize" components of the low-molecular
pool. The ability of PJ to pass ion fluxes, comparable in values with those
through the plasma membrane, makes these structures a paramount mechanism
of self-organization in a multicellular system. In considering the functional
role of PJ, the concept of Mitchell and Crane on the energetic function
of ionic gradients across biological membranes proved to be extremely useful.
On
the grounds of analytical calculations and experimental evaluations, we
have shown that inorganic ion fluxes across PJ, moving along corresponding
electrochemical gradients, can maintain "energy cooperation" on a scale
comparable with the total energy production (energy consumption) of an
individual cell. The existence of such energy cooperation of cells is revealed
in electrophysiological experiments by the presence of intercellular currents
and/or electrical gradients across PJ. The studies of fungus Neurospora
crassa allow not only to document the chemiosmotic theory by direct
electrophysiology, but, on the another band, they illustrate the power
of chemiosmotic logic to illuminate the physiology and energetics of multicellular
system possessing PJ. Consideration of the results on cyanobacteria, fungi
and animal indicates, that the values close to the normal of membrane potentials
and intracellular ion content can be maintained in a cell population with
a large share (up to 50%) of cells with inactivated ion-ATPases. The participation
of PJ in the "energy support" of one type of cell of the population by
others has been described in ischemia of the myocard. The notions of an
intratissue substructue such as the information-energy module, developed
for Neurospora, can be useful in detailed studies not only of the laws
of interactions between materials and driving forces in the course of polarized
growth, but in studies of many other functions being realized through combined
activity of a multicellular ensemble. For details see:
T.V.
Potapova. Biologicheskie membrane (in Russian), 2004, V. 21, ?3, P.P.163-191.
Modified
ADP/ATP antiporter is involved in fatty acid-induced and GDP-suppressed
uncoupling in rat kidney mitochondria
E.A.
Prikhodko1,2,
L.S. Khailova2, V.I. Dedukhova2, E.N. Mokhova2
1Faculty
of Bioengineering and Bioinformatics, Moscow State University, 2Department
of Bioenergetics, A.N. Belozersky Institute of Physico-Chemical Biology,
Moscow State University, Moscow 119992, Russia.
In
recent years (2001-2004) Brand, Murphy and colleagues investigated activated
by superoxide and suppressed by GDP uncoupling in the presence of fatty
acids or lipid peroxidation products (LPP). They proposed that GDP-sensitive
uncoupling is mediated by uncoupling proteins: UCP2 in kidney
and UCP3 in skeletal muscle mitochondria. We suggested that
the modified ADP/ATP antiporter is involved in this GDP-sensitive uncoupling,
that LPP decrease the specificity of its nucleotide-binding site(s), and
GDP binding results in suppression of the ADP/ATP antiporter-mediated uncoupling.
To
verify this assumption we studied effects of GDP and specific inhibitor
of the ADP/ATP antiporter carboxyatractylate (CAtr) on oleic or palmitic
acid-induced uncoupling in kidney mitochondria isolated from rats with
mass about 180 g. Our experimental conditions were different from those
used in the above-mentioned publications. (1) To increase CAtr coupling
effect incubation medium pH was changed from 7.2 to 7.4. (2) In the most
of assays sucrose incubation medium was used instead of salt incubation
medium (to decrease the fatty acid-induced swelling). (3) In the most assays
pyruvate was used as oxidation substrate; the rate of its oxidation was
low, so even a weak uncoupling effect resulted in an essential decrease
in ??. (4) Palmitic
acid concentration in ethanol stock solution was about 5 mM; at the higher
concentration palmitic acid-induced inhibition of respiratory activity
was increased. (5) Oleic acid and palmitic acid (the less potent uncoupler)
were added at low concentrations to produce essential stimulation of respiration
in the presence of oligomycin and only small inhibition of the uncoupled
respiration.
Under
these conditions in some assays with pyruvate only 0.2 mM GDP or only CAtr
(being added after oligomycin but before fatty acid) practically completely
prevented the oleic acid-induced ??
decrease. In other assays when GDP only partially prevented oleic acid-
or palmitic acid-induced uncoupling CAtr increased the GDP coupling effect.
GDP and CAtr being added after fatty acid produced less pronounced coupling
effect (probably due to lower ??).
These and other data support our proposal concerning mechanism of the GDP-suppressed
uncoupling.
'Mild'
uncoupling revisited
A.V.
Pustovidko,
M.Yu. Vyssokikh
A.N.Belozersky
Institute of Physico-Chemical Biology, Moscow State University, Build 'A',
1/40, GSP-2, Moscow,119992, Russia
Under
resting (respiratory control or State 4) conditions mitochondrial respiratory
chain is known to form reactive oxygen species (ROS). The mechanism of
ROS generation based on its strong activation when the electrochemical
H+-potential difference (DmH+)
is high and the rate of electron transport is limited by discharge of DmH+.
Earlier it was proposed that the cellular defense system against ROS include
the thyroid hormone-mediated uncoupling in mitochondria. This phenomenon
was named 'mild' uncoupling.
In
present work we investigated the effect of a chemical compound ('Novonordisk
0005') on respiration and membrane potential of rat heart mitochondria.It
was shown that named compound induced uncoupling of succinate supported
respiration in concentration-dependent manner (in presence of rotenone
and oligomycin).The uncoupling action
was observed from 10-8M, the maximal effective uncoupling concentration
was from 10-7M to 10-4M. In contrast to other uncouplers
studied, excess of Novonordisk 005 does not inhibit respiration.
We
have shown that uncoupling induced by '0005' can be prevented or suppressed
by 6-ketocholestanol.
We
proposed that '0005' is a 'mild' uncoupler and can be used as the potential
drug for protection from ROS-induced injury without a risk of inhibition
of respiration when concentration of uncoupler increases.
Unusual
ultrastructural alterations of mitochondria in HeLa cells induced by inhibitors
of bioenergetic functions
V.B.
Saprunova,
L.E.Bakeeva
Belozersky
Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
Moscow 119992, Russia. E-mail: bakeeva@imec.msu.ru
The
electron microscopical observations of HeLa cells treated for 48-72 h with
uncouplers in combination with antimycine or myxothiazole showed surprising
unknown in literature cell ultrastructure. Mitochondrial apparatus of the
cells was reduced to several clusters of mitochondrial fragments. There
were no mitochondria of native ultrastructure in these cells and we did
not observe accumulation of autophagosomes in cytoplasm. But in the same
time our electron microscopic investigations revealed clearly that in cytoplasm
of the cells there were many unusual electron-dense organelles. These organelles
had spherical or elongate shape surrounded with a single membrane. Sections
at different angles showed that their interior is filled with closely packed
tubulose structures. These unusual organelles unknown in the literature,
we believe that it's newly formed in the cells by stressful conditions
indused by prolonged incubation with mitochondrial poisons. Probably these
organells provide viable of the cells which devoid of mitochondria. The
work was supported by grant from the Russian Foundation for Basic Research
(04-04-48121)
and grant 'Leading Scientific Schools' from the Ministry
of Education and Science Russia (1710.2003.04).
LDL
modificatiom in the molecular mechanisms of atherosclerosis and diabetes
development
G.S.
Shepelkova, N.E.
Arzamastseva, A.K. Tikhaze, V.Z. Lankin
Objectives:
It is known that oxidative stress as well ascarbonyl
stress development may contribute to the pathogenesis of atherosclerosis
and diabetes. We investigated the levels of peroxy-LDL (oxidative derivations
of free radical peroxidation) in the blood plasma of patients with atherosclerosis
and diabetes mellitus. We suggested that aldehydes which were formed during
lipoperoxidation(malondialdehyde)
and which accompanied atherosclerosis development as well as glucose autoxidation
(glyoxal and methilglyoxal) during diabetes both could modify LDL.
Methods: The Cu2+-mediatedsusceptibility
of human LDL (prepared by ultracentrifugation) to oxidation has been studied
by means UV-spectrophotometry (absorption max at 233 nm). The concentration
of lipid hydroperoxides in LDL was estimated by modified Fe3+-xylenol
orange method before and after treatment with triphenylphosphine for lipohydroperoxides
reduction. Also, the level of fluorescent products (the Schiff bases),
which were produced during interaction between amino-groups of lysine and
different aldehydes (malondialdehyde, glyoxal and methilglyoxal), was estimated.
Results:
We observed, that susceptibility of LDL, from plasma of type 2 diabetes
coupled with coronary heart disease (CHD) patients, to Cu2+-mediated
oxidation was significantly higher than susceptibility of LDL from plasma
of patients with atherosclerosis. The level of peroxy-LDL during diabetes
also was much higher than it was during CHD with hypercholesterolemia and
especially higher than it was during CHD without hypercholesterolemia.
Therefore diabetes mellitus as well as CHD with hypercholesterolemia was
accompanied by intensification of free radical LDL peroxidation, but diabetes
- in significantly more extent. Compensation of carbohydrate metabolism(treatment
with sulfonylureas as monotherapy or in combination with metformin) led
to decreasing of peroxy-LDL level that confirmed increasing of the rate
enzymatic utilization of reactive oxygen species and lipohydroperoxides
(increasing of SOD and GSH-Px activities) as well as decreasing of the
primary and secondary products of free radical peroxidation levels (lipohydroperoxides
and malondialdehyde)in
LDL. We found that the rate of fluorescent
products (the Schiff bases) formed
during reaction of lysine amino-groups with glyoxal or methilglyoxal was
much higher than during reaction with malondialdehyde.
Conclusions:
Therefore may be suggested that both intensification of free radical lipoperoxidation
during atherosclerosis and lipoperoxide-mediated glucose autooxidation
during diabetes lead to accumulation of modified peroxy-LDL in blood of
patients.
Expression
of cytochrome P450scc in Escherichia coli: intracellular forms and
interaction with endogenous redox proteins
V.M.
Shkumatov1,
V.G. Radyuk1, Ya.V. Falertov1, A.A. Vinogradova2,
L.A. Novikova2, V.N. Luzikov2
1Institute
of Physico-Chemical Problems, State University, Minsk, Belarus, 2Belozersky
Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
Russia.
Cytochrome
P450scc is a component of the cholesterol side-chain cleavage system transforming
cholesterol into pregnenolone. This reaction proceeds in the inner membrane
of adrenocortical mitochondria with the involvement of mitochondrially-made
NADPH. In this work we used
E.
coli cells
producing cytochrome P450scc to answer two questions: 1) whether this enzyme
can exist in a soluble form and 2) whether this enzyme can be engaged in
bacterial metabolic pathways.
Homogenization
of the E. coli cells producing mature form of bovine CYP11A1 (cytochrome
P450scc) followed by centrifugation of cell homogenates at 105000g resulted
in membraneous and 'soluble' fractions both containing cytochrome P450scc.
Upon gel-permeating HPLC of the 'soluble' fraction, cytochrome P450scc
was detected amongst 400-2000-kDa particles. Affinity column chromatography
with immobilized adrenodoxin showed that cytochrome P450scc can not be
extracted from the 'soluble' fraction by binding with its ligand. Such
extraction can only be achieved after pretreating this fraction with sodium
cholate and ammonium sulfate. These data suggest that the 400-2000-kDa
particles including cytochrome P450scc constitute some lipoprotein structures.
As the 'soluble' fraction exhibited succinate dehydrogenase activity proportional
to the cytochrome P450scc content, these structures are likely to be fragments
of the bacterial cytoplasmic membrane.
In
the 'soluble' fraction cytochrome P450scc binds added cholesterol and 22R-hydroxycholesterol,
with 60-80% of its substrate-binding sites being accessible for these sterols.
In this fraction, cytochrome P450scc is also undergoes one-electron reduction
with the involvement of bacterial endogenous electron-transfer proteins
and catalyzes transformation of the above sterols under the rate amounting
up to 10% of that of the reaction in a reconstituted system composed of
isolated cytochrome P450scc, adrenodoxin and adrenodoxin reductase. Thus,
we have for the first time found evidences of compatibility of mammalian
cytochrome P450scc with bacterial redox systems.
This
work was supported by the Russian Foundation for Basic Research (grants
02-04-81001 and 02-04-48204 to V.N. Luzikov)
Study
of electrogenic proton transfer in cytochrome c oxidase
S.
Siletsky1,
A. Pawate2, K. Weiss2, R. Gennis2 , A.
Konstantinov1
1
A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Moscow, Russia, 2 Department of Biochemistry, University of
Illinois, 600 S. Mathews Street, Urbana, IL 61801.
Photo
injection of an electron into cytochrome c oxidase results in multiphase
kinetics of membrane potential generation. The electrogenic phase compositions
depend on initial state of oxidase and consist of an electron transfer
into binuclear centre and coupled proton translocation steps. Forth electron
reduction stage (F to O state transition) in catalytic cycle of bovine
heart and Rhodobacter sphaeroides cytochrome oxidases revealsthree
phases of vectorial charge movement: 'rapid', 'intermediate' and 'slow'.
In the 'rapid' phase, electron is transferred from CuA to heme a. During
the 'intermediate' and 'slow' phases,proton
transport across the membrane is driven by the energy ofheme
a reoxidation by the binuclear center. The N139D mutant of cytochrome c
oxidase from Rhodobacter sphaeroides retains full steady oxidase activity
but completely lacks proton transmembrane translocation coupled to turnover
in liposomes. The wild type 'intermediate' electrogenic phase is missing
in the non-pumping N139D mutant and probably reflects translocation of
proton to the outer aqueous side of the membrane. The 'slow' electrogenic
phase in wild type and in N139D mutant report proton transfer from the
inner aqueous phase to E286, replacing the 'chemical' proton transferred
from E286 to the heme a3/CuB centre. Significantly, with the wild type
oxidase, the proton pumping precedes the electrogenic phase associated
with the oxygen chemistry. This work was supported by Russian Fund for
Basic Research Grants ? 04-04-48856.
Regulation
of colicin E1 ion channel activity by lipid bilayer curvature
A.A.
Sobko1,
E.A. Kotova1,
S.D. Zakharov2,3,
W.A. Cramer2, Y.N.
Antonenko1
1
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Moscow 119992, Russia; 2Department of Biological Sciences, Purdue
University, West Lafayette, Indiana 47907; 3Institute of Basic
Problems of Biology, Russian Academy of Sciences, Pouschino, Moscow Region,
142290, Russia.
Spontaneous
curvature (SC) of lipid membranes is known to be critical for function
of a variety of membrane proteins. With ionic channels, a study of the
dependence of the channel activity on SC allows to elucidate the mechanism
of pore formation. The present study deals with the effect of SC on colicin
E1 channels. The mechanism of action of this pore-forming bacteriocin remains
obscure despite a great number of studies performed so far. We found that
both the colicin-induced current across a planar bilayer lipid membrane
(BLM) and carboxyfluorescein release from unilamellar lipid vesicles were
augmented by lysophosphatidylcholine and reduced by oleic acid, agents
promoting positive and negative SC, respectively. Dramatic stimulation
of colicin E1 channel activity was also observed after photodynamic or
chemical pretreatment of BLM before colicin addition. It is supposed that
the channel activation results from oxidizing modification of lipids that
leads to an increase in positive SC. Experiments with membranes of different
lipid compositions revealed a correlation between the membrane-permeabilizing
potency of colicin E1 and the bending propensity of a lipid bilayer. Models
of colicin E1 channel formation are discussed in view of the new data.
Design
and synthesis of DNA construction for expression and purification of human
apoptosis-inducing factor (AIF)
T.A.
Sysoeva, A.V.
Pustovidko, E.Y. Plotnikov, M.Y. Vyssokikh, D.B. Zorov
A.N.
Belozersky Institute of Physico-Chemical Biology,
Moscow State University
Human
apoptosis-inducing factor (AIF) is a flavoprotein with relative molecular
mass 57 kDa. This protein shares relatively high homology with several
members of flavine-dependent oxidoreductases family such as monodehydroascorbate
and byphenyl oxidoreductases. Mature protein normally confined in the mitochondrial
intermembrane space but translocates to the nucleus when apoptosis in cell
is induced. Recombinant AIF causes chromatin condensation and large-scale
fragmentation of DNA.
There
are several conflicting reports about AIF properties. We proposed that
such contradiction is a result of different protein folding followed by
isolation non-proceeded recombinant AIF from prokariotic cells.
That
is why we designed DNA-construction based on pYES2-vector for expressing
and purification AIF in yeast. In yeast cells recombinant protein has a
chance to find mitochondria for proper folding and transformation from
apo- to holoenzyme form. This construction contains HisTag-sequence between
two parts of AIF-gene. First part of the construction is mitochondrial
localization sequence (MLS); it should be processed by mitochondrial protease.
Second part is sequence corresponding to mature AIF. We propose that such
construction provides expression needed recombinant protein in yeast cells
- processed mature AIF that is fused with HisTag. These constructions based
on pET15b vector were synthesized and E.coli cells (BL21Rossetta strain)
were transformed by it. Gene construction was expressed and two proteins
were purified from E.coli by affine chromatography. Obtained proteins were
analysed by MALDI analysis. One of purified proteins is AIF-HisTag-fusion.
Second protein is product of degradation or proteolysis of recombinant
protein in bacterial cells.
Structural
mechanisms of the NF-?B redox sensibility
M.
Timchenko, A.
Lomakin,
K. Evlakov, E. Kubareva, M. Ivanovskaya
A.N.
Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Russia. E-mail: alexelomakin@yandex.ru
Nuclear
factor-kappaB (NF-?B)
is one of the key regulatory molecules in oxidative stress-induced cell
activation. The cellular redox status can modify the function of NF-?B,
whose DNA-binding activity can be inhibited by oxidative, nitrosative,
and nonphysiological agents. This inhibitory effect has been proposed to
be mediated by the oxidation of a conserved cysteine in p50 subunit of
NF-kB DNA-binding domain (Cys62) through unknown mechanisms. Earlier it
has been shown, that in oxidative stress conditions Cys62 of p50 NF-?B
is responsible for activation NF-?B
in nucleus. In the cytoplasm Cys62 of p50 exists in the oxidized form,
while in the nucleus occurs the reduction of Cys62 with participation of
redox factor Ref-1. This stage of transition Cys62 from the oxidized form
in reduced is a key in the regulation of antioxidative protection genes
transcription by NF-?B.
The aim of the work was to investigate the structural basis of redox sensibility
of NF-?B. Mutant
form of p50 subunit of NF-?B
where Cys62 was replaced with Ser was obtained by site-directed mutagenesis.
By the method of circular dichroism and cross-linking with synthetic DNA
fragments containing protein recognition site it was shown that the sensibility
of NF-?B to redox
status of Cys62 is stipulated with involvement of this Cys in formation
of unique 3D-structure of protein binding center in NF-?B-DNA
complex. The replacement of Cys62 with Ser leads to a full destruction
of the spatial organization of NF-?B
DNA-binding domain. This work was supported in part by the RFBR projects
no. 05-03-32813 andno.04-04-97255.
Relevance
of fatty acid oxidation in regulation of the outer mitochondrial membrane
permeability for ADP
A.
Toleikis, S. Trumbeckaite,
D. Majiene, L. Kursvietiene, A. Dagys
Institute
for Biomedical Research, Kaunas University of Medicine, Eiveniu Str. 4,
50009
Kaunas, Lithuania.
Our
previous studies on saponin-permeabilized rat heart muscle fibers revealed
that fatty acid (FA) oxidation induces a dramatic increase of the outer
mitochondrial membrane (OMM) permeability for ADP. The aim of this study
was further elucidation ofthe role
of FA in the regulation of oxidative phosphorylation in rat heart muscle
fibers. Oxidation
of octanoyl-L-carnitine, decanoic acid (in combination with pyruvate+malate
(PM)) and oleoyl-CoA+L-carnitine decreased app. KmADP
up to 76 +
1 mM,
66.0 +
13 mM
and 55.7 +
5 mM,
respectively, as compared to this value characteristic for PM (217 +
4 mM).
Thus, saturated (long and medium chain), unsaturated, activated or not
activated FA produce the similar effect. Furthermore, we showed that even
low concentration (2 mM)
of palmitoyl-L-carnitine (PC) caused the large decrease in the app. KmADP
(up to 108 +
17 mM
compared to 243 +
19 mM
for PM). The low app. Km value was also observed (59 +
26 mM)
when PC oxidation was titrated with exogenous ATP. Thus, the effect of
FA did not depend on the way of delivery of ADP to mitochondria (exogenous,
or endogenous - produced by ATP-ases of the cell). The low app. KmADP
value observed in case of palmitoyl- or octanoyl-L-carnitine oxidation
increased more than 3 times in medium supplemented with 5%
dextran T-70. However, the
oncotic pressure had no effect on high KmADP value
characteristic for PM. Noteworthy,
dextran diminished (by 21-25%) the State 3 respiration rate with all three
substrates. We also
demonstrated that the functional coupling between ADP/ATP translocator
and creatine kinase is preserved in mitochondria despite the significant
increase in the OMM permeability for ADP induced by FA oxidation. The findings
that FA oxidation reduces app. KmADP and, thus, increases
OMM permeability for ADP was also confirmed on human cardiac fibers.
In
conclusion, the data demonstrate the impact of the FA oxidation system
in the regulation of the OMM permeability for ADP which can be related
to the fine structural changes of mitochondria. However, the precise mechanism
of this phenomenon remains unclear.
Anomalous
binding of cytochrome c in mitochondria of heart tissue in apoptosis at
anoxia conditions
A.A.
Tonshin, N.V.
Lobysheva
A.N.
Belozersky Institute of Physico-Chemical Biology
,
Moscow State University, Moscow, 119899, Russia.Fax
(095) 939-0338; E-mail:
1.Mannella
CA, Marko M, Buttle K. Reconsidering mitochondrial structure: new views
of an old organelle.
Trends Biochem Sci. 1997 Feb;22(2):37-8.
Voltage
changes on the reducing side of photosystem II complex
A.A.
Tyunyatkina
and M.D. Mamedov
A.N.Belozersky
Institute of Physico-Chemical Biology of M.V. Lomonosov Moscow State University,
119992 Moscow, Leninskie gory; Fax: (095) 939 3181, email:mahir@genebee.msu.ru
The
generation of transmembrane electric potential difference (??) in quinone
acceptor complex of proteoliposomes containing spinach core complexes of
photosystem II were studied by direct electrometrical technique. Besides
the fast increase in the photoelectric response associated with electron
transfer reaction between redox-active tyrosine-161 (YZ) in
D1 polypeptide and the primary quinone acceptor QA, an additional
electrogenic phase with ? ~0.85 ms at pH 7.3 and maximal relative amplitude
of ~11% was observed after the second light pulse. The sensitivity of this
phase to diuron - an inhibitor of electron transfer between the QA
and secondary QB quinone acceptors, the flash-number dependency
of its amplitude as well as the rate constant decrease with increasing
pH indicate that it is due to the dismutation of QA-
and QB- and to subsequent protonation of a doubly
reduced quinone molecules: QA-QB-
+ 2 H+?
QAQBH2. Key
words: core complex of photosystem
II, oxygen evolving complex, secondary quinone acceptor, proteoliposome,
membrane potential, electrogenicity, direct electrometrical technique.
Apoptosis
in wheat seedling coleoptiles under influence of 'external' superoxide
A.A.
Vorobjev1,
E.G. Smirnova2, L.E. Bakeeva1, L.S. Yaguzhinsky1
1Belozersky
Institute of Physico-Chemical Biology, Lomonosov Moscow State University,Moscow
119899, Russia, fax: (095) 939-3181;
E-mail:yag@genebee.msu.ru,
2Institute of Agricultural Biotechnology, Russian Academy of
Agricultural Sciences, Timiryazevskaya ul., 42, Moscow 127550, Russia
Influence
of 'external' superoxide on apoptosis in wheat seedling coleoptiles was
investigated. A specific feature of apoptosis in wheat seedlings coleoptiles
during the normal morphogenesis such as a chromatin margination and formation
of vesicles containing mitochondria were enhanced by addition of 10-4
M gametocide - dibutylphthalate (DBP). At the same concentration
DBP also activates the superoxide excretion in the external environment
in wheat seedlings coleoptiles. It is important that lower and higher concentrations
DBP (10-5 M and 10-3 M) do not influence one of two
specified processes practically. Obtained correlation of DBP influence
on 'external' superoxide generation speed and mitochondria vesicles formation
(and, accordingly, an apoptosis) speaks well for function interrelation
of these processes. Supported
by RFFI grants 04-04-48121 & 00-04-48257
Primary
Charge Separation between P* and BA: Electron-Transfer
Pathways in Native and Mutant GM203L Bacterial Reaction Centers
A.G.
Yakovlev1, M.R. Jones2, L.G. Vasilieva3,
A.Ya. Shkuropatov3 and V. A. Shuvalov1,3
1Department
of Photobiophysics, Belozersky Institute of Chemical and Physical Biology,
Moscow State University, Moscow, 1Institute of Basic Biological
Problems, Russian Academy of Sciences, Pushchino, Moscow Region 142290,
Russian Federation, 3Department of Biochemistry, School of Medical
Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United
Kingdom.
Coherent
components in the dynamics of decay of stimulated emission from the primary
electron donor excited state P* and of population of the product
charge-separated states P+BA- and P+HA-
were studied in GM203L mutant reaction centers (RCs) of Rhodobacter
sphaeroides by measuring oscillations in the kinetics of absorbance
changes at 940 nm (P* stimulated emission region), 1020 nm (BA-
absorption region) and 760 nm (HA bleaching region). Absorbance
changes were induced by excitation of P (870 nm) with 18 femtosecond pulses
at 90 K. In the GM203L mutant, replacement of Gly M203 by Leu results in
exclusion of the crystallographically-defined water molecule (HOH55) located
close to the oxygen of the 131-keto carbonyl group of BA
and to His M202, which provides the axial ligand to Mg of the PB
bacteriochlorophyll. The results of femtosecond measurements were compared
with those recently reported for Rb. sphaeroides R-26 RCs containing
an intact water HOH55. The main consequences of the GM203L mutation were
found to be as follows: (i) The low-frequency oscillation at 32 cm-1,
which is characteristic of the HOH55-containing RCs, disappears from the
kinetics of absorbance changes at 1020and
760 nm in the mutant RCs; (ii) The dominant faster (80%; time constant
of 1.1 ps) component of electron transfer from P* to BA
observed in HOH55-containing RCs is not seen in the mutant RCs, being replaced
by a slower (time constant of about 4.3 ps) component that is also present
in water-containing RCs as a minor component (20%). The previously postulated
rotation of water HOH55 with a fundamental frequency of 32 cm-1,
triggered by electron transfer from P* to BA, was
confirmed by observation of an isotopic shift of the 32 cm-1
oscillation in the kinetics of P+BA- population
in deuteratedpheophytin-modified
RCs of Rb. sphaeroides R-26 by a factor of 1.6. These data are discussed
in terms of the involvment of the HOH55 molecule in charge separation via
a sequence of polar groups N-Mg(PB)-N-C-N(HisM202)-HOH55-O=(BA)
linking PB and BA, and providing the most effective
electron-transfer route for the primary reaction P* ? P+BA-.Authors
gratefully acknowledge the support by the Russian Basic Research Foundation
and RAS grants.
About proton transfer through interfaces of inner membrane of the uncoupled mitochondria
V.I. Yurkov, M.S. Fadeeva
The
new phenomenon - the increasing of the maximal rate of uncoupled respiration
by nonpenetreting buffer in incubation media was founded. The two kinetic
stages in the process of the proton transfer through the interfaces of
the mitochondrial membrane in the presence of uncouplers was identified.
The analysis of the experimental data showed that changes in uncoupler
concentration from low to high result in the spatial shift of limiting
kinetic stage of the proton transfer process from outer surface of inner
membrane to inner surface. The respiratory inhibition mechanism by high
concentration of uncouplers was discussed. The inhibition of the mitochondrial
respiration by the high uncouplers concentration resulted in pH decrease
in the matrix space. The existence of the local Н+-
gradient on uncoupled mitochondria was detected. The experiments with FITC-labelled
mitochondria demonstrated that the buffer effect is coupled with the decreasing
of the localН+-
gradient on the outer side of the inner mitochondrial membrane.
Kinetic
model of mitochondrial Krebs cycle
Kinetic
model of mitochondrial Krebs cycle with glutamate, malate and alpha-ketoglutarate
as oxidized substrates has been developed. In this model Krebs cycle operates
via aspartate aminotransferase shunt connecting oxaloacetate and alpha-ketoglutarate.
In vivo this shunt functions when the energy needs of the cell increase
[1].Substrates enter mitochondria
via two translocators - aspartate/glutamate carrier and alpha-ketoglutarate/malate
carrier. Reducing equivalents (NADH) formed in Krebs cycle reactions are
utilized by respiratory chain which is presented in the model by complex
I. With the enzymes cited above our model also includes the description
of the following enzymes: alpha-ketoglutarate dehydrogenase, succinate
thiokinase, succinate dehydrogenase, fumarase, malate dehydrogenase.
Our
model is based on the information of separate enzymes functioning. Rate
law of each enzyme was derived according kinetic mechanism found in literature.
Rate equations contain a number of parameters. The values of unknown parameters
were estimated via fitting the rate equation to the available in vitro
experimental data using computer program DBSolve7. Some parameters (e.g.,
the mitochondrial enzymes concentrations) cannot be estimated from in
vitro data. We estimated their values using experimental data describing
substrates oxidation by the suspension of mitochondria. In this case the
whole model with unknown parameters was fitted to the two sets of experiments:
in the first glutamate and malate [2] and in the second alpha-ketoglutarate
[3] were oxidized by mitochondria. So we estimated enzymes concentrations
and other parameters. Constructed model of Krebs cycle contains detailed
descriptions of enzymes functioning rates with estimated parameters and
can be applicated for the investigation of drugs influence on energy metabolism
of mitochondria.
References:
1.
Biofizika, 1989, 34(3), 450-458.
2.
Archives of Biochemistry and Biophysics, 1979, 195, 578-590.
3.
Archives of Biochemistry, 1954, 51, 47-61.
The
study of streptolydigin effects on the bacterial RNA polymerase activities
S.D.
Zorov1,
N.S. Zenkin2, K.V. Severinov2, V.G. Nikoforov3
1A.N.
Belozersky Institute of Physico-Chemical Biology, Moscow State University,
Moscow, Russia, 2Waksman Institute, Department of Molecular
Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA, 3Public
Health Research Institute, Newark, NJ, USA.
The
tetramic-acid antibiotic streptolydigin inhibits prokaryotic RNA polymerase.
Here, we investigated the influence of streptolydigin on kinetic parameters
of Thermus aquaticus RNA polymerase activities.
The
kinetics of single-step transcript elongation was measured in the presence
and in the absence of streptolydigin. The addition of streptolydigin resulted
in the appearance of two types of transcription elongation complexes: fully
active and fully inactive, the latter bound to streptolydigin. Because
of the dynamic equilibrium between the active, streptolydygin free, and
inactive, streptolydigin-bound, complexes, the observed effect of streptolydigin
on elongation is slowing down of average rate of transcript elongation.
We also showed that other activities of RNA polymerase in transcription
elongation complexes, such as intrinsic or factor-dependent cleavage of
3'-terminal fragments of RNA are also strongly inhibited by streptolydigin.
Our data suggest that binding of streptolydigin renders transcription complex
fully inactive. The inhibition does not occur at the level of substrate
binding, since the apparent Km value for the incoming nucleoside triphosphate
is not changed in the presence of streptolydigin.
To
further investigate the mechanism of streptolydigin action we generated
T. aquaticus RNA polymerase mutant harboring a deletion of the b'
subunit amino acids 932 to 1136. The region removed by the deletion is
in close proximity to the proposed streptolydigin-binding site. We showed
that the elongation activity of the mutant enzyme is not inhibited by streptolydigin.
Moreover, a stimulatory effect of streptolydigin on the rate of transcript
elongation was observed. However, the intrinsic cleavage of 3'-terminal
fragments of RNA in the mutant enzyme elongation complexes was inhibited
by streptolydigin. Thus, the mutation allowed to separate the effects of
streptolydigin on two types of reactions catalyzed by the RNA polymerase
active center, which opens way for detailed studies of the molecular mechanism
of inhibition.