Reversibility of nucleoside diphosphate kinase solubilization from the surface of the outer mitochondrial membrane
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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.
Key wordsnucleoside diphosphate kinase mitochondria outer compartment solubilization liver
nucleoside diphosphate kinase
outer mitochondrial membrane bound NDPK
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