The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump.

Y. Moriyama, N. Nelson

Research output: Contribution to journalArticle

111 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)9175-9180
Number of pages6
JournalJournal of Biological Chemistry
Volume262
Issue number19
Publication statusPublished - Jul 5 1987
Externally publishedYes

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Chromaffin Granules
Proton Pumps
Anions
Adenosine Triphosphatases
Protons
Membranes
Nitrates
formic acid
Enzymes
Sulfates
Adenosine Triphosphate
Ethylmaleimide
Centrifugation
Secretory Vesicles
Ammonium Sulfate
Durapatite
Protease Inhibitors
Glycerol
Purification
Phospholipids

ASJC Scopus subject areas

  • Biochemistry

Cite this

The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump. / Moriyama, Y.; Nelson, N.

In: Journal of Biological Chemistry, Vol. 262, No. 19, 05.07.1987, p. 9175-9180.

Research output: Contribution to journalArticle

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