Phosphorus metabolite distribution in skeletal muscle: Quantitative bioenergetics using creatine analogs

The functional coupling of contractile activity to metabolic processes in skeletal, cardiac and smooth muscle has been extensively examined in the intact cell through the advent of P-31-NMR spectroscopy. The near-equilibrium formulation for creatine kinase (CK) has been used for the calculation of ADP(free) and ATP chemical potential in many of these studies. However, control of the bulk cytoplasmic PCr/Cr ratio by the ATP/ADP ratio through CK implies that the ATP/ADP ratio is the same in all loci within the cell. Alternatively the cytoplasmic fraction of ATP and ADP must be so large that other 'compartments' do not influence the physicochemical properties of the bulk cytoplasm. By feeding creatine analogs to rodents, it is possible to test whether these synthetic analogs and the endogenous substrates obey simple rules of enzyme kinetics and equilibration. Two important concepts can be tested: (1) Are phosphorus metabolises fully visible to in vivo P-31-NMR measurements? (2) Does CK equilibrate with its substrates in the cytosol? It will be shown that in spite of localized enzymatic activity and microcompartments in the cell (such as mitochondria), creatine kinase equilibrates with its substrates in fast and slow skeletal muscles at rest, Therefore, the physicochemical properties of the cytosol are best described as freely mixing with respect to the bioenergetically important metabolites (PCr, ATP and phospho-analog) and fully quantifiable by P-31-NMR spectroscopy. It follows that only a narrow range of intercellular heterogeneity with respect to chemical potential is acceptable if bioenergetic processes (e.g. CK fluxes or regulation of oxidative phosphorylation) are to be meaningfully interpreted in a rigorous biochemical framework.