Äîêóìåíò âçÿò èç êýøà ïîèñêîâîé ìàøèíû. Àäðåñ îðèãèíàëüíîãî äîêóìåíòà : http://www.biochem.bio.msu.ru/assets/docs/diploma/2008/Brodiagina.pdf Äàòà èçìåíåíèÿ: Sun Oct 23 15:44:13 2011 Äàòà èíäåêñèðîâàíèÿ: Mon Oct 1 20:16:41 2012 Êîäèðîâêà:

. . .





ADP F1Fo-ATP Paracoccus denitrificans


: . .

2008




..................................................................................................................... 3 ................................................................................................................................................ 4 ............................................................................................................................ 5 F1·FO- ................................................... 5 ......................................................................................................................... 7 F1·F- ...................................................... 8 F1·FO- ........................................................................................................ 9 F1·F- ARACOCCUS DENITRIFICANS ......................................................................................... 15 .............................................................................................................. 19 ................................................................... ..................................................................................................................... 1. ATP .................................................................................................. 2. NADH-. ..................................................................................... 3. ........................................................................................ 4. P. DENITRIFICANS ................... 5. P. DENITRIFICANS, ADP ..................................... 6. .................................................................................................... 19 20 20 22 22 22 23 24

......................................................................................................................................... 25 1. ­ ITP GTP ­ P. DENITRIFICANS ................................................................................................ 25 2. , ADP, ................................................................................................................ 30 . ....................................................................................................... 42 .................................................................................................................................................. 49 ..................................................................................................................... 50

2



F1·Fo-ATP ­ +- F1 ­ Fo ­ F ­ -4- NTP ­ HEPES ­ N-2--N- ­ ­ Pi ­

~
H

-

­ ­ ­ ­ () ­ ­ U ­

3



F1·Fo- / ­ , / , ­ , . , , ­ . , . , , , - . F1·Fo- Paracoccus denitrificans. 1-2 % . , - . , -/ / ADP. , ADP (1-2 ), . , , ADP , , , ADP . F1·F-. ADP ,

ADP, .

4



F1·Fo-
+-F1·Fo- / , . , ( ~
H

), -

. F1·Fo- , ~
H

,







,







:





, . F1·F- : F1 F. F1- 5 :

,

,

,





55, 51,5, 30, 21, 15 , , 3:3:1:1:1. F- , : a, b 30, 17, 8 , , 1:2:10-12 ( E. coli (E F1·F ) [1,2]. F1·F- (CF1·F) . -, - -

(MF1·F) ; - - , - ­ , (OSCP). . F1-, . 10-12

-

-, , ,

, -, . - .

- F- . -, , 5


. .

- b- -

; b , . , F1 F- , -. [2] - -. - b-

.1. / [3].

, F1- . ADP ; , . , , 6


- . -. -,

, , , - [4].

, ,


() « ». : 1) , , ; 2) , . - F F1 [2]. F1 F · . - F1- , . , F- , - . - - . . , F1- , [5]. 1990 , , , «» F1 ( -) - - [6]. : (close), (tight) (open). 7


. . . - .



-

-. , - -. · -. -,
3 3-

,

, «» () [7].

F1·F-
F1·F- ATP ADP Pi, . ADP Mg2+. ATP :

n ( 2 4) ­ Fo , m - , [8]. ATP, F1·F-, F1·F-, . , GDP IDP [9,10]. F1·F- ATP, . ·Mg2+. , =10
-4

.







,







·Mg2+,





8



,


ADP·Mg2+·Pi. Mg2+·Pi Mg2+ , . ADP, [8]. , F1·F- , ITP GTP. , . , . F1 , G I 25% [11]. F1·Fo-

UTP [11].

F1·Fo-
F1·Fo-.

F1·Fo- ; . 1) 1963 (. IF1.

, , - [12]). F1·F- 1:1. , IF1 F1. IF1 , (open) ( ) IF1 , , ,

. , . , IF1. , - [. 12].

9


2) S-S

- . ,

-

(SH) , (S-S) ­ [13]. 3) - . F1. - -



. (), Ki 10 . , -, , F1 F . F1, Fo. , - F1·Fo, - - . , - EFC1 EFC1 [14]. , - F1-ATP Bacillus strain PS3 . Mg2+ . , F1-Fo . . -, - F1·Fo [15]. , - , . - , , , . , ­ [16]. . -,

10


4) ADP , , [17]. C . , ADP Mg2+ (F1), , - [18]. F1, . , (, , ) Mg2+. . Mg2+ ADP . [17]. , ADP Mg2+, , - . ( ADP Mg2+, ), , - ADP ADP·Mg2+- . , I . C ADP Mg2+, I, , -. I . , ADP , IDP . [19]. , ADP . , - . ­ , 11 . ADP·Mg2+- . [18]. ,
-7

)

ADP (Ki 10


. , , ADP, Mg2+ (E*·ADP), . (E) (E*·ADP) . , . , Km, , [19]. , E*·ADP ADP, [19]. ­ ­ . , Pi [8]. (10 ) Ki ADP ADP·Mg2+- 7·10
-7

5·10

-6

[20]. ,



ADP-

, +. , - . C , . - , ADP·Mg2+- . , [21]. , . , ADP -, ADP- [22]. C . ADP·Mg2+- , , [21]. . 2 ADP·Mg2+- . 12


E·ADP, E ADP, , E·ADP E*·ADP. - ADP (E*·ADP). E*·ADP- , E·ADP (. 2).

. 2. , , [21]. ,

ADP. ADP . . [23] ADP . , ADP, , [19] (. ). , , . . . [24] DP , ADP . [25] D 13


. , ADP 2 . . . 5)

~
H

-



[26] ,

~
H



ADP·Mg2+-, . - ADP 20 . F1·F- . [27, 28] Rhodspirillum rubrum [29] . - . capsulatis, - F1·F-. Rhodobacter ,

,

~
H



. Rhodobacter capsulatis , , . 30 . 3 Pi 80 , 1 ADP 15 [30]. , F1·F , . , , , , [8]. . 3. .

14


.3. F1·F-. F1·F : ( · ), (-) (a- b- F F1). - ~ . , , . ­ , ­ [8]. F1·F-, ADP, Mg2+
H

, [31]. , ­ [21]. , , . Paracoccus denitrificans.

F1·F- aracoccus denitrificans
Paracoccus denitrificans ­ . ( CO2), ( ). , , N2O . - . P. denitrificans . , CO2 . 15


, P. denitrificans [32]. , F1·F- . denitrificans , , [32]. P. denitrificans, . K ADP 12 , K - 190 [33]. . , , [34,35]. - . Km AP 30-100 [36]. - 90 % ­ [35]. : , , , . , , , , , . . Vmax. [34,37]. , [34]. . , ADP, . ADP, , . , , , 2 , [34]. , -

~
H

- :

16


[37]. [35,38]. ,

~
H

-

F1·F-

F1·F- . ADP, - ­ ADP - . , 1 ADP - , ADP [38]. - : , . - [38]. , , F1·F-ATP ADP. , ADP , . ADP ( - : +), . - . [39]. , ADP . , ADP - . , ADP . ADP F1·F- P. denitrificans.

17


­ ADP F1·F-. : ITP GTP; , ADP; F1·F- , ADP.

18



[32, 40]
P. denitrificans , 33° , 60 , 67 - 7,0, 35 , 5,5 , 30 NH4Cl, 0,6 Na2MoO4, 0,3 MgSO4 ( ). (200 rpm 21 30°). 1,175 , A 0,1 KNO3. 33° 17 . 660 0,8-1,0, . . (6000 rpm, 10 ), , 1 (10 Hl, 50 NaCl pH 7,3), . 1 800 (6500 rpm, 10 ). , 30° 2 (0,5 , 10 -Hl, 50 NaCl pH 7,3) . 2 D550=0,3 ( 300 ), ( 1 ), (25 100 ) 30 , D550 0,03 (10-15 ). . 10 15000 rpm. - 25 100 - , 7,3 ( 3). (10 /) 10 . 12 , 5 , 0,1 . , 19


(1 / 50 Mg ). 0,5 100 15 . 10 15000 rpm. Mg 5 , 10 , 40 15000 rpm. . . - , , 4 (10 , 5 Mg- 1 , 7,3). . 4 20 /. -196º . ( 5,0) NADH- , 1,6 ­ 1,9 /· . , .


, 0,25 , 0,1 KCl, 2,5 HEPES-KOH (pH 8,0), 5 MgCl2 0,1 . 2 . «Hitachi-557» 30°.

1. ATP
1.1. ATP AP-

. NADH 340 ( = 6220 -1·-1).

20


2,5 , 2,0 ( ), 1 , 1,5 , 0,15 NADH, ( ). NADH- . 7,5 / . 2 1 30 . 1.2. ATP H+- ATP :

8,0 [H+]/[ADP]=1 [41]. pH ; , 557 . 557 618 , 2,5 , 2,0 ( ) 25 . 1 20 . HCl .

21


1.3. ITP ITP , , ITP 2 , , .

2. NADH-.
, 0,25 , 0,1 KCl, 5 ( 8,0), 5 MgCl2, 0,2 150 NADH NADH 340 . 2 (V); (0,2 /) 15 , , NADH- (V


).


V

V ,

.

3.
624 602 2 , 1,5 - VI [42]. . VI , .

4. P. denitrificans
6,75 / , 2,5 HEPES 10 , 10 . (240 ) 10 U/ . 40 , 2 , 2 , 2,5 25 . 22


100 1 I 20 P. denitrificans, , ( , . . 2.2). 3 2 FCCP I . , , ITP . , « ICN» 150034. 1 U , (1 U 1 - 25°, =9,6).

5. P. denitrificans, ADP
0,2 / 10 ­ 20 . (1 , 15000 rpm). . 1) . , «Sigma» P7923. 1 U , (1 U 1 - 37°, =9,8). 4 : 1) ; , 2) 25 U/ , 3) 50 4) 25 U/ 50 . 20 . 10 . . 250 . 2) . , «Sigma» A6535. 1 U , (1 U 1 - 37°, =4,8).

23


10 40 , 3,3 U/ 3,3 U/ 2 . 40 , , . 300 . , , 2

. 75%.

6.
. : 1% (Na ), 2% Na2CO3·H2O, 0,95% NaHCO3, 0,16% Na, 0,4% NaOH, 11,25. ( NaOH ). : 4% CuSO4·5H2O ( ). , 1 50 . (20 100 ) 100 . 2 30 37º. . 562 «LKB-Ultraspec III». 20-100 . .

24



, , ADP 1-2 [39]. , , ATP, ADP. , , ­ ITP GTP ­ . ATP ITP .

1. ­ ITP GTP ­ P. denitrificans
P. denitrificans, ITP GTP . 4 . , , , 100 . 4, . ITP ­ . , , . 4, . ITP , . 4 , . ITP , , . GTP, , ITP ( ). . 4, ITP , . .

25


1. 1. , ITP GTP P. denitrificans


,
/·

-
,
/·

NDP,


NP, 30-100 300

ITP GTP

0,42 0,21 0,23

13 36 41

0,15-0,22 0,08-0,15 0,08

-

, « ». ..

1 , ­ . . 1 F1·Fo- P. denitrificans, ADP, IDP GDP . , P. denitrificans , ­ ITP GTP. , ITP. ­ NADH NTP (. « »). , ADP , , F1·Fo- . . 5 ITP, NTP- [39], (+).

ADP 26


. 5. , , 10 U , . 160 U ( 0,17 0,06 /· ). , ITP . ITP. 5, ITP 10 U , . . 5 . . 5, . - . ITP . , , IDP ITP, . , ITP , , . , P. denitrificans - [35]. , [37]. ITP. 6 ITP P. denitrificans. , ­ NADH NTP- . , , ITP, [33]. 27


. NTP . , ITP. , . , , ITP. . 7 ITP. 7 ITP , ­ NADH NTP . . 7, , [37]. ITP. , , , , ITP ( 7). 7, NADH NTP- , . , , . ( 0,14 0,06 /· ). 7, , NTP- ITP , . ITP . , ITP. , , NTP- . , ADP . , 28


~0,5% ADP, , , 1 ~5 ADP. , ADP , ADP·Mg- (. « »). , - - - - ADP [38]. , ADP [20]. [19], ADP IDP GDP. , , ITP P. denitrificans. (. 6 7), ITP . , , [43, 44], , , [29]. , ADP, , . ADP ITP . , , ITP P. denitrificans . I , , I, I (. 6 7). , I D, , . , D .

29


2. , ADP, .
, ADP, , . ITP, ADP . , ITP ADP . , ADP. : 1) - [45] 2) 3) , ADP [46]. , , . , ADP. , . P. denitrificans . , . 2. , . . , NTP 2,5 . .

30


2. .

Vmax, / ADP ITP IDP 2,4 3,8 2,0 2,0 20,6 16,5 -

, , (. « »). 1 . 21 8 .









ADP



. 80 . , , ADP , ADP . , ADP . 8.

31


8. , ADP. , ADP ( -); , ADP ( ); , ADP ( -). D ADP, D ­ ADP, . P. denitrificans , ADP. , , , , . , ITP. , .

32


ITP , . ITP . ITP , . , , . ITP , ADP. . . 9, ITP: 1) ITP ; 2) - ITP . , 9, I . , ITP (, ), . , . 9, , (5, 20 50 U/), ( ). , . , ADP [38]. ADP ( 6 ) , . ADP , - . ,

33


DP ( 10, ). . 10, , , ( ) 1 50-60%. , (+) . 10, -. . , ADP . ADP . IT F1·Fo-ATP P. denitrificans. , . , , , , . , ADP, , (. « »). : 1) ( ), 2) , 3) 4) .

34


, 3,0. : 1) ; 2) - ; 3) I - ( 1 1 I, ). 4) I ( 100 , ); 5) I ( 100 , , 3 5 FCCP). , . . ITP ADP- . ADP, , . , ITP - , . , . 3, , ( 3). , .

35


3. P. denitrificans . 2,4 1,3 2,5 + 1,1

, (. « »). 25 ­ 35 /.













. 11, , , . , , , , , , , , . , ADP . - . . 12, 3 - (. 12 ). . . , . . 12 , , , . - . . . 12 , 5-6 , .. . , , , . , 36


, , . ITP , ITP (. 13). . 13 , , - I. I (. 13, ) (. 13, ) . , , . 12 13, : 1) ITP . 2) - ITP, , , . 3) ITP, . , , . , ADP . , , ADP, , , , . , ITP , . . 11, , , , .

37


, ADP . , , ADP , , . , ADP, , . ADP , . , . 14, , . . 2 : , 100 ( 0,25 U/ ), , 10-12 . , , (. 10). , ADP + . (. 14). , , F1·Fo-. F1·Fo- . . 15, . ADP , , -, . , ADP , . . 16, . , 38 .


, , , , . , ADP (. 16 17). . , ADP . . 17, 0,5 -2 ­ 20 . ADP , ADP, ( ). , (. . 11), , . 18, , , . . , 4 -5 , . , , , . 10 . , , , . , . , , . , , ADP (. 15). 39


, . , . , . , . 5,3. 1,8-1,9. ( 4). 4. P. denitrificans 5,3 (+ ) 1,9 (­ ) 1,8



, 2 , (. « »). 25 ­ 35 /. .

4, , . - , , . . 19 , , , , . . . . 20 , - 40 , ,


(. 20 ). , , (. 20 ). , , -, -, (.. ITP (. 20 21),

) (. 20 21 ), , ( ) (. 20 21 ). , . 12 13, . 20 21, , , , . , : ADP. , . ITP . . 22, , , AD, .

41


.
, . denitrificans : , - . ADP , , , - - [38]. ADP, : , ADP , E·D . , ADP (1-2 ), [39]. , , , ADP , , , ADP . , ­ ­ - , .. . ADP. , , ( ) (~ 1 0-20 ) ( ), . ( ), ITP . , (. . 5), ( )

42


ITP, IDP. , [39]. . 5, , , , -. , . . , . ITP , ( . 5), . - , «» ADP [37, 38]. . 6 7 ( ), . ITP . ITP- . denitrificans , . ADP , . +, ADP 2 ­ 4 [39]. , ADP - . ITP ADP . . denitrificans 0,6-0,9 / [43]. , ~ 0,1 M ADP. , - . , , ADP , i. , , ATP ADP. , ADP, , 43


, . , ADP. , ADP : E*·D + D ( E*·D AD, - ) , ADP , . , (. 9) - (. 10), ADP - (. 9) (. 10). , - . : . , ( ) (. 11, 12 13). , ­ . , . , UCP3 [47]. , , , 5- . - , , , E·D.

44


, ( ) . , (. 12 13). , . , (. 11 19). . denitrificans, ADP. , ADP, , , ­ , ADP . (. 15, 16, 17). , , . ADP (. 14). , , . . , ADP , . , F1 Fo . , , . , , ADP, ITP, .. ADP . , ADP, . , ITP ITP - - ITP (. 21). , 45


(. 20). , ADP, . , , (. 22). , ADP . , ADP- - . , , , ADP. . , , , , , - . , , F1·Fo- P.denitificans 150 [36]. , / DP, . , - -, , , . , - F1-ATP Bacillus strain PS3 . Mg2+ D [15]. , , . [16]. , ADP , , , - -. , , . ADP , , ( « »). , F1·Fo- E. coli [48]. , F1ATP Schizosaccharomyces pombe. , 46


ADP [48 Jault BBA 2004]. . . , . . Rhodobacter capsulatis [50]. , <1, . 0,25 ADP 1 . F1 F. ADP >1 [50].

. 23. ADP . , ADP. , ADP. ­ , ADP

47


, ADP , - . (. 23), ADP. , , ADP, . , . , , . , ADP, , . . - , ( «» ADP). . , , ADP , . ADP .

48



1. () ITP F1·F-ATP P. denitrificans, +-. (ITP)- ( + ) , ITP () . 2. ADP , , , ITP; , . 3. F1·F-ATP P. denitrificans, ADP. ADP ADP, denitrificans. 4. ADP F1·F-ATP a. ; b. 5. , ADP F1·F-ATP P. denitrificans, . P.

49



1. 2. Senior A.E. (1988) ATP synthesis by oxidative phosphorylation. Physiol. Rev., 68, 177-231. Walker J.E., Fearnley I.M., Gay N.J., Gibson B.W ., Northrop F.D., Powell S.J., Runswick M.J., Saraste M., Tybulewicz V.L. (1985) Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria. J. Mol. Biol. , 184(4), 677-701. 3. 4. Weber J., Senior A. E. (2003) ATP synthesis driven by proton transport in F1·Fo -ATP synthase. FEBS Letters, 545, 61 ­ 70. Capaldi R.A., Aggeler R., Turina P., W ilkens S. (1994) Coupling between catalitic sites and the proton channel in F1·Fo-type ATPases. TIBS, 19, 284289. 5. 6. Boyer P.D. (1993) The binding change mechanism for ATP synthase ­ some probabilities and possibilities. Biochim. Biophys. Acta, 1140, 215-250. LÝcken U., Gogol E.P., Capaldi R.A. (1990) Structure of the ATP synthase complex (ECF1·Fo) of Escherichia coli from cryoelectron microscopy. Biochemistry, 29, 5339 ­ 5343. 7. Cross R.L., Duncan T.M. (1996) Subunit rotation in F1·F- synthases as a means of coupling proton transport through Fo to the binding changes in F1. J. Bioenrg. and Biomembr., 28, 403 ­ 407. 8. 9. A.. (1999) : . , 64, 1443 ­ 1456. Perlin D.S., Latchney L.R., W ise J.G., Senior A.E. (1984) Spec ifity of the proton adenosinetriphosphatase of Escherichia coli for adenine, guanine and inosine nucleotides in catalysis and binding. Biochemistry, 23, 4999-5003. 10. Pedersen P.L. (1976) ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity. J. Biol. Chem., 251, 934 ­ 940. 11. Fernandez-Belda F.J., Garcia-Carmona F., Garcia-Canovas F., Lozano J.A., Gomez-Fernandez J.C. (1982) Mitochondrial ATPase inactivation by interaction with its substrate. Arch. Biochem. Biophys., 215, 40-46. 12. Green D.W ., Grover G.J. (2000) The IF1 inhibitor protein of the mitochondrial F1·F0-ATPase. Biochim. Biophys. Acta, 1458, 343 ­ 355.

50


13.

Hisabori T., Ueoka-Nakanishi H., Konno H., Koyama F. (2003) Molecular evolution of the modulator of chloroplast ATP-synthase: origin of the conformational change dependent regulation. FEBS Letters, 545, 71 ­ 75.

14.

Sternweis P.C., Smith J.B. (1980) Characterization of the inhibitory (epsilon) subunit of the proton-translocating adenosine triphosphatase from Escherichia coli. . Biochemistry, 19, 526 ­ 531.

15. 16.

Kato-Yamada Y., Yoshida M. (2003) Isolated

Subunit of Thermophilic F1-

ATPase Binds ATP. J. Biol. Chem., 278, 36013-36016 Suzuki T., Murakami T., Iino R., Suzuki J., Ono S., Shirakihara Y., Yoshida M. (2003) F1·Fo-ATPase/Synthase Is Geared to the Synthesis Mode by Conformational Rearrangement of 17. Subunit is Response to Proton Motive Force and ADP/ATP Balance. J. Biol. Chem., 278, 46840-46846. Fitin A. F., Vasilyeva E. A., Vinogradov A. D. (1979). An inhibitory high affinity binding site for ADP in the oligomycin-sensitive ATPase of beef heart submitochondrial particles. Biochem. Biophys. Research Communications, 86, 434 ­ 439. 18. Minkov I.B., Fitin A.F., Vasilyeva E.A., Vinoqradov A.D (1979) Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1. Biochem. Biophys. Research Communications, 89, 1300 ­ 1306. 19. Vasilyeva E.A., Minkov I.B., Fitin A.F., Vinogradov A.D. (1982) Kinetic mechanism 20. of mitochondrial adenosine triphosphatase. ADP -specific inhibition as revealed by the steady-state kinetics. Biochem. J., 202, 9-14. Yalamova M.V., Vasilyeva E.A., Vinogradov A. D. (1982) Mutually dependent influence of ADP and Pi on the activity of mitochondrial adenosine triphosphatase. Biochemistry International, 4, 337 ­ 344. 21. Vasilyeva E.A., Minkov I.B., Fitin A.F., Vinogradov A.D. (1982) Kinetic mechanism of mitochondrial adenosine triphosphatase. Inhibition by a zide and activation by sulphite. Biochem. J., 202, 15-23. 22. Walker J.E., Leslie A.G.W ., Montgomery M.G., Bowler M.W . (2006) How azide inhibits ATP hydrolysis by the F-ATPases. J. Biol. Chem., 103, 86468649. 23. Vogel P.D, Cross R.L, (1991) Adenine nucleotide-binding sites on mitochondrial F1-ATPase. Evidence for an adenylate kinase-like orientation of catalytic and noncatalytic sites. J. Biol. Chem., 266, 6101-6105 51


24.

Milgrom Y.M, Boyer P.D. (1990) The ADP that binds tightly to nucleotidedepleted mitochondrial F1-ATPase and inhibits catalysis is bound at a catalytic site. Biochim. Biophys. Acta, 1020, 43 ­ 48.

25.

Chernyak B.V., Cross R.L. (1992) Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site. Arch. Biochem. Biophys., 295, 247 ­ 252.

26.

Galkin M. A., Vinogradov A. D. (1999) Energy-dependent transformation of the catalytic activities of the mitochondrial F1·Fo-ATPsynthase. FEBS Lett., 448, 123 ­ 126.

27.

Strotmann H., Bickel S., Huchzermeyer B. (1976) Energy-dependent release of adenine nucleotides tightly bound to chloroplast coupling factor CF 1. FEBS Lett., 61, 194 ­ 198.

28.

Shoshan V., Selman B.R. (1979) The Relationship between Light-induced Adenine Nucleotide Exchange and ATPase Activity in Chlo roplast Thylakoid Membranes. J. Biol. Chem., 254, 8801 ­ 8807.

29.

Slooten L., Nuyten A. (1981) Activation-deactivation reactions in the ATPase enzyme in Rhodospirillum rubrum chromatophores. Biochim. Biophys. Acta, 638, 305 ­ 312.

30.

Turina P., Rumberg B., Melandri B.A., GrÄber P. (1992) Activation of the H(+)-ATP synthase in the photosynthetic bacterium Rhodobacter capsulatus. J. Biol. Chem., 267, 11057 ­ 11063.

31.

Syroeshkin A.V., Vasilyeva E.A., Vinogradov A.D. (1995) ATP synthesis catalyzed by the mitochondrial F1·Fo ATP synthase is not a reversal of its ATPase activity. FEBS Lett., 366, 29-32.

32.

John P., W hatley F. R. (1970) Oxidative phosphorylation coupled to oxygen uptake and nitrate reduction in Micrococcus denitrificans. Biochim. Biophys. Acta, 216, 342 ­ 352.

33.

Perez J. A., Ferguson S.J. (1990) Kinetics of Oxidative Phosphorylation in Paracoccus denitrificans. Mechanism of ATP Synthesis at the Active Site(s) of FoF1-ATPase, Biochemistry, 29, 10503-10518.

34.

Pacheco-MoisÈs F., Garcia J. J., Rodriguez-Zavala J. S. and MorenoSÀnchez R. (2000). Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase. Eur. J. Biochem. 267, 993 ­ 1000.

52


35.

.., .. (2003) - Paracoccus .. (2005) denitrificans: - . , 68, 1370 ­ 1380.

36.





F1·Fo- Paracoccus denitrificans . . , . 37. Zharova T.V., Vinogradov A.D. (2006) Energy-linked binding of Pi is required for continuous steady-state proton-translocating ATP hydrolysis catalyzed by F·F1 ATP synthase. Biochemistry 45, 14552 ­ 14558. 38. Zharova T.V., Vinogradov A.D. (2004) Energy-dependent transformation of F1·Fo-ATPase in Paracoccus denitrificans plasma membranes. J. Biol. Chem., 279, 12319 ­ 12324. 39. Zharova T.V., Vinogradov A.D. (2006) Requirement of medium ADP f or the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo·F1-ATP synthase. Biochim. Biophys. Acta, 1757, 304-310. 40. 41. 42. John P., Hamilton W.A. (1970) Respiratory control in membrane particles of Micrococcus denitrificans. FEBS Lett., 10, 246 ­ 248. Chance B., Nishimura M. (1967) Sensitive measurements of changes of hydrogen concentration. Methdods Enzymol., 10, 641 ­ 650. Waggoner A.S. (1979) The use of cyanine dyes for the determination of membrane potentials in cells, organelles, and vesicles. Methods Enzymol., 55, 689 ­ 695. 43. Harris D.A., John P., Radda G.K. (1977) Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation . 1. The Paracoccus denitrificans system. Biochim. Biophys. Acta, 459, 546-559 44. Harris D.A., Radda G.K., Slater, E.C. (1977) Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. 2. The beef heart mitochondrial system. Biochem.Biophys.Acta., 459, 560-572. 45. Garret N.E., and Penefsky H.S. (1975) Interaction of Adenine Nucleotides with Multiple Binding Sites on Beef Heart Mitochondrial Adenosine Triphosphatase, J. Biol. Chem., 250, 6640 ­ 6647.. 46. Digel J.G., Kishinevsky A., Ong A.M., McCarty R.E. (1996) Differences between two tight ADP binding sites of the chloroplast coupling factor 1 and their effects on ATPase activity. J. Biol. Chem. 271, 19976­19982

53


47.

Parker N., Affourtit C., Vidal-Puig A., Brand M.D. (2008) Energizationdependent endogenous activation of proton conduct ance in skeletal muscle mitochondria. Biochem. J., 412, 131 ­ 139.

48.

Weber J., Senior A.E. (1995) Location and properties of pyrophosphate binding sites in Escherichia coli F1-ATPase. J. Biol. Chem., 270, 12653 ­ 12653.

49.

Falson P., Goffeau A., Boutry M., Jault J.M. (2004) Structural insight into the cooperativity between catalytic and noncatalytic sites of F1 -ATPase. Biochim. Biophys. Acta, 1658, 133 ­ 140.

50.

Feniouk, B.A., Mulkidjanian, A.Y., Junge, W. (1995) Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence. Biochim. Biophys. Acta, 1706, 184 ­ 194.

54




4.





ATP



ITP





P. denitrificans. , 2,5 2,0 (. « »). I +- - ; ­ VI (. « »). 1 2 ITP, 20 (37,5 /). ­ NTP / 1 .

55


5. ATP ITP P. denitrificans, NTP . , « ». 2,5 2,0 , 20 U : 10 U ( ), 160 U ( ) ( ). 1,5 M VI ( ). ATP ITP 1 2 , . 20 . ­ 67,5 /.

56


7. ATP ITP P. denitrificans. ­ 2 , ­ . ( ), . 4, NTP ( ), . 5. 20 U , 12 U .

57


6. ATP ITP P. denitrificans. ( ), , . 4, NTP ( ), , . 5. 20 U , 12 U , 1 2 ITP. , 5 FCCP.

58


V, ITP/*

0,08

V0
0,04

V1
0 10 20 30

,

9. I . ( . « »). , . , 2,5 2,0 , 100 ITP (. . 1) ( ) (). 20 2 ITP ITP ( ). 1 FCCP ( ). .

59


V,/*

5 0,15 4

0,10

3

2 0,05 1

0

50

100

150

200

250

,

300

10. - - +- . , 0,5 , 10 -Hl ( 8,0), 0,2 KCl, 10 MgCl2, 1 , 10 , 0,2 , (100 U/, ) (20 U/) (1,5 ), ), 1:2, 5 /. 2 20 ( ). 5 ADP. , , , 2,5 , 2,0 , 1 , 20 . ­ 62,5 /. ADP ( ­ , - ).

60

ADP, M


11. P. denitrificans, , 1) (); 2) 50 (), 3) 25 U/ (), 4) 25 U/ + 50 ( + ) , , 2 , 2,5 , 2,0 , 1,5 M VI. ­ 75 /.

61


.
, ATP/*
0,20

0,15

0,10

0,05

0,00







+

.
, ATP/*
0,20

0,15

0,10

0,05

0,00







+

12. ~ () () H P. denitrificans , 2,0 , - . ( ) - . 100 , 2,5 . 20 1 () . 100 . 1 . () TP , 3 . 100 , 2,5 . 20 1 , 2 5 CCP (. . 6). ( . . 11) ­ 45 /. 62


.
, ITP/*

0,10

0,05

0,00







+

.

, ITP/*

0,10

0,05

0,00







+

13. ~ () () I P. H denitrificans , . 12, 1 , 2 I 90 /.

63


14. , + - P. denitrificans. 2,5 , 1,5 M VI. . ­ 0,5 U , ­ 50 U 1,5 , - 50 U . ­ 77,5 /. , 5 FCCP.

64


15. ADP, P. denitrificans. , 2,5 . ­· , 2,5 0,5 U . , 2,0 (Pi), 100 ADP (0,25 /). ­·77,5 /. , 5 FCCP.

65


16. ADP P. denitrificans. , . 15. , 100 ADP (0,25 /), 5 FCCP. ­ 77,5 /.

66


.

.

17. () () P. denitrificans. , . 15. . , 2 . . , (). ­ 77,5 /. , 5 FCCP.

67


18. - P. denitrificans. , (0,25 U/) ( ) ( ). 0, 3 7 2,5 . , 5 FCCP. ­77,5 /.

68


.
, ATP/*

0,25

0,20

0,15

0,10

0,05

0,00



+ Pi

- Pi

0,25

, ATP/*

.

0,20

0,15

0,10

0,05

0,00



+ Pi

- Pi

20. ~ () () P. H denitrificans , 1) (); 2) 25 U/ + 2 ( + Pi) 4) 25 U/ ( - Pi) , , , . , . 12. ­ 40-50 /.

69


.
, ITP/*
0,15

0,10

0,05

0,00



+ Pi

- Pi

.
, ITP/*
0,15

0,10

0,05

0,00



+ Pi

- Pi

21. ~ () () I P. H denitrificans . 21. I , . 13. 80 ­ 90 /.

70


19. P. denitrificans, , 1) (- ); 2) 25 U/ + 2 ( + Pi) 4) 25 U/ ( - Pi) , , 2 , 2,5 , 1,5 M VI. . ­ 90 /. , (+Pi) (-Pi) . ­ 81 84 / .

71


.
, TP /*
0,20

0,15

0,10

0,05

0,00





.

, ITP /*

0,20

0,15

0,10

0,05

0,00





22. () I (), P. denitrificans . - I. , 2,5 2,0 , . 100 20 1 2 I. NTP , 3 (0,25 /). 45 ­ 90 /.

72