Shenolikar S, Fischer K, Chang L, Weinman E J
Department of Pharmacology, University of Texas Medical School at Houston 77025.
Second Messengers Phosphoproteins. 1988;12(2-3):95-104.
Photolabelling with 32P-8-azido-cAMP identified a major cAMP-binding protein (54 kDa) in isolated rabbit renal apical membranes, whose labelling was competitively inhibited by cAMP. Membrane associated cAMP-binding polypeptides were extensively purified by affinity chromatography on cAMP-Sepharose. The 54 kDa polypeptide represented 70-80% of the total protein eluted with cAMP. This protein was rapidly phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, with a shift in its apparent mobility on SDS-PAGE to Mr 56/58,000. The phosphopeptide maps of autophosphorylated rat skeletal muscle RII and rabbit kidney 56/58 kDa proteins were essentially identical. Western immuno-blot analysis, using antibodies generated against purified rat RI and RII, indicated preferential cross-reactivity of rabbit kidney 54 kDa protein with anti-RII antibodies. The data demonstrates the specific association of the regulatory subunit of type II cAMP dependent protein kinase with rabbit renal brush border membranes.
用³²P - 8 - 叠氮基 - cAMP进行光标记，在分离的兔肾顶端膜中鉴定出一种主要的cAMP结合蛋白（54 kDa），其标记被cAMP竞争性抑制。通过在cAMP - 琼脂糖上进行亲和层析，对膜相关的cAMP结合多肽进行了广泛纯化。54 kDa的多肽占用cAMP洗脱的总蛋白的70 - 80%。该蛋白被cAMP依赖性蛋白激酶的催化亚基迅速磷酸化，其在SDS - PAGE上的表观迁移率转变为Mr 56/58,000。自磷酸化的大鼠骨骼肌RII和兔肾56/58 kDa蛋白的磷酸肽图谱基本相同。使用针对纯化的大鼠RI和RII产生的抗体进行的Western免疫印迹分析表明，兔肾54 kDa蛋白与抗RII抗体具有优先交叉反应性。数据表明II型cAMP依赖性蛋白激酶的调节亚基与兔肾刷状缘膜存在特异性关联。
