Poole R C, Halestrap A P
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Biochem J. 1994 Nov 1;303 ( Pt 3)(Pt 3):755-9. doi: 10.1042/bj3030755.
An improved purification for the rabbit erythrocyte lactate transporter, using aminoethyl-Sepharose chromatography, is described. The process of purification of the 40-50 kDa transporter, labelled with 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), was followed by Western blotting with anti-DIDS antibodies [Poole, R. C. and Halestrap, A. P. (1992) Biochem. J. 283, 855-862]. Fractions highly-enriched in transporter were further purified by SDS/PAGE and the 40-50 kDa DIDS-labelled polypeptide was subjected to N-terminal protein sequencing. This analysis identified the first 16 amino acids of the protein. With the exception of one conservative substitution, this protein sequence is identical to the N-terminal protein sequence predicted from a cDNA isolated from Chinese hamster ovary cells that encode a monocarboxylate transporter, MCT1 [Kim Garcia, C., Goldstein, J. L., Pathak, R. K., Anderson, R. G. W. and Brown, M. S. (1994) Cell 76, 865-873]. This observation, along with similarities in functional properties, leads us to conclude that lactate transport in rabbit erythrocytes is mediated by the MCT1 monocarboxylate transporter isoform.
本文描述了一种使用氨乙基琼脂糖层析法对兔红细胞乳酸转运蛋白进行的改进纯化方法。用4,4'-二异硫氰基芪-2,2'-二磺酸盐（DIDS）标记的40 - 50 kDa转运蛋白的纯化过程，随后用抗DIDS抗体进行蛋白质印迹分析[普尔，R.C.和哈利斯特雷普，A.P.（1992年）《生物化学杂志》283卷，855 - 862页]。富含转运蛋白的组分通过SDS/PAGE进一步纯化，对40 - 50 kDa的DIDS标记多肽进行N端蛋白质测序。该分析确定了该蛋白质的前16个氨基酸。除了一个保守替换外，该蛋白质序列与从中国仓鼠卵巢细胞分离的编码单羧酸转运蛋白MCT1的cDNA预测的N端蛋白质序列相同[金·加西亚，C.，戈尔茨坦，J.L.，帕塔克，R.K.，安德森，R.G.W.和布朗，M.S.（1994年）《细胞》76卷，865 - 873页]。这一观察结果，连同功能特性的相似性，使我们得出结论，兔红细胞中的乳酸转运是由MCT1单羧酸转运蛋白同工型介导的。
