Measurement of an individual rate constant in the presence of multiple exchanges: application to myocardial creatine kinase reaction.

Abstract

Forward [creatine phosphate (CP)----adenosine 5'-triphosphate (ATP)] and reverse (ATP----CP) fluxes of myocardial creatine kinase (CK) measured by using 31P nuclear magnetic resonance (NMR) and conventional saturation transfer (CST) methods are unequal; this is a paradoxical result because during steady state fluxes into and out of the CP pool must be the same. These measurements, however, treat the CK reaction as a two-site exchange problem and ignore the presence of the ATP gamma in equilibrium Pi exchange involving the ATPases. We have applied a method [Uğurbil, K. (1985) J. Magn. Reson. 64, 207] based on the saturation of multiple resonances, by which a single unidirectional rate constant can be measured unequivocally in the presence of multiple exchanges, to the measurement of CK fluxes in isovolumic rat hearts perfused under three different conditions; two of the three perfusion conditions showed a large discrepancy in the CK fluxes determined by CST, and one did not. In contrast, when the effect of the ATP gamma in equilibrium Pi exchange on the CK rate measurements was eliminated, multiple saturation transfer (MST) measurements on the same hearts yielded equal forward and reverse fluxes in all cases. The rate constant for the ATP gamma----CP conversion measured by MST was larger than the value obtained by the conventional methodology whereas both methods gave the same rate constant in the CP----ATP direction. These results demonstrate that the cause of the paradoxical data obtained by CST measurements of CK kinetics is the ATP gamma in equilibrium Pi exchange and that CK rates when determined rigorously are consistent with the CK reaction being in equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)