Rate equation for creatine kinase predicts the in vivo reaction velocity: phosphorus-31 NMR surface coil studies in brain, heart, and skeletal muscle of the living rat

Abstract

Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase rection under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques ot meausre reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 .+-. 2.8, 62.4 .+-. 4.5, and 224 .+-. 16 mM/s) and exceeded the NMR-determined in vivo reactions velocities by an order of magnitude (4.1.+-. 1.2, 5.1 .+-. 1.6, and 18.4 .+-. 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues; >99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98%MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p < 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.