title: Nucleotide and bivalent cation specificity of the insulin-granule proton translocase.
abstract: 1. The nucleotide and bivalent cation specificity of the proton translocase activity of insulin secretory granules was investigated by assessing the inhibitor-sensitive rates of nucleotide hydrolysis by these organelles in relation to their chemiosmotic properties. 2. The relative rates of nucleotide hydrolysis by freeze/thawed granule preparations were: Mg2+ATP (100%) greater than Mg2+GTP (55%) greater than Mg2+UTP (48%) greater than Mg2+ITP (44%) greater than Mg2+CTP (23%) greater than Mg2+TTP (20%), and by intact granules were: Mg2+ATP (100%) greater than Mg2+ITP (74%) greater than Mg2+GTP (60%) greater than Mg2+CTP (35%). Mg2+ATP, Mg2+GTP and Mg2+ITP hydrolyses were inhibited by tributyltin and stimulated, in intact granules, by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Mg2+CTP hydrolysis was not markedly affected by these compounds. Correspondingly, only Mg2+ATP, Mg2+GTP and Mg2+ITP produced large changes in the delta psi and delta mu H+ across the granule membrane. 3. The relative rates of maximal ATPase activity stimulated by bivalent cations in freeze/thawed granule preparations were: Mg2+ (100%) greater than Mn2+ (82%) greater than Ca2+ (40%) greater than Co2+ (36%) greater than Zn2+ (0%), and in intact granules were: Mg2+ (100%) greater than Mn2+ (85%) greater than Co2+ (61%) greater than Ca2+ (42%). Tributyltin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone affected Mg2+-, Mn2+- and Co2+-activated, but not Ca2+-activated, ATP hydrolysis. Correspondingly, only Mg2+, Mn2+ and Co2+ supported the generation of a delta psi and delta mu H+ across granule membranes in the presence of ATP. 4. The results were consistent with a single proton translocase that had its catalytic site exposed on the external face of the granule membrane. The indicated specificity (Mg2+ATP = Mn2+ATP greater than Co2+ATP greater than Mg2+GTP greater than Mg2+ITP) was similar to that of enzymes described in membrane fractions prepared from adenohypophyseal tissue, adrenal chromaffin granules and yeast vacuoles. The insulin-granule activity thus appears to be a type of proton translocase, which is characteristic of intracellular storage vesicles in eukaryotic cells.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000201:enzymology; D004563:Electrochemistry; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000201:enzymology; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D009374:Neoplasms, Experimental / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000201:enzymology; D006180:Proton-Translocating ATPases; D011522:Protons; D051381:Rats; D014220:Trialkyltin Compounds / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D007328:Insulin; D008565:Membrane Proteins; D009711:Nucleotides; D011522:Protons; D014220:Trialkyltin Compounds; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C011559:tributyltin; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases