Maddox A M, Steiner A L, Shenolikar S
Division of Hematology-Oncology, University of Texas Health Science Center, Houston, Texas.
Second Messengers Phosphoproteins. 1988;12(2-3):83-94.
Polyclonal antibodies were generated against regulatory subunits (RI and RII) of type-I and type-II cAMP-dependent protein kinases from rat skeletal muscle. Western immunoblot analyses showed specific cross-reactivity of rat and bovine RI with anti-RI. Similarly, RII from both species was specifically recognized by anti-RII. Quantitative immunoassays, using antisera against proteins from either species, indicated selectivity towards regulatory subunits from the same species. Molecular basis for this selectivity was examined by comparison of peptide maps of 32P-8-azido-cAMP-labelled or autophosphorylated peptides. Detailed analysis of two-dimensional peptide fingerprints demonstrated extensive homology between either RI or RII from the two species. The data suggests that the overall protein-chemical and functional determinants characterizing type-I and type-II regulatory subunits of cyclic AMP dependent protein kinase from different species are substantially similar. However, minor differences in structure, also predicted by amino-acid sequences for RI and RII obtained by molecular cloning, may account for the distinct immunological properties of the proteins from rat and bovine tissues.
制备了针对大鼠骨骼肌I型和II型环磷酸腺苷（cAMP）依赖性蛋白激酶调节亚基（RI和RII）的多克隆抗体。蛋白质免疫印迹分析显示，大鼠和牛的RI与抗RI具有特异性交叉反应。同样，两种物种的RII均被抗RII特异性识别。使用针对任一物种蛋白质的抗血清进行的定量免疫测定表明，对来自同一物种的调节亚基具有选择性。通过比较32P-8-叠氮基-cAMP标记的肽或自磷酸化肽的肽图，研究了这种选择性的分子基础。二维肽指纹图谱的详细分析表明，两种物种的RI或RII之间具有广泛的同源性。数据表明，来自不同物种的环磷酸腺苷依赖性蛋白激酶I型和II型调节亚基的整体蛋白质化学和功能决定因素基本相似。然而，通过分子克隆获得的RI和RII氨基酸序列也预测到的结构上的微小差异，可能解释了大鼠和牛组织中蛋白质不同的免疫特性。
