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 ((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.