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 demons tated by an apparent discrepancy between the forward (F-f) and reverse (F-r ) 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 gamma ATP (inv-ATP), and the dis tribution of metabolites between mitochondria and cytosol was studied by su bcellular fractionation. Physiological conditions were designed to modify t he concentration and distribution of CK metabolites (control, adenylate dep letion, inhibition of respiration, KCl arrest). Depending on cardiac activi ty, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased t ogether with mito-ATP (F-f/F-r ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP), Under conditions masking the influence of the AT P-P-i exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential too l to assess metabolite compartmentation in the whole organ.