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Biochimie. 1996;78(3):195-200.

Characterization, purification and properties of the yeast mitochondrial dicarboxylate carrier (Saccharomyces cerevisiae).

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  • 1Université de Rouen, Faculté des Sciences, Laboratoire des Transports intracellulaires, URA-CNRS 203, Mont-Saint-Aignan, France.

Abstract

The dicarboxylate carrier has been characterized and purified from mitochondria of wild strain Saccharomyces cerevisiae. The mitochondria were solubilized with Triton X-100 and the detergent extract was chromatographed on hydroxylapatite. SDS-PAGE of the hydroxylapatite pass-through showed five protein bands with M(r)s ranging from 28,000 to 35,000, by silver nitrate staining. The n-butylmalonate-sensitive succinate(out)/malate(in) exchange activity of the hydroxylapatite pass-through reconstituted into liposomes, was increased nine-fold with respect to the activity of the Triton X-100 extract. The exchange activity was inhibited by p-chloromercuriphenylsulfonate (PMPS), 4.4'diisothiocyanostilbene-2.2'-disulfonate (DIDS) and pyridoxal-phosphate, suggesting that one or more thiol groups and basic residues are implicated in the binding mechanism. The purification of the carrier was achieved by affinity chromatography on Sepharose-immobilized malate dehydrogenase. The purified protein presented the same properties as the dicarboxylate carrier in native mitochondria and displayed a single protein band with an M(r) of 28,000 as determined by SDS-PAGE. The specific activity of the purified carrier showed a 53-fold increase compared to that of the initial material. The Km for the reconstituted exchange was 2 mM for succinate with a V of 1.5 mumol min-1 mg-1 protein at 22 degrees C. The high purification state achieved for the yeast dicarboxylate carrier should allow the study of its molecular properties.

PMID:
8831951
[PubMed - indexed for MEDLINE]
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