de Koning H P, Jarvis S M
Research School of Biosciences, University of Kent, Canterbury, United Kingdom.
Eur J Biochem. 1997 Aug 1;247(3):1102-10. doi: 10.1111/j.1432-1033.1997.01102.x.
The mechanism of purine nucleobase transport in procyclic cells of the protozoan parasite Trypanosoma brucei brucei was investigated. Hypoxanthine uptake at 22 degrees C was rapid and saturable, exhibiting an apparent Km of 9.3 +/- 2.0 microM and a Vmax of 4.5 +/- 0.8 pmol x (10(7) cells)(-1) x s(-1). All the natural purine nucleobases tested (Ki 1.8-7.2 microM), as well as the purine analogues oxypurinol and allopurinol, inhibited hypoxanthine influx in a manner consistent with the presence of a single high-affinity carrier. Nucleosides and pyrimidine nucleobases had little or no effect on hypoxanthine influx. The uptake process was independent of extracellular sodium, but inhibited by ionophores inducing cytosolic acidification (carbonyl cyanide chlorophenylhydrazone, nigericin, valinomycin) or membrane depolarisation (gramicidin) as well as by the adenosine triphosphatase inhibitors N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. Using the fluorescent dyes bisoxonol and 2',7'-bis-(carboxyethyl)-5,6-carboxy-fluorescein to determine membrane potential and intracellular pH (pHi), the rate of hypoxanthine uptake was shown to be directly proportional to the protonmotive force. Similarly, under alkaline extracellular conditions hypoxanthine uptake was reversibly inhibited alongside a reduction in protonmotive force. In addition, hypoxanthine accelerated the rate of pH, recovery to pH 7 after base-loading with NH4Cl, indicative of a proton influx concurrent with hypoxanthine transport. Finally, after pretreatment of cells with N-ethylmaleimide, hypoxanthine induced a slow membrane depolarisation, demonstrating that hypoxanthine transport is electrogenic. These data show that hypoxanthine uptake in T. b. brucei procyclic cells is dependent on the protonmotive force, and are consistent with a nucleobase/H+-symporter model for this transporter.
对原生动物寄生虫布氏布氏锥虫前循环细胞中嘌呤核碱基转运机制进行了研究。22℃下次黄嘌呤摄取迅速且具有饱和性，表观Km为9.3±2.0μM，Vmax为4.5±0.8 pmol×(10⁷个细胞)⁻¹×s⁻¹。所有测试的天然嘌呤核碱基（Ki 1.8 - 7.2μM）以及嘌呤类似物氧嘌呤醇和别嘌呤醇，均以与单一高亲和力载体存在相一致的方式抑制次黄嘌呤内流。核苷和嘧啶核碱基对次黄嘌呤内流几乎没有影响。摄取过程不依赖细胞外钠，但受到诱导胞质酸化的离子载体（羰基氰化物间氯苯腙、尼日利亚菌素、缬氨霉素）或膜去极化剂（短杆菌肽）以及腺苷三磷酸酶抑制剂N - 乙基马来酰亚胺和N,N'-二环己基碳二亚胺的抑制。使用荧光染料双羟萘酚和2',7'-双（羧乙基）-5,6-羧基荧光素来测定膜电位和细胞内pH（pHi），结果表明次黄嘌呤摄取速率与质子动力直接相关。同样，在碱性细胞外条件下，次黄嘌呤摄取随着质子动力的降低而被可逆抑制。此外，次黄嘌呤加速了用NH₄Cl进行碱加载后pH恢复到7的速率，表明质子内流与次黄嘌呤转运同时发生。最后，用N - 乙基马来酰亚胺预处理细胞后，次黄嘌呤诱导缓慢的膜去极化，表明次黄嘌呤转运是生电的。这些数据表明布氏布氏锥虫前循环细胞中次黄嘌呤摄取依赖于质子动力，并且与该转运体的核碱基/H⁺同向转运体模型一致。
