Regulation of salt and fluid transport by a novel G-protein in kidney medulla

大鼠肾髓质前列腺素（PG）E_1受体经蔗糖梯度离心法部分纯化后，在鸟嘌呤核苷酸存在下，其与激动剂的结合活性增强。PGE_1结合的促进作用不是由于B_2的增加，而是<max>由于Kd值的降低。PGE_1受体用6%毛地黄皂苷溶解后，经SepharoseCL-6 B、小麦胚芽凝集素（WGA）-琼脂糖和DEAE-Toyoprotein三步柱层析纯化，并检测各组分<35>与GTPrS的结合。在每个柱层析上共洗脱^3 H-PGE_1结合受体的放射活性和^<35>S-GTPrS结合活性。纯化的PGE_1受体含有百日咳毒素ADP核糖基化的底物。PGE_1受体与鸟嘌呤核苷酸结合蛋白进行非变性梯度聚丙烯酰胺凝胶电泳。^3 H-PGE_1结合活性和^<35>S-GTPrS结合活性的迁移分子量约为400 KD。通过SDS-聚丙烯酰胺凝胶电泳分析从凝胶洗脱的蛋白质，以检测分子量为140、80和50 KD的三种肽。这些结果表明，大鼠肾髓质PGE_1受体与百日咳毒素敏感的鸟嘌呤核苷酸结合蛋白结合，并与百日咳毒素敏感的底物结合。

Prostaglandin (PG)E_1 receptor from rat kidney medulla, partially purified through a sucrose gradient centrifugation, was shown to increase its binding activity to agonist in the presence of guanine nucleotides. The stimulation of PGE_1 binding was not due to the increase in B_<max> but due to the decrease in Kd value. Pertussis toxin but not cholera toxin abolished the stimulatory effect of PGE_1 binding by guanine nucleotides.The PGE_1 receptor, solubilized with 6% digitonin, was purified through three successive steps of column chromatographies (Sepharose CL-6B, Wheat germ agglutinin (WGA)-Agarose and DEAE-Toyopearl), and fractions were examined for ^<35>S-GTPrS binding. The radioactivities of ^3H-PGE_1-bound receptor and ^<35>S-GTPrS binding activity were co-eluted on each column chromatography. The purified PGE_1 receptor contained a substrate for ADP-ribosylation by pertussis toxin. The PGE_1 receptor associated with a guanine nucleotide binding protein was subjected to a native gradient polyacrylamide gel electrophoresis. The activities of ^3H-PGE_1, binding and of ^<35>S-GTPrS binding migrated with a molecular weight of about 400 KD. The protein eluted from the gel was analyzed by a SDS-polyacrylamide gel electrophoresis to detect three peptides with molecular weights of 140, 80 and 50 KDs. These results suggest that PGE_1 receptor of rat kidney medulla exists as a associated form with a pertussis toxin-sensitive guanine nucleotide binding protein and that PGE_1 receptor associated with the pertussis toxin sensitive substrate is responsible for a guanine nucleotide-induced stimulation of PGE_1 binding.