Moffett J, Jones M, Englesberg E
Biochemistry. 1987 May 5;26(9):2487-94. doi: 10.1021/bi00383a013.
Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.9, derived from alar4, may be the result of amplification of a gene coding for an A-system transporter. Under conditions in which the same membrane potential (interior negative) and Na+ gradient were employed, the mutant vesicles show increases in the A system over that of the parental CHO-K1 cell line, paralleling, but not equivalent to, that found in whole cells. L-system and 5'-nucleotidase activities of these vesicles were similar, indicating that the increased A-system activity of the mutant vesicles is not due to the differential enrichment of the A system in these vesicles. The membrane potential was produced by a K+ diffusion gradient (internal greater than external) in the presence of valinomycin or by the addition of a Na+ salt of a highly permeant anion such as SCN-. Monensin was employed to study the effect of the Na+ gradient on transport and membrane potential. The latter was determined by measuring the uptake of tetraphenylphosphonium ion. A negative membrane potential determines the concentrative ability and the initial velocity of the A system in these vesicles. The concentration of external Na+ has a stimulatory effect on the initial velocity of this system. However, the Na+ gradient (external greater than internal) has no effect on the initial velocity or the membrane potential when the potential is set by valinomycin and high internal K+. Little if any ASC system could be detected in vesicles from CHO-K1.
从CHO - K1细胞以及丙氨酸抗性转运突变体alar4和alar4 - H3.9制备了膜泡。Alar4是A系统的组成型突变体，而源自alar4的alar4 - H3.9可能是编码A系统转运蛋白的基因扩增的结果。在采用相同膜电位（胞内为负）和Na⁺梯度的条件下，突变体膜泡的A系统活性相较于亲本CHO - K1细胞系有所增加，这与在完整细胞中发现的情况平行，但并不等同。这些膜泡的L系统和5'-核苷酸酶活性相似，表明突变体膜泡中A系统活性的增加并非由于该系统在这些膜泡中的差异富集。膜电位由缬氨霉素存在下的K⁺扩散梯度（胞内大于胞外）产生，或者通过添加高渗透性阴离子（如SCN⁻）的钠盐产生。莫能菌素用于研究Na⁺梯度对转运和膜电位的影响。后者通过测量四苯基鏻离子的摄取来确定。负膜电位决定了这些膜泡中A系统的浓缩能力和初始速度。外部Na⁺浓度对该系统的初始速度有刺激作用。然而，当通过缬氨霉素和高浓度胞内K⁺设定电位时，Na⁺梯度（胞外大于胞内）对初始速度或膜电位没有影响。在CHO - K1细胞的膜泡中几乎检测不到ASC系统。
