MONOCARBOXYLATE AND ALPHA-KETOGLUTARATE CARRIERS FROM BOVINE HEART-MITOCHONDRIA - PURIFICATION BY AFFINITY-CHROMATOGRAPHY ON IMMOBILIZED 2-CYANO-4-HYDROXYCINNAMATE

  • 25 October 1989
    • journal article
    • research article
    • Vol. 264  (30) , 18024-18030
Abstract
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 0.75 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 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 moleuclar 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 818, 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.