Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain

X. S. Xie, D. K. Stone, E. Racker

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Abstract

Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X.S., and Stone, D.K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 μM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 μmol of P(i)·mg of protein-1·min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.

Original languageEnglish (US)
Pages (from-to)1710-1714
Number of pages5
JournalJournal of Biological Chemistry
Volume264
Issue number3
StatePublished - 1989

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Clathrin-Coated Vesicles
Clathrin
Purification
Adenosine Triphosphatases
Brain
Ethylmaleimide
Vanadates
Ouabain
Proton-Translocating ATPases
Centrifugation
Acidification
Egtazic Acid
High performance liquid chromatography
Molecular mass
Enzymes
Electrophoresis
Oligomers
Sodium Dodecyl Sulfate
Gel Chromatography
Sucrose

ASJC Scopus subject areas

  • Biochemistry

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Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain. / Xie, X. S.; Stone, D. K.; Racker, E.

In: Journal of Biological Chemistry, Vol. 264, No. 3, 1989, p. 1710-1714.

Research output: Contribution to journalArticle

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