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

通过蔗糖梯度离心部分纯化的大鼠肾髓质前列腺素（PG）E_1受体在鸟嘌呤核苷酸存在下增加了其与激动剂的结合活性。PGE_1结合的刺激不是由于B_<max>的增加，而是由于Kd值的降低。百日咳毒素能抑制PGE_1与鸟嘌呤核苷酸结合的刺激作用，而霍乱毒素不能。PGE_1受体用6%洋地黄苷溶解，通过连续三个柱层析（Sepharose CL-6B、小麦胚芽凝集素(WGA)-琼脂糖和DEAE-Toyopearl）纯化，并检测组分的S-GTPrS结合^<35>。^ 3h - pge_1结合受体的放射性和^<35>S-GTPrS结合活性在各柱层析上共洗脱。纯化的PGE_1受体含有被百日咳毒素进行adp核糖基化的底物。对鸟嘌呤核苷酸结合蛋白相关的PGE_1受体进行天然梯度聚丙烯酰胺凝胶电泳。^3H-PGE_1结合和^<35>S-GTPrS结合的活性迁移分子量约为400 KD。用sds -聚丙烯酰胺凝胶电泳对凝胶洗脱的蛋白进行分析，检测到分子量分别为140、80和50 kd的三种肽段。这些结果表明，大鼠肾髓质PGE_1受体以一种与百日咳毒素敏感鸟嘌呤核苷酸结合蛋白相关的形式存在，与百日咳毒素敏感底物相关的PGE_1受体参与了鸟嘌呤核苷酸诱导的PGE_1结合刺激。

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.