Inhibition of Na+-dependent phosphate transport by group-specific covalent reagents in rat kidney brush border membrane vesicles. Evidence for the involvement of tyrosine and sulfhydryl groups on the interior of the membrane.
Inhibition of Na+-dependent phosphate transport by group-specific covalent reagents in rat kidney brush border membrane vesicles. Evidence for the involvement of tyrosine and sulfhydryl groups on the interior of the membrane.
复制标题
大鼠肾刷状缘膜囊泡中基团特异性共价试剂对 Na 依赖性磷酸盐转运的抑制。
DOI：
10.1016/0003-9861(89)90559-6
复制
发表时间：
1989
影响因子：
3.9
通讯作者：
Pedersen,PL
中科院分区：
文献类型：
--
作者：
Pratt,RD;Pedersen,PL
研究了酪氨酸和巯基专一性试剂对大鼠肾皮质刷状缘膜囊泡Na+依赖的磷酸盐转运的影响。这项研究首次表明，酪氨酸专一性试剂7-氯-4-硝基苯并-2-氧-1，3-二唑和四硝基甲烷以浓度和时间依赖的方式使转运蛋白失活，而膜上不重要的酪氨酸试剂N-乙酰咪唑对磷酸盐的吸收没有影响。膜试剂N-乙基马来酰亚胺对磷酸盐的转运过程也有时间和浓度依赖性的钝化作用，而膜试剂7-氯-4-磺基-苯并-2-氧-1，3-二唑和曙红-5-马来酰亚胺对磷的吸收几乎没有影响。酪氨酸和巯基特异性试剂的抑制作用是相加的，但通过将小泡与转运蛋白的底物或运输过程的竞争性抑制剂预先孵育，不能防止酪氨酸特异性试剂的失活。这些结果表明，经这些试剂修饰的氨基酸位于转运蛋白的膜内或胞浆表面。这些残基可能不参与底物结合，但对于磷酸盐跨膜转运所需的转运蛋白的构象变化可能是重要的。这项研究还表明，依赖于Na+的磷酸盐运输可以被其他共价修饰蛋白质上的组氨酸、羧基和氨基的试剂灭活。
The effects of tyrosine- and sulfhydryl-specific reagents on the Na+-dependent transport of phosphate in brush border membrane vesicles prepared from rat renal cortex were investigated. This study is the first to show that the tyrosine-specific reagents 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane inactivate the transporter in a concentration- and time-dependent fashion while the membrane impermeant tyrosine reagent,N-acetylimidazole, has no effect on phosphate uptake. The membrane permeant sulfhydryl reagentN-ethylmaleimide also caused a time and concentration-dependent inactivation of this transport process but the membrane impermeant reagents 7-chloro-4-sulfobenzo-2-oxa-1,3-diazole and eosin-5-malemide had little effect on phosphate uptake. The inhibitory effects of both tyrosine- and sulfhydryl-specific reagents were additive, but no protection from inactivation by tyrosine-specific reagents could be achieved by preincubation of the vesicles with the substrates of the transporter or with competitive inhibitors of the transport process. These results suggest that the amino acids modified by these agents are located either within the membrane or on the cytosolic surface of the transporter. These residues may not participate in substrate binding, but may be important for the conformational change of the transporter necessary for the translocation of phosphate across these membranes. This study also shows that Na+-dependent phosphate transport can be inactivated by other reagents which covalently modify histidine, carboxyl, and amino groups on proteins.