Monocarboxylate and alpha-ketoglutarate carriers from bovine heart mitochondria. Purification by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate.
2-Cyano-4-hydroxycinnamate was covalently linked, through a diazo bond, to Sepharose 4B, which had been elongated with a hydrophobic spacer. A Triton X-100 extract from bovine heart mitochondria was pre-purified by hydroxylapatite chromatography and passed through the 2-cyano-4-hydroxycinnamate affinity resin in the presence of 0.7% deoxycholate. At pH 6 and in the presence of 0.2 M sodium chloride, a single polypeptide with an Mr of 34,000 was eluted. Subsequently, at pH 8 and in the presence of 2-cyano-4-hydroxycinnamate, another single protein with an Mr of 31,500 was released. Both proteins were reconstituted into phospholipid vesicles and their transport activities were measured. High, delta pH-dependent, 2-cyanocinnamate-sensitive pyruvate uptake was measured in vesicles containing only the 34-kDa protein. alpha-Ketobutyrate and other alpha-ketomonocarboxylic acids were competitive inhibitors of the pyruvate uptake, whereas di- and tricarboxylates had only small effects. alpha-Ketoglutarate-alpha-ketoglutarate exchange could only be measured in vesicles containing the 31.5-kDa protein. The molecular weight of this protein and its functional properties were similar to those of the alpha-ketoglutarate carrier isolated by a different method (Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369). 2-Cyano-4-hydroxycinnamate inhibited the alpha-ketoglutarate exchange in a noncompetitive manner with an apparent Ki of 0.7 mM. It is concluded that by the described affinity chromatography procedure, two mitochondrial carriers transporting alpha-ketoacids, i.e. the monocarboxylate and the alpha-ketoglutarate carrier, could be purified in a functionally active state.
Bolli
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Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Nałecz
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KA
Azzi
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D000818:Animals; D001692:Biological Transport; D002264:Carboxylic Acids / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D003373:Coumaric Acids; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism
D002264:Carboxylic Acids; D002352:Carrier Proteins; D003373:Coumaric Acids; D007656:Ketoglutaric Acids; D008081:Liposomes; D026901:Membrane Transport Proteins; D011773:Pyruvates; C021723:oxoglutarate translocator; C007175:alpha-cyano-4-hydroxycinnamate