Askenasy, Nadir, and Alan P. Koretsky. Transgenic livers expressing mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels. Am J Physiol Cell Physiol 282: C338-C346, 2002. First published October 3, 2001 10.1152/ajpcell.00404.2001.-The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination.31 P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 mol ⅐ g wet wt Ϫ1 ⅐ min Ϫ1 ), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 Ϯ 9 to 38 Ϯ 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920-1,635 mol ⅐ g wet wt Ϫ1 ⅐ min Ϫ1 ) were 52 Ϯ 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 Ϯ 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations. mitochondrial creatine kinase; cytosolic creatine kinase; phosphorus-31 nuclear magnetic resonance spectroscopy; magnetization transfer; liver adenosine 5Ј-diphosphate concentration