A comparison of 31P-NMR saturation transfer and isotope-exchange measurements of creatine kinase kinetics in vitro

Abstract

31P-NMR measurements of saturation transfer have been used to measure the exchange of phosphate between phosphocreatine and the γ-phosphate of ATP in the reaction catalysed by creatine kinase in vitro. The similarity of the calculated exchange flux with the flux estimated from an isotope-exchange experiment, in which exchange of 15N label between creatine and phosphocreatne was measured, showed that the two-site-exchange model, normally used in the analysis of saturation-transfer data, is valid in this case. 15N label exchange was monitored using a heteronuclear 31P/ 15N spin-echo NMR experiment in which the incorporation of 15N label into phosphocreatine was detected by following the phase modulation of the spin-spin coupled 31P resonance. The isotope-exchange experiment should prove to be useful in studies of creatine kinase in systems where the low concentration of the enzyme precludes saturation-transfer measurements, for example in muscle mitochondria preparations.