Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance

Abstract

31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.