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

J Biol Chem. 1986 Sep 25;261(27):12767-73.

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 (alpha, beta) or a single subunit (beta) form. Significantly, both forms display a similar high magnitude N-ethylmaleimide (NEM)-sensitive Pi/Pi 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 (beta) or a 33-kDa (beta) plus a 35-kDa (alpha) 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 Pi 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 mumol/min/mg of protein. The exchange is characterized by a first order rate constant of 0.85 min-1, a t1/2 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-dimethylaminoaphthalene-1-sulfonyl chloride. In addition to providing a simple, rapid method for preparing the NEM-sensitive phosphate carrier in nearly homogeneous form, these studies provide new information about the catalytically active species of the carrier, its kinetic properties, and its inhibitor sensitivities.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arginine / metabolism
  • Biological Transport, Active / drug effects
  • Cardiolipins / pharmacology
  • Carrier Proteins / isolation & purification*
  • Electrophoresis, Polyacrylamide Gel
  • Ethylmaleimide / pharmacology
  • Imidazoles / pharmacology
  • Lysine / metabolism
  • Mersalyl / pharmacology
  • Mitochondria, Liver / analysis*
  • Phencyclidine / analogs & derivatives
  • Phencyclidine / pharmacology
  • Phosphate-Binding Proteins
  • Rats

Substances

  • Cardiolipins
  • Carrier Proteins
  • Imidazoles
  • Phosphate-Binding Proteins
  • 1-(1-phenylcyclohexyl)-3-methylpiperidine
  • Mersalyl
  • Arginine
  • Phencyclidine
  • Lysine
  • Ethylmaleimide
  • N-acetylimidazole