Cardiac creatine kinase metabolite compartments revealed by NMR magnetization transfer spectroscopy and subcellular fractionation.

In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstated by an apparent discrepancy between the forward (Ff) and reverse (Fr) CK flux if this compartment was neglected in the analysis [Joubert et al. (2000) Biophys. J. 79, 1−13]. To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or γATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (Ff/Fr ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP). Under conditions masking th...