Interaction of creatine kinase and hexokinase with the mitochondrial membranes, and self-association of creatine kinase: crosslinking studies.

Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes. The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers. Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase. Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu++-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization. In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.