Purification and characterization of the reconstitutively active phosphate transporter from rat liver mitochondria

R. S. Kaplan, R. D. Pratt, P. L. Pedersen

Research output: Contribution to journalArticlepeer-review

Abstract

Procedures have been developed for the purification of a nearly homogeneous, highly active phosphate transport system from rat liver mitochondria in either a two-subunit ((α,β) or a single subunit (β) form. Significantly, both forms display a similar high magnitude N-ethylmaleimide (NEM)-sensitive P(i)/P(i) exchange activity upon incorporation into phospholipid vesicles. The transport system is extracted from hypotonically shocked mitoplasts with Triton X-114 and purified in the presence of cardiolipin by sequential chromatography on hydroxylapatite, DEAE-Sepharose CL-6B, and Affi-Gel 501. Depending on the conditions used to elute the transporter from Affi-Gel 501, preparations are obtained which, when analyzed by high resolution sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis, consist of either a single 33-kDa protein (β) of a 33-kDa (β) plus a 35-kDa (α) component. In preparations yielding the latter result, both bands display a nearly equivalent Coomassie staining intensity. Furthermore, after alkylation with NEM, the two protein bands co-migrate. Fluorography indicates that the coalesced band contains [3H]NEM. Upon reconstitution of the purified P(i) carrier into liposomes, direct measurement of both the initial transport rate and the amount of protein that actually incorporates into the phospholipid vesicles yields a specific transport activity of 22.6 μmol/min/mg of protein. The exchange is characterized by a first order rate constant of 0.85 min-1, a t( 1/2 ) of 49 s, and is inhibited by sulfhydryl reagents (i.e., NEM, p-chloromercuribenzoate, and mersalyl). It is also substantially inhibited by diethyl pyrocarbonate, N-acetylimidazole, phenylglyoxal, and 5-dimethylaminonaphthalene-1-sulfonyl chloride. In addition to providing a simple, rapid method for preparing the NEM-sensitive phosphate carried in nearly homogeneous form, these studies provide new information about the catalytically active species of the carrier, its kinetics properties, and its inhibitor sensitivities.

Original languageEnglish (US)
Pages (from-to)12767-12773
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number27
StatePublished - Dec 1 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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