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 -guanidopropionate (GPA) to test if mass action ratios () for CK in muscle could be predicted from combined equilibrium constants (K) measured in solutions mimicking the intracellular environment. Mice were fed cCr or GPA and their muscles assayed for substrates and products of the CK reaction by P NMR spectroscopy and high performance liquid chromatography. After three weeks of feeding, was indistinguishable from K 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 GPA-treated muscles, was smaller than K due to a higher content of muscle GPA. Feeding GPA for 9-12 weeks resulted in a closer agreement between K and , 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.

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