IN-VITRO DETERMINATION OF CREATINE-KINASE SUBSTRATE FLUXES USING P-31-NUCLEAR-MAGNETIC-RESONANCE

Forward (k(f)) and reverse (k(r)) ratWe measured k, > k, for all enzym e activities below 600 U ml-l. This obsenration could partially be exp lained by the presence of ATPase contamination in the enzyme. These fi ndings are relevant for the in vive studies of creatine kinase activit y in the presence of multi-site phosphate exchange in cellular ATP-poo ls. As mitochondrial creatine kinase is not in equilibrium these resul ts are also ofe constants and the corresponding flux rates of the crea tine kinase catalysed reaction between creatine phosphate (CrP) and ad enosine triphosphate (ATP); CrP + ADP [GRAPHICS] ADP + Cr were measure d in vitro at 295 K. Both rate constants were determined using magneti c resonance saturation transfer techniques. To study the dependence of k(r) and k(f) on the creatine kinase concentration, the ceatine kinas e activity was varied from 2400 to 75 U.ml(-1). At equilibrium and hig h creatine kinase activities, the forward to reverse flux rate ratios are close to 1. A dispersion in the reaction rate constants was observ ed at activities less than or equal to 600 U.ml(-1) We measured k(r) > k(f) for all enzyme activities below 600 U.ml(-1). This observation c ould partially be explained by the presence of ATPase contamination in the enzyme. These findings are relevant for the in vivo studies of cr eatine kinase activity in the presence of multi-site phosphate exchang e in cellular ATP-pools. As mitochondrial creatine kinase is not in eq uilibrium these results are also of interest in this area.