Ballarin-Denti A, Den Hollander J A, Sanders D, Slayman C W, Slayman C L
Biochim Biophys Acta. 1984 Nov 21;778(1):1-16. doi: 10.1016/0005-2736(84)90442-5.
Interactions between intracellular pH (pHi) and H+-coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and 'energy starved' cells of the yeast Saccharomyces cerevisiae. The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K1/2 values near 14 and 0.4mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pHi in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pHi, imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H+ concentration appears to be third order, showing Hill slopes of 2.7-3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pHi produced by various metabolic inhibitors and uncouplers. Extracellular pH (pHo), by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a pK near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pHi and pHo, and accommodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.
利用31P - NMR技术，对酿酒酵母的需氧细胞和“能量饥饿”细胞进行了研究，以探讨细胞内pH值（pHi）与甘氨酸的H⁺偶联跨膜转运之间的相互作用。所用酵母菌株（NCYC 239）中甘氨酸转运的一般特征与已报道的卡尔斯伯酵母和酿酒酵母相似，存在两个动力学上不同的甘氨酸摄取系统，其pH值不依赖的K1/2值分别接近14和0.4mM，但最大速度依赖于pH值。在需氧细胞中，甘氨酸转运本身对pHi没有可测量的影响，在能量饥饿细胞中只有轻微影响，但通过细胞外添加丁酸引起的pHi变化强烈影响甘氨酸转运。实际上，（在能量饥饿细胞中）甘氨酸内流对细胞质H⁺浓度的依赖性似乎是三级的，希尔斜率为2.7 - 3.0。各种代谢抑制剂和解偶联剂导致的通量下降与pHi下降之间的比例关系进一步表明了细胞质pH在甘氨酸转运中起关键的动力学作用。相比之下，细胞外pH（pHo）对甘氨酸内流只有微弱影响，希尔斜率为0.5。主要观察结果可以用一个简单的循环载体模型来解释，即2个或更多质子与甘氨酸一起转运，但在测试范围内只有一个细胞外质子结合位点解离，pK接近5.5。该模型需要一个有限的膜电位，它必须对pHi和pHo都有点敏感，并通过酵母膜中的其他质子传导途径分流来解释测量的净质子通量（每个甘氨酸一个）与动力学所需的质子通量（每个甘氨酸两个或更多）之间的差异。
