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. 1982 Apr 15;204(1):161–170. doi: 10.1042/bj2040161

Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules

John C Hutton 1,*, Mina Peshavaria 1
PMCID: PMC1158328  PMID: 6126182

Abstract

Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg2+-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2–3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K2SO4 or by the addition of a detergent. Permeant anions also increased Mg2+-dependent ATPase activity under iso-osmotic conditions when combined with K+ and nigericin, or NH4+. It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H+, K+, NH4+ and SO42− but permeable, in increasing order, to phosphate or acetate, Cl−, I− and SCN−. Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN′-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.

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Selected References

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