Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat.
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 reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure 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 reaction 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: greater than 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 less than 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.
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
J A
JA
Bittl
J
J
DeLayre
J S
JS
Ingwall
D000818:Animals; D001921:Brain / Q000378:metabolism*; D003402:Creatine Kinase / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D009132:Muscles / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D009928:Organ Specificity; D010758:Phosphorus; D051381:Rats; D011919:Rats, Inbred Strains
D010758:Phosphorus; D003402:Creatine Kinase