Creatine kinase-catalyzed ATP-phosphocreatine exchange:: Comparison of 31P-NMR saturation transfer technique and radioisotope tracer methods

Abstract

Unidirectional fluxes from ATP to phosphocreatine, catalyzed by the MM isoenzyme of creatine kinase, were measured by both the 31P-NMR saturation transfer technique and radioisotope tracer ([γ- 32P]ATP) method. It was found that at 30–37°C and pH 7.4, over a wide range of[phosphocreatine]/[creatine] (from 0.2 to 5.0) ratios, both methods gave the same results, showing that magnetization transfer allows determination of real fluxes under ‘physiological’ conditions. However, at[PCr]/[Cr] ratios higher than 5 ([ADP] free < 30 μM) or at lower temperatures (t < 15°C,[PCr]/[Cr] ∼ 1), the fluxes assessed by saturation transfer were somewhat faster than those detected by the radioisotope tracer method. These data imply that under physiological conditions phosphoryl group transfer is actually the rate-determining step of the creatine kinase reaction. In contrast, at high[PCr]/[Cr] ratios or at lower temperatures, control may be shifted from phosphoryl group transfer or distributed among other steps of the reaction.