Purification and characterization of the reconstitutively active phosphate transporter from rat liver mitochondria.
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.
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D002308:Cardiolipins / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D008634:Mersalyl / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000032:analysis*; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology; D028044:Phosphate-Binding Proteins; D051381:Rats
D002308:Cardiolipins; D002352:Carrier Proteins; D007093:Imidazoles; D028044:Phosphate-Binding Proteins; C036590:1-(1-phenylcyclohexyl)-3-methylpiperidine; D008634:Mersalyl; D001120:Arginine; D010622:Phencyclidine; D008239:Lysine; D005033:Ethylmaleimide; C012160:N-acetylimidazole