Creatine kinase and protein kinase reactions of cardiac cell membranes

Abstract

This report summarizes the results of our recent research on the creatine kinase isozymes associated with the cardiac mitochondrial and sarcolemmal membranes and also describes some unique properties of the sarcolemmal protein kinase reaction. In heart mitochondria, the kinetics of creatine kinase is influenced by oxidative phosphorylation, with alterations in the substrate dissociation parameters of the creatine kinase reaction being the dominant effect. The change is specific for the dissociation of ATP from the ternary enzyme-substrate complex, expressed by an order of magnitude decrease in K a . This tight binding of MgATP to the E·MgATP·Cr complex also limits the degree of product inhibition by phosphocreatine. Thus, the isozyme can catalyze the rapid synthesis of phosphocreatine even in the presence of physiological and inhibitory concentrations of this product. The results emphasize the importance of the functional coupling between creatine kinase and the adenine nucleotide translocase, which may be viewed as a potential multi-step complex integrated for the aerobic synthesis of phosphocreatine. With respect to cytoplasmic forms, creatine kinase MM isozyme bound to the cardiac sarcolemma effectively utilized phosphocreatine for energizing the active transport of sodium and potassium. Therefore, both mitochondrial and sarcolemmal creatine kinase reactions are clearly affected by their localization at membranes and by reactions in their intimate microenvironments. The sarcolemmal membranes also exhibited endogenous protein kinase activity, phosphorylating the membranes of the vesicles. Both the endogenous sarcolemmal protein, with a molecular weight of 11 500 daltons, and an exogenous protein (histone) were used as substrates for the phosphorylation. In contrast to the histone phosphorylation reaction, the endogenous phosphorylation reaction was insensitive to cAMP. Kinetic analysis of the protein kinase reaction associated with the sarcolemma, using histone and Mg γ- 32P-ATP as substrates, showed that this reaction may occur by a sequential, quasi-equilibrium Bi-Bi mechanism, characterized by the following kinetic constants: K m (MgATP) = 12.1 μ m; K m (histone) = 0.47 mg/ml; K i (MgADP) = 15.6 μ m. A comparison of the results of these experiments with data cited in the literature for soluble protein kinase indicated that these enzymes are similar with respect to their kinetic mechanisms.