Activity of nucleoside diphosphate kinase α (NDPK α) capable of binding to outer mitochondrial membrane accounts for less than 10% of total NDPK activity present in cytoplasm of liver cells.

During incubation of a constant volume of rat liver cytosol with an increasing quantity of mitochondrial protein in the presence of 3.3 mM MgCl(2), the binding of nucleoside diphosphate kinase (NDPK) from the cytosol to mitochondrial membranes is described by a saturation curve. The highest bound NDPK activity accounts for less than 9% of the added activity. Analysis of the results suggests that only one NDPK isozyme is bound to the membranes. Western blotting showed it to be NDPK α, a homolog of human NDPK-B. Substrates of NDPK, hexokinase, and glycerol kinase, as well as N,N'-dicyclohexylcarbodiimide and palmitate, did not influence the association of NDPK with mitochondrial membranes. We conclude that the sites of NDPK binding to the outer mitochondrial membrane are not identical to those of hexokinase and glycerol kinase.

Reversibility of nucleoside diphosphate kinase solubilization from the surface of the outer mitochondrial membrane.

It was found that in medium with low ionic strength nucleoside diphosphate kinase (NDPK) solubilization from the outer membrane of liver mitochondria could be partially reversed by the addition of 3.3 mM MgCl2. Complete rebinding of the enzyme after the addition of MgCl2 was observed when the mitochondrial washing and storage medium contained leupeptin, an inhibitor of cathepsins. It was demonstrated that leupeptin and another inhibitor of cysteine proteinases, E-64, do not influence the rate of NDPK solubilization as well as its solubilized and membrane-associated activity. We conclude that NDPK becomes sensitive to proteolysis only after its solubilization; proteolysis does not affect the part of the enzyme molecule that is responsible for catalysis. After solubilization of NDPK in the absence of leupeptin, cathepsins damage sites of its binding on the membranes. The rate of the enzyme solubilization is dependent on the pH of the storage medium (pH 6.0-8.0); it decreases with increase in pH. It was shown that in the medium with high ionic strength, MgCl2 does not reverse pH-dependent NDPK solubilization, but solubilization could be reversed by increase in medium pH in the presence of E-64 and BSA. The physiological importance of these results is discussed.

Mitochondrial nucleoside diphosphate kinase: mode of interaction with the outer mitochondrial membrane and proportion of catalytic activity functionally coupled to oxidative phosphorylation.

In the present study, we found that ionic interactions are not essential for the binding of nucleoside diphosphate kinase of liver mitochondria outer compartment to outer mitochondrial membrane and that the proportion of the enzyme activity involved in functional coupling with oxidative phosphorylation (we demonstrated the existence of functional coupling earlier) is only 17%. Additional evidence was obtained that functionally coupled activity of nucleoside diphosphate kinase is associated with the outer surface of mitochondria. Dextran (10%) did not increase functional coupling. The physiological importance of these effects is discussed.

Functional coupling between nucleoside diphosphate kinase of the outer mitochondrial compartment and oxidative phosphorylation.

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane.

Data on localization of nucleoside diphosphate kinase (NDPK) in the outer mitochondrial compartment are contradictory. We have demonstrated that repeated quintuple wash of a mitochondrial pellet (protein concentration is about 2 mg/ml) solubilized only 60% of total NDPK activity. Since no release of adenylate kinase, the marker enzyme of the intermembrane space, was observed, it was concluded that the solubilized NDPK activity was associated with the outer surface of the outer mitochondrial membrane. Treatment of mitochondria with digitonin solutions in low (sucrose, mannitol) or high (KCl) ionic strength media revealed that solubilization of remaining NDPK activity basically coincided with the solubilization curve of monoamine oxidase, the marker enzyme of the outer mitochondrial membrane, but differed from solubilization behavior of adenylate kinase and malate dehydrogenase. We concluded that the remaining NDPK activity was also associated with the outer mitochondrial membrane and electrostatic interactions were not essential for NDPK binding to mitochondrial membranes. Results of polarographic determination of remaining adenylate kinase and NDPK activities of mitochondria incubated in ice for different time intervals and subjected to subsequent centrifugation suggest that all NDPK activity of the outer compartment of rat liver mitochondria is associated with the outer surface of the outer mitochondrial membrane. We suggest the existence of at least three NDPK fractions. They represent 70, 15, and 15% of total NDPK activity of the outer compartment and differ by tightness of membrane binding.

Once again about the functional coupling between mitochondrial creatine kinase and adenine nucleotide translocase.

The synthesis of creatine phosphate (CP) by mitochondrial creatine kinase during oxidative phosphorylation was terminated when the mass action ratio of the creatine kinase reaction Gamma = [ADP]*[CP]/[ATP]*[Cr] became equal to the apparent equilibrium constant (K(eq)(app))of this reaction. Subsequent excess of Gamma over the K(eq)(app) was due to an increase in the ADP concentration in the medium. A comparable increase in the ADP concentration also occurred in the absence of creatine (Cr) in the incubation medium. Increase in the ADP concentration was shown to be associated with a decrease in the rate of oxidative phosphorylation and with a relative increase in the ATPase activity of mitochondria during the incubation. A low concentration of ADP (<30 micro M) and relatively high concentrations (1-6 mM) of other components of the creatine kinase reaction prevented the detection of the reverse reaction within 10 min after Gamma exceeded the K(eq)(app), but the reverse reaction became evident on more prolonged incubation. The reverse reaction was accompanied by a further increase in Gamma. Low ADP concentration in the medium was also responsible for the lack of an immediate conversion of the excess creatine phosphate added although Gamma > K(eq)(app). The findings are concluded to be in contradiction with the concept of microcompartment formation between mitochondrial creatine kinase and adenine nucleotide translocase.