The physiological role of the nucleoside diphosphate kinase from the outer compartment of rat liver mitochondria

Senior scientist, Doctor of Science

Group leader - Tatiana Lipskaya and
junior research worker V. Voinova

Under physiological conditions, nucleoside diphosphate kinase (EC 2.7.4.6, NDPK) catalyzes the reactions of nucleoside triphosphates synthesis from ATP and the respective nucleoside diphosphates. These nucleoside triphosphates, but not ATP, are involved in the main anabolic processes and creation of DNA and RNA molecules. In humans, ten NDPK isoenzymes are recognized. NDPK isoenzymes differ in intracellular localization and tissue specificity. The enzyme also reveals regulatory properties. These properties are not due to the enzyme catalytic activity. It means that the catalytic and regulatory functions of NDPK may exist independently from each other. NDPK is involved in regulation of such processes as cell motility, growth and development, malignant growth, and apoptosis. NDPK is indeed a universal regulator of intracellular processes. Therefore, it is not astonishing that the enzyme attracts the attention of many scientists. However, in spite of progress achieved in studies of NDPK by means of the molecular biology approaches, specific biochemical mechanisms of regulation in which NDPK is involved still remain unknown. It was assumed that regulatory properties of NDPK are mostly due to its ability to form complexes with many intracellular proteins and thus change their properties. Study of these regulatory mechanisms is of interest not only for fundamental science but also for medicine.

During the last years, we have studied properties and functions of NDPK in the outer compartment of rat liver mitochondria. Earlier, our group studied properties and functions of creatine kinase from heart mitochondria. On the basis of that research eight PhD theses were defended.

Why did we decide to study NDPK of the outer compartment of rat liver mitochondria? In rat liver mitochondria, the enzyme was found in the outer compartment and in the matrix. The functions of matrix NDPK are well understood, whereas a physiological role of NDPK in the outer mitochondrial compartment remains unknown, though its activity may account for nearly the maximal rate of oxidative phosphorylation. The enzymes of the outer mitochondrial compartment play an important but not sufficiently described role in the development of apoptosis and in the regulation of metabolic pathways. Disturbances in these pathways may lead to many metabolic diseases. Well-known regulatory properties of NDPK lend support to the suggestion that this enzyme also plays an important role in the regulation of the processes which occur in the outer compartment of mitochondria. Meanwhile the questions still remain unanswered. First of all, we studied the exact localization of NDPK in the outer mitochondrial compartment. Knowledge of the exact localization of the enzyme is important as functions and the ways of the activity regulation are different for the enzymes with different intracellular localization. Evidence has been obtained that NDPK is bound to the outer surface of the outer mitochondrial membrane (omNDPK), and ionic interactions are not essential for the enzyme binding to the membranes.

As known, the major function of mitochondria in normal cells is providing of them with energy. However, the outer mitochondrial membrane represents a diffusion barrier for ADP and potentially may limit the rate of intracellular energy transport. The problem is solved as the result of so called functional coupling between kinases of the outer mitochondrial compartment (creatine kinase, adenylate kinase, hexokinse, glycerol kinase) and the oxidative phosphorylation system. Earlier, we formulated a hypothesis about a mechanism of participation of creatine kinase in functional coupling and physiological role of this enzyme in the intermembrane space of heart and skeletal muscle mitochondria. During the study of omNDPK it was found that oxidative phosphorylation is also accompanied by appearance of functional coupling between omNDPK and the oxidative phosphorylation system. In rat liver mitochondria, omNDPK is the primary enzyme which ensures functional coupling with that system. We have shown that functional coupling involves only a small fraction of the omNDPK molecules, which are most tightly bound to mitochondria and are responsible for about 25% of the total enzyme activity. The role of other omNDPK molecules remains unknown. The goal of our study is to elucidate this role.

We have demonstrated that omNDPK molecules not involved in functional coupling can be solubilized from mitochondria and that solubilization is reversible. This allows for the possibility that reversible solubilization can occur under physiological conditions. It was found that only a small part (' 9%) of free NDPK present in the cytoplasm of rat liver cells can be bound to the outer mitochondrial membrane, and it is in a state of equilibrium with the bound enzyme. The constant of NDPK binding to the membranes was determined. Western blotting showed the bound enzyme to be NDPK-α, a homolog of human NDPK-B. Evidence was obtained that the sites of NDPK binding to the outer mitochondrial membrane are not identical to those of hexokinase and glycerol kinase.

On the basis of this research one candidate of science thesis was defended. This work was supported in part by ALSAM Foundation (Los-Angeles, USA).

In the future, studies we intend to work out a method of isolation and purification of omNDPK from rat liver mitochondria; to find out what differs these molecules from other NDPK-α molecules present in cytoplasm of liver cells. Solution of this problem can help to recognize another function(s) of omNDPK. Besides, we intend to estimate kinetic parameters of omNDPK, solubilized and bound to mitochondria.

Key publications:

• T.Y. Lipskaya, P.J. Geiger, S.P. Bessman (1995) Biochem. Mol. Medicine, 55, 81 - 89.
• T.Y. Lipskaya (2001) Biochemistry (Mosc), 66, 115 - 129.
• T.Y. Lipskaya (2001) Biochemistry (Mosc), 66, 1098 - 1111.
• T.Y. Lipskaya, M.S. Savchenko (2003) Biochemistry (Mosc), 68, 68 - 79.
• T.Y. Lipskaya, K.N. Plakida (2003) Biochemistry (Mosc), 68, 1136 - 1144.
• T.Y. Lipskaya, V.V. Voinova (2005) Biochemistry (Mosc), 70, 1354 - 1362.
• T.Y. Lipskaya, V.V. Voinova (2008) Biochemistry (Mosc), 73, 321 - 331.
• T.Y. Lipskaya, V.V. Voinova (2009) Biochemistry (Mosc), 74, 578 - 587.
• T.Y. Lipskaya, V.V. Voinova (2012) Biochemistry (Mosc), 77, 593 - 602.

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Last updated 20.12.2013