Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria

Janna P. Wehrle, Peter L Pedersen

Research output: Contribution to journalArticle

Abstract

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.

Original languageEnglish (US)
Pages (from-to)477-483
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume223
Issue number2
DOIs
StatePublished - 1983

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Phosphate Transport Proteins
Mitochondria
Ethylmaleimide
Liver Mitochondrion
Liver
Rats
Proton-Translocating ATPases
Membranes
Liposomes
Purification
Phospholipids
Chloromercuribenzoates
Active Biological Transport
Octoxynol
Mitochondrial Membranes
Durapatite
Therapeutics
Electrophoresis
Sodium Dodecyl Sulfate
Powders

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria. / Wehrle, Janna P.; Pedersen, Peter L.

In: Archives of Biochemistry and Biophysics, Vol. 223, No. 2, 1983, p. 477-483.

Research output: Contribution to journalArticle

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