McCormick J, Johnstone R M
Department of Biochemistry, McGill University, Montreal, Que., Canada.
Biochem Cell Biol. 1990 Feb;68(2):512-9. doi: 10.1139/o90-073.
Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)
用胆酸溶解的艾氏腹水癌细胞质膜悬浮液，用于研究巯基试剂对Na⁺依赖性氨基酸转运的影响。在重构以测定转运活性之前，这些悬浮液用巯基结合剂对氯汞苯磺酸或N - 乙基马来酰亚胺处理。用对氯汞苯磺酸处理溶解膜形成的蛋白脂质体，未显示出Na⁺依赖性α - 氨基异丁酸转运，而经N - 乙基马来酰亚胺预处理的膜保留了约90%的原始活性。为避免N - 乙基马来酰亚胺成分的干扰，在用对氯汞苯磺酸处理之前，对用200μM N - 乙基马来酰亚胺预处理的膜进行了进一步研究。25μM对氯汞苯磺酸的浓度可抑制Na⁺依赖性α - 氨基异丁酸转运50%。在存在A转运系统特异性底物的情况下，抑制程度显著降低。使用抑制指数来评估在有和没有底物时抑制的效果，可以表明，在存在对氯汞苯磺酸时1.0的指数，单独使用（甲基氨基）异丁酸时降至0.84，在存在100 mM Na⁺和5 mM（甲基氨基）异丁酸时降至0.05。单独的Na⁺没有提供保护作用。结果表明，氨基酸载体上的巯基可能直接参与底物结合，并且底物结合位点在解聚的膜状态下是有功能的。此外，Na⁺直接影响（甲基氨基）异丁酸盐的结合能力，因为氨基酸类似物对氯汞苯磺酸抑制的保护程度受Na⁺存在的影响。（摘要截短于250字）
