Johanson R, Maddox A M, Washington J, Steiner A L
Division of Endocrinology, University of Texas Medical School, Houston.
J Cyclic Nucleotide Protein Phosphor Res. 1986;11(6):411-20.
Cyclic AMP-dependent protein kinase (cAMP-PrK) regulatory subunits, RI and RII, and cyclic GMP-dependent protein kinase (cGMP-PrK) have been simultaneously purified from skeletal muscle, utilizing sequential affinity chromatography on cyclic AMP-Sepharose. Rat skeletal muscle extract was chromatographed over DEAE-cellulose. Appropriate fractions, enriched in RI, RII or cGMP-PrK were further purified by affinity chromatography on cAMP-Sepharose. The protein kinase units were specifically eluted with cAMP or cGMP. A novel procedure, using two affinity columns, differing in their linkage of cAMP via either N6 or C-8 bonds, was developed to obtain RII free of other cyclic nucleotide binding proteins. In all cases, affinity chromatography was followed by HPLC gel exclusion chromatography to remove residual contaminating proteins. Proteins were purified to essential homogeneity as judged by silver stained SDS polyacrylamide gels. This procedure yields protein kinase subunits of high purity, and may be applicable to the isolation of these proteins from other sources.
环磷酸腺苷依赖性蛋白激酶（cAMP-PrK）调节亚基RI和RII以及环磷酸鸟苷依赖性蛋白激酶（cGMP-PrK）已通过在环磷酸腺苷-琼脂糖上的顺序亲和层析从骨骼肌中同时纯化出来。大鼠骨骼肌提取物在DEAE-纤维素上进行层析。富含RI、RII或cGMP-PrK的适当级分通过在环磷酸腺苷-琼脂糖上的亲和层析进一步纯化。蛋白激酶单位用环磷酸腺苷或环磷酸鸟苷特异性洗脱。开发了一种新方法，使用两个亲和柱，它们通过N6或C-8键连接环磷酸腺苷的方式不同，以获得不含其他环核苷酸结合蛋白的RII。在所有情况下，亲和层析后进行HPLC凝胶排阻层析以去除残留的污染蛋白。通过银染SDS聚丙烯酰胺凝胶判断，蛋白质被纯化至基本同质。该方法可产生高纯度的蛋白激酶亚基，并且可能适用于从其他来源分离这些蛋白质。
