L'Hostis C, Geindre M, Deshusses J
Department of Biochemistry, University of Geneva, Switzerland.
Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):297-301. doi: 10.1042/bj2910297.
The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.
利用L-[14C]脯氨酸以及通过油混合物离心的快速分离技术，研究了布氏锥虫前循环型中L-脯氨酸的转运特性。L-脯氨酸摄取呈现典型的米氏动力学，在27℃时，Km为19微摩尔，每10(8)个细胞的最大转运速度为17纳摩尔/分钟。在0.02毫摩尔脯氨酸中孵育1分钟后获得的最大浓度梯度因子为270倍。用80微摩尔洋地黄皂苷通透化的细胞仍能够积累14C标记，但程度较低。脯氨酸摄取的温度依赖性给出了74.9千焦·摩尔-1的表观活化能。在与10倍过量的结构类似物的竞争研究中，发现L-丙氨酸、L-半胱氨酸和L-氮杂环丁烷-2-羧酸抑制L-脯氨酸摄取。pH的变化或添加质子载体羰基氰化物间氯苯腙（“CCCP”）不影响脯氨酸转运，表明其不是由质子动力驱动的。无论有无莫能菌素，Na+的缺失均不影响脯氨酸转运。K+的缺失以及添加Na+，K(+)-ATP酶抑制剂哇巴因对脯氨酸摄取活性无显著影响。硫醇修饰试剂碘乙酸盐（10毫摩尔）使脯氨酸摄取降低一半。KCN（1毫摩尔）对脯氨酸摄取的抑制程度较小，且抑制程度与细胞内ATP浓度成正比。使用过滤技术对细胞的质膜囊泡中脯氨酸转运进行的初步实验表明，囊泡摄取脯氨酸（0.67纳摩尔/毫克蛋白质），但仅在囊泡内存在ATP时。由此获得的结果表明，布氏锥虫中的脯氨酸载体系统是由ATP驱动的，且独立于Na+、K+或H+共转运。
