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PMID: 2904677 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 85 ·No. 24 ·1988-12-00 ·Pages 9590-4

Young GP, Qiao JZ, Al-Awqati Q

Abstract

Kidney cortex microsomes enriched in Golgi markers and probably also containing endosomes were isolated by cell fractionation and found to contain a proton-translocating ATPase that was inhibited by N-ethylmaleimide (NEM). This NEM-sensitive ATPase was solubilized with n-octyl glucoside and purified using anion-exchange sievorptive chromatography on sequential DEAE-Sephadex and QAE-Sephadex columns followed by a final hydroxyapatite HPLC column. The purified enzyme, with a specific activity of 4.4 mumol.mg-1.min-1 was completely inhibited by NEM. Addition of asolectin and removal of the detergent by dialysis resulted in reconstitution of NEM-sensitive electrogenic proton transport. This vacuolar ATPase is composed of five polypeptides with apparent molecular masses of 68, 58, 40, 37, and 16 kDa.

MeSH Terms

Animals Cattle Chromatography, High Pressure Liquid Ethylmaleimide/pharmacology Golgi Apparatus/enzymology,ultrastructure Intracellular Membranes/enzymology Kidney Cortex/ultrastructure Molecular Weight Proton-Translocating ATPases/analysis

Chemicals

Proton-Translocating ATPases Ethylmaleimide

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Young G P

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032.

Qiao J Z

Al-Awqati Q

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24 references, click to expand

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Article Info

Journal

Proceedings of the National Academy of Sciences of the United States of America

Abbr.

Proc Natl Acad Sci U S A

ISSN

0027-8424

DOI

10.1073/pnas.85.24.9590

Published

1988-12-00

Pages

9590-4

Language

English

Region

United States

NLM ID

7505876

PMCID

PMC282806

Subset

Grants

NIDDK NIH HHS · DK 20999 · United States

NINDS NIH HHS · NS 07258 · United States

External Links

PubMed source DOI: 10.1073/pnas.85.24.9590 PMC Full Text View journal details

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Purification and reconstitution of the proton-translocating ATPase of Golgi-enriched membranes.

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