Determination of mitochondrial creatine kinase fluxes in intact heart mitochondria using 31P-saturation transfer nuclear magnetic resonance spectroscopy

Abstract

Forward (→ATP) and reverse (→CrP) fluxes through the creatine kinase reaction were determined in isolated rat and bovine heart mitochondria and with soluble MM-CK from rabbit skeletal muscle, using 31P-saturation transfer NMR. With soluble MM-CK forward and reverse fluxes were identical in the absence and presence of BSA or rat liver mitochondria. Addition of liver mitochondria decreased fluxes with increasing mitochondria concentration. The flux f/ V max (f) ratio was 0.006 with 10 mg BSA and 0.04 with 10 mg rat liver mitochondria, respectively. With heart mitochondria, flux r was considerably higher than flux f and the flux f/ V max (f) ratio was 1.7 for rat heart and 0.22 for bovine heart. It is concluded that in the presence of isolated mitochondria, the flux through the creatine kinase is driven by the mitochondrial ATP–ADP turnover. Therefore the flux f/ V max (f) ratio is highest for rat heart mitochondria with a high ATP–ADP turnover, intermediate for bovine heart mitochondria and low for MM-CK in the presence of liver mitochondria. It is lowest with MM-CK alone, where the creatine kinase reaction is at equilibrium and external ATP–ADP turnover is absent. The higher reverse than forward fluxes of mitochondrial creatine kinase determined at steady state by saturation transfer NMR, are caused mainly by a high ATP↔P i exchange in heart mitochondria preparations, having a high ATPase activity, compared to liver mitochondria.