SUBSTRATE CYCLING BETWEEN 5-AMINO-4-IMIDAZOLECARBOXAMIDE RIBOSIDE ANDITS MONOPHOSPHATE IN ISOLATED RAT HEPATOCYTES

AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5'-p hosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of t he de novo synthesis of purine nucleotides. We investigated ii, in the se cells, a cycle analogous to the adenosine-AMP substrate cycle opera tes between AICAriboside and ZMP. When 50 mu M ITu, an inhibitor of ad enosine kinase, was added to hepatocytes that had metabolized AICAribo side for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at diffe rent concentrations of ZMP, were first order, up to the highest concen tration of ZMP (approx. 5 mu mol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic k inetics, with a V-max of 0.65 mu mol/min per g protein and an apparent K-m of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GM P 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most l ikely by IMP-GMP 5'-nucleotidase, into AICAriboside, which is rephosph orylated into ZMP by adenosine kinase. Substrate cycling was also show n to occur between other nucleoside analogs and their phosphorylated c ounterparts.