title: The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump.
abstract: The proton-ATPase of chromaffin granules was purified so as to maintain its proton-pumping activity when reconstituted into phospholipid vesicles. The purification procedure involved solubilization with polyoxyethylene 9 lauryl ether, hydroxylapatite column, precipitation by ammonium sulfate, and glycerol gradient centrifugation. The protease inhibitor mixture used in previous studies inhibited the proton-pumping activity of the enzyme; therefore, the protein was stabilized by pepstatin A and leupeptin. The enzyme was purified at least 50-fold with respect to both ATPase and proton-pumping activity. The ATP-dependent proton uptake activity of the reconstituted enzyme was absolutely dependent on the presence of Cl- or Br- outside the vesicles, whereas sulfate, acetate, formate, nitrate, and thiocyanate were inhibitory. Sulfate inhibition seems to be due to competition with Cl- on the anion-binding site outside the vesicles, whereas nitrate and thiocyanate inhibited only from the internal side. As with the inhibition by N-ethylmaleimide, the proton-pumping activity was much more sensitive to nitrate than the ATPase activity. About 20 mM nitrate were sufficient for 90% inhibition of the proton-pumping activity while 100 mM inhibited only 50% of the ATPase activity both in situ and in the reconstituted enzyme. The possible regulatory effect of anions on the ATP-dependent proton uptake in secretory granules is discussed.
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology*; D008564:Membrane Potentials; D009566:Nitrates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D009566:Nitrates; D014634:Valinomycin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017301:Ca(2+) Mg(2+)-ATPase; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid