Creatine kinase equilibration follows solution thermodynamics in skeletal muscle. 31P NMR studies using creatine...

The hypothesis tested was whether creatine kinase (CK) equilibrates with its substrates and products in the cytosol as if in solution. We used the creatine analogs cyclocreatine (cCr) or beta-guanidopropionate (beta GPA) to test if mass action ratios (gamma) for CK in muscle could be predicted from combined equilibrium constants (Kcomb) measured in solutions mimicking the intracellular environment. Mice were fed cCr or beta GPA and their muscles assayed for substrates and products of the CK reaction by 31P NMR spectroscopy and high performance liquid chromatography. After three weeks of feeding, gamma was indistinguishable from Kcomb in cCr-treated muscles demonstrating both PCr/Cr and phospho-analog/analog must have equilibrated with a constant and uniform cellular ATP/ADP ratio. In beta GPA-treated muscles, gamma was smaller than Kcomb due to a higher content of muscle beta GPA. Feeding beta GPA for 9-12 weeks resulted in a closer agreement between Kcomb and gamma, suggesting ATP/ADP ratios are not uniform within the muscle perhaps due to transient metabolic stress in some cells. From this analysis it follows that calculation of free ADP from the CK equilibrium for a heterogeneous population of cells with respect to total Cr and ATP content is correct only if chemical potentials of these cells are uniform.