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

Abstract

Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-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 K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). 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(+), NH(4) (+) and SO(4) (2-) 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.

Isolation and characterisation of insulin secretory granules from a rat islet cell tumour

“…The large number of proteins present in the insulin granule membrane presumably reflects a diversity of biochemical processes. Some of these properties, like the ability to accumulate protons [28,29], Ca 2+ [30] and amines [31] are found in other granule types such as that of the chromaffin cell of the adrenal medulla. No striking similarities, however, were evident between the observed spectrum of proteins in the insulin granule membrane and that published for the chromaffin granule [321.…”

Histochemical and cytochemical studies of neutral pH p-NPPase on lysosomal membrane in rat liver.

“…Recently the ATP-driven proton pump has been studied extensively in biochemistry (2,3,8,10,31,34). It has been reported that there are three general categories of proton ATPase in eukarytic cells, such as the mitochondrial F0F1 ATPase, the plasma membrane proton ATPase and the vacuolar ATPase (main member of vacuoles is lysosome), and that there are some structural and mechanistic similarities between the lysosomal proton pump and mitochondrial F0F1-ATPase (17,23,24).…”

The insulin secretory granule

“…Evidence for the presence of a proton-translocating ATPase in the insulin secretory granule membrane has come from studies of the ATPase activity in freshly-isolated osmotically-intact insulin granules combined with determination of the pH gradient across the secretory granule membrane, measured by the space of distribution of a radiolabelled permeant base, methylamine, and the transmembrane voltage difference, determined from the distribution of a radiolabelled anion, thiocyanate [24][25][26]. The results of such experiments are readily interpreted within a chemiosmotic model which postulates firstly, that there is a catalytic activity in the membrane which transports hydrogen ions vectorially from the cytosol into the granule interior at the expense of ATP hydrolysis, and secondly that the passive conductivity of the membrane to protons is low.…”