CHARACTERIZATION OF NUCLEOSIDE DIPHOSPHOKINASE ACTIVITY IN HUMAN AND RODENT PANCREATIC BETA-CELLS - EVIDENCE FOR ITS ROLE IN THE FORMATION OF GUANOSINE TRIPHOSPHATE, A PERMISSIVE FACTOR FOR NUTRIENT-INDUCED INSULIN-SECRETION

We have recently demonstrated a permissive role for GTP in nutrient-induced insulin secretion. One of the possible loci at which GTP might exert its regulatory effects include one (or more) of the GTP-binding proteins which we have identified in subcellular fractions (including secretory granules) of pancreatic islets. Herein, we characterize nucleoside diphosphokinase (NDP kinase) activity, which catalyzes the transphosphorylation of nucleotide diphosphate (e.g., GDP) to nucleotide triphosphates (e.g., GTP) in insulin-secreting cells. The presence of NPP kinase activity in normal rat and human islets, and pure beta (RIN and HIT) cells, was verified by three distinct approaches: first, its catalytic activity (formation of GTP or GTP gamma S from GDP and ATP or ATP gamma S); secondly, by immunologic detection; and third, by quantitating the phosphoenzyme intermediate of NDP kinase, which is involved in a ping-pong phosphotransfer mechanism. Subcellularly, NDP kinase is predominantly cytosolic (with a tetrameric molecular mass of 85-90 kDa) and requires divalent metal ions and thiols for its activity. UDP, which forms an abortive complex with the enzyme, inhibited its activity in a Concentration-dependent manner (K-i = 2 mM). The phosphorylated intermediate of NDP kinase was differentially sensitive to heat, acidic pH, and a histidine-selective reagent, diethyl pyrocarbonate, suggesting that (one of) the phosphoamino acid(s) may be histidine. These data demonstrate that in beta cells NDP kinase undergoes transient phosphorylation and suggest that this phosphate, in turn, is transferred to GDP. If the GTP which is formed thereby is bound to, or channelled to, relevant GTP-binding proteins, it would facilitate the formation of active form of these proteins. NDP kinase might therefore couple mitochondrial oxidative events to extramitochondrial sites critical to stimulus-secretion coupling.