Glutamate production in islets of Langerhans: properties of phosphate-activated glutaminase.
Homogenates of rat pancreas, pancreatic islets, and HIT-T15 cells (a clonal line derived from B cells) catalyzed the breakdown of glutamine to glutamate. This activity was markedly stimulated by the addition of orthophosphate and was much greater in homogenates from islets and the B-cell-derived clonal cell line than in those from whole pancreas. Islet glutaminase was half-maximally stimulated with 40 mmol/L phosphate. Kinetic analyses of the rates of glutamine hydrolysis showed that the Vmax for the reaction increased with the increase in phosphate concentration, whereas the Km for glutamine (2.6 +/- 0.2 mmol/L) was unaltered. The pH optimum for enzyme activity was 8.0 to 8.5 at all phosphate concentrations studied. Glutamine breakdown was enhanced by adenosine triphosphate ([ATP] approximately 100% at 10 mmol/L) and citrate (approximately 30% at 10 mmol/L), but it was unaffected by malate, 2-oxoglutarate, lactate, and ammonia. Glutamate significantly inhibited glutamine hydrolysis. Freshly isolated islets had a low content of both glutamate and glutamine. After culturing for 1 hour in an amino acid-containing medium, the concentrations of glutamine and glutamate increased. Subsequent perifusion without amino acids caused a loss of glutamine and a concomitant increase in glutamate level. Perifusion with 1 mmol/L glutamine led to an increase in both internal glutamine and glutamate. The addition to the perifusion medium of either 10 mmol/L glutamine, 10 mmol/L orthophosphate, or both substantially enhanced insulin release evoked by 10 mmol/L leucine.(ABSTRACT TRUNCATED AT 250 WORDS)
Michalik
M
M
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Nelson
J
J
Erecińska
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D002951:Citrates / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000378:metabolism* / Q000502:physiology; D018698:Glutamic Acid; D005972:Glutaminase / Q000378:metabolism* / Q000502:physiology; D005973:Glutamine / Q000378:metabolism / Q000493:pharmacokinetics / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism*; D007773:Lactates / Q000494:pharmacology; D008293:Malates / Q000494:pharmacology; D008297:Male; D010179:Pancreas / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D010710:Phosphates / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D000596:Amino Acids; D002951:Citrates; D005971:Glutamates; D007328:Insulin; D007773:Lactates; D008293:Malates; D010710:Phosphates; D005973:Glutamine; D018698:Glutamic Acid; D005972:Glutaminase