[Determination of molar content of creatine kinase in heart mitochondria by SH-reagents].

The maximal content of mitochondrial isoenzyme of creatine kinase (CK) in rat heart mitochondria does not exceed 12.5 moles per mole of ATP-ADP translocase. This value was obtained by titration of mitochondrial CK activity in aged mitochondria by 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and 2,4-dinitrofluorobenzene (DNFB) and by a more complex and accurate method. The essential thiol groups of membrane-bound mitochondrial CK (and its enzymic activity) can be specifically protected by phosphocreatine (12 mM) + ADP (1-5 mM) against inactivation by DTNB. Mitochondria with protected SH-groups of CK and with groups inactivated by DTNB were repeatedly incubated with DTNB under identical conditions and the number of additionally reacted sulfhydryl groups and the changes in CK activity were measured. The differences in the number of additionally reacted SH-groups correlated with the changes in the CK activity, which made it possible to calculate the molar ratios of mitochondrial CK to cytochrome c oxidase and ATP-ADP translocase (2.16 +/- (0.4): 1:2, respectively).