Studies on Mg2+-dependent ATPase in bovine adrenal chromaffin granules. With special reference to the effect of inhibitors and energy coupling.
The Mg2+-ATPase activities of bovine adrenal chromaffin granules were studied in highly purified preparations of granule ghosts and in intact organelles. The overall ATPase activity (150-250 nmol ADP min-1 mg-1) of the granule ghost preparations was inhibited less than 5% by the bathophenanthroline chelate of Fe(II), a potent inhibitor of mitochondrial F1-ATPase. This small inhibition can be accounted for by a very minor contamination with mitochondria or mitochondrial fragments. The overall ATPase activity of native granule ghosts was inhibited about 75% by N-ethylmaleimide, with half-maximal inhibition at about 20 microM. The titration curve was slightly shifted towards higher concentrations as compared to the inhibition curve for the proton pump activity, which was completely inhibited at 25 microM. N,N'-Dicyclohexylcarbodiimide inhibited the overall ATPase activity by 75-80% at 1.1 mumol/mg protein, a concentration that completely abolished the proton pump activity. Low concentrations (10 microM) of vanadate inhibited the overall ATPase activity by about 15% but had no effect on the proton pump activity, which was partly inhibited only at higher vanadate concentrations. Our attempts to assign a function to the vanadate-sensitive and N-ethylmaleimide-insensitive ATPase have so far been unsuccessful. In particular, our assay for ATP diphosphohydrolase activity was negative, although the chromaffin granule ghosts revealed a low Mg2+-ADPase activity (11.8 nmol AMP min-1 mg-1 protein). In intact chromaffin granules the specific Mg2+-ATPase activity (50-70 nmol ADP min-1 mg-1) was stimulated 2-fold by uncouplers, as compared to 1.6-1.7-fold in granule ghosts. The degree of energy coupling was rather independent of the external pH (6.5 less than pH less than 8.0) and temperature (20-45 degrees C). As expected, partial inhibition (about 15%) of the overall ATPase activity by 10 microM vanadate increased the ATPase control ratio. ADP was found to be a potent inhibitor of the proton pump activity with MgATP as the substrate, and the effect can partly be explained by a competitive type of inhibition of the hydrolytic reaction. This effect of ADP explains some of the kinetic data reported for MgATP-dependent (H+-ATPase-dependent) reactions in this organelle, notably the energy-dependent accumulation and storage of catecholamines.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000166:cytology* / Q000201:enzymology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010618:Phenanthrolines / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D013696:Temperature; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D010618:Phenanthrolines; D014639:Vanadium; D014638:Vanadates; C006686:bathophenanthroline; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide