title: Characterization, purification and properties of the yeast mitochondrial dicarboxylate carrier (Saccharomyces cerevisiae).
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.
authors.affiliation: Université de Rouen, Faculté des Sciences, Laboratoire des Transports intracellulaires, URA-CNRS 203, Mont-Saint-Aignan, France.
authors.forename: J
authors.initials: J
authors.lastname: Lançar-Benba
authors.forename: B
authors.initials: B
authors.lastname: Foucher
authors.forename: M
authors.initials: M
authors.lastname: Saint-Macary
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000838:Anions / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry*; D008970:Molecular Weight; D011510:Proteolipids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*
chemical_list: D000838:Anions; D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; D002731:4-Chloromercuribenzenesulfonate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid