GABA production in rat islets of Langerhans.
Homogenates of pancreatic islets catalyzed breakdown of L-glutamate to GABA with a rate of 0.24 +/- 0.04 nmol.min-1 x mg-1 protein at 37 degrees C. The formation of GABA was stimulated by addition of pyridoxal phosphate in the range 0.05-1 microM (0.97 +/- 0.02 nmol.min-1 x mg protein-1 at a saturating cofactor concentration), which indicates that the process was catalyzed by glutamic acid decarboxylase. The half-maximal effect was obtained with 0.1 microM PLP. Kinetic analyses of the results showed that the Vmax and Km for the reaction were 1.12 nmol.min-1 x mg protein-1 and 0.66 mM, respectively. The pH optimum was 7.0. Subcellular fractionation revealed that 51% of GAD activity was present in the cytosol, 17% in microsomes, 9% in secretory granules, 5% in mitochondria, and 11% in cell debris. Comparison of the kinetic properties of the cytosolic and microsomal forms of the enzyme showed that their Km for glutamate was the same, but that the cytosolic GAD had a lower Km for PLP. GABA synthesis in the nominal absence of PLP was enhanced by malate (twofold increase at 5 mM) and citrate (threefold increase at 5 mM), but was unaffected by ATP and chloride. However, if the islet homogenate was prepared and incubated in the presence of PLP, neither malate nor citrate influenced enzyme activity. Aspartate and AOA were powerful inhibitors of glutamate breakdown. Freshly isolated islets contained approximately 4 mM GABA, whereas the concentration was < 0.1 mM in whole pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmacology, University of Pennsylvania, School of Medicine, Philadelphia 19104.
M
M
Michalik
J
J
Nelson
M
M
Erecińska
D000244:Adenosine Diphosphate / Q000032:analysis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D002951:Citrates / Q000494:pharmacology; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D005968:Glutamate Decarboxylase / Q000032:analysis / Q000378:metabolism / Q000502:physiology; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000097:blood; D007515:Islets of Langerhans / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008293:Malates / Q000494:pharmacology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D005680:gamma-Aminobutyric Acid / Q000032:analysis / Q000378:metabolism*
D002951:Citrates; D003509:Cyclohexanecarboxylic Acids; D005971:Glutamates; D007328:Insulin; D008293:Malates; D000625:Aminooxyacetic Acid; D001224:Aspartic Acid; C012987:gabaculine; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005968:Glutamate Decarboxylase