Hammerman M R
Am J Physiol. 1986 Apr;250(4 Pt 2):F659-66. doi: 10.1152/ajprenal.1986.250.4.F659.
We have previously demonstrated cAMP-dependent 32P phosphorylation and dephosphorylation of a 62,000 relative molecular weight (Mr) protein in autoradiograms of sodium dodecyl sulfate polyacrylamide gels originating from canine renal brush border membranes. In the current studies 32P phosphorylation of the 62,000 Mr protein that was independent of cAMP was noted in the presence of Zn2+. Under these conditions, cAMP inhibited the 32P phosphorylation of this protein. Concentration-dependent photoaffinity labeling of a band with Mr 60,000 in autoradiograms of gels resulted from incubation of membranes with cyclic 8-azidoadenosine-3',5'-monophosphate (8-N3-[32P]cAMP) followed by exposure to light. In the presence of Zn2+ and ATP, an apparent shift in the Mr of a portion of the photoaffinity-labeled band to 62,000 was seen. The 62,000 Mr phosphoprotein in detergent-solubilized supernatants of brush border membranes was immunoprecipitated with antibodies directed against the regulatory subunit of type II cAMP-dependent protein kinase. Our observations strongly suggest that the 62,000 Mr protein is the regulatory subunit.
我们之前已经在源自犬肾刷状缘膜的十二烷基硫酸钠聚丙烯酰胺凝胶的放射自显影片中证明了一种相对分子量（Mr）为62,000的蛋白质的cAMP依赖性32P磷酸化和去磷酸化。在当前的研究中，在存在Zn2+的情况下，观察到了与cAMP无关的62,000 Mr蛋白质的32P磷酸化。在这些条件下，cAMP抑制了该蛋白质的32P磷酸化。用环状8-叠氮腺苷-3',5'-单磷酸（8-N3-[32P]cAMP）孵育膜，然后曝光，导致凝胶放射自显影片中出现一条Mr为60,000的条带的浓度依赖性光亲和标记。在存在Zn2+和ATP的情况下，观察到一部分光亲和标记条带的Mr明显向62,000移动。用针对II型cAMP依赖性蛋白激酶调节亚基的抗体对刷状缘膜去污剂溶解上清液中的62,000 Mr磷蛋白进行免疫沉淀。我们的观察结果强烈表明，62,000 Mr蛋白质是调节亚基。
