Bolli R, Nałecz K A, Azzi A
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
J Biol Chem. 1989 Oct 25;264(30):18024-30.
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.
2-氰基-4-羟基肉桂酸通过重氮键与已用疏水间隔臂延长的琼脂糖4B共价连接。牛心线粒体的Triton X-100提取物先用羟基磷灰石色谱法预纯化，然后在0.7%脱氧胆酸盐存在下通过2-氰基-4-羟基肉桂酸亲和树脂。在pH 6和0.2 M氯化钠存在下，洗脱得到一条Mr为34,000的单一多肽。随后，在pH 8和2-氰基-4-羟基肉桂酸存在下，释放出另一条Mr为31,500的单一蛋白质。将这两种蛋白质都重组到磷脂囊泡中并测量其转运活性。在仅含有34-kDa蛋白质的囊泡中测量到高的、依赖于ΔpH的、对2-氰基肉桂酸敏感的丙酮酸摄取。α-酮丁酸和其他α-酮单羧酸是丙酮酸摄取的竞争性抑制剂，而二羧酸和三羧酸的影响较小。α-酮戊二酸-α-酮戊二酸交换只能在含有31.5-kDa蛋白质的囊泡中测量。该蛋白质的分子量及其功能特性与通过不同方法分离的α-酮戊二酸载体相似（Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362 - 369）。2-氰基-4-羟基肉桂酸以非竞争性方式抑制α-酮戊二酸交换，表观Ki为0.7 mM。结论是，通过所述的亲和色谱程序，可以纯化两种转运α-酮酸的线粒体载体，即单羧酸载体和α-酮戊二酸载体，并使其处于功能活性状态。
