Structural comparisons of cAMP-dependent protein kinases I and II from porcine skeletal muscle.
Structural comparisons of cAMP-dependent protein kinases I and II from porcine skeletal muscle.
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猪骨骼肌 cAMP 依赖性蛋白激酶 I 和 II 的结构比较。
DOI：
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发表时间：
1979
影响因子：
4.8
通讯作者：
S. Taylor
中科院分区：
文献类型：
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作者：
M. Zoller;A. Kerlavage;S. Taylor
为了最大限度地减少物种和组织差异，从猪骨骼肌中纯化camp依赖性蛋白激酶II的调控和催化亚基，使其具有同质性，并与来自同一组织的I型酶进行对比。催化亚基的计算分子量为38,000（十二烷基硫酸钠聚丙烯酰胺凝胶为42,000），在大小方面与I型催化亚基无法区分。氨基酸组成虽然不完全相同，但非常相似。色氨酸的二维图证实了多肽链是非常同源的，尽管显然不完全相同。相反，解离的调节亚基有很大的不同。原生II型亚基的计算分子量为109,000，而I型调节亚基的计算分子量为92,500。基于十二烷基硫酸钠凝胶电泳的分子量分别为55,000和47,000。从deae -纤维素中两个亚基的洗脱反映出显著的离子差异，II型代表更酸性的蛋白质。这种酸性特性反映在两个亚基的氨基酸组成上，它们不同，但都异常富含谷氨酸和天冬氨酸残基。在Tris/甘氨酸缓冲液中进行凝胶电泳后，II型调控亚基以双分子形式迁移，这似乎是由于蛋白质的磷酸化和去磷酸化形式。在I型调控亚基中没有观察到类似的双重结构。此外，色氨酸肽的图谱显示初级结构存在实质性差异。与催化亚基形成鲜明对比的是，很少（如果有的话）肽是重叠的，这表明，至少在一级结构方面，这两个分子没有共享一个共同的高度同源的核心。显然，I型调控亚基不能被解释为源自II型亚基的蛋白水解片段。调控亚基也被证明对内源性蛋白水解不稳定，产生一个M = 37,000的单体camp结合蛋白。
In order to minimize species as well as tissue differences, both the regulatory and catalytic subunit of CAMP-dependent protein kinase II from porcine skeletal muscle have been purified to homogeneity and are contrasted to the type I enzyme from the same tissue. The catalytic subunit had a calculated molecular weight of 38,000 (42,000 on sodium dodecyl sulfatepolyacrylamide gels) and was indistinguishable in terms of size from the type I catalytic subunit. Amino acid compositions were very similar although not identical. Two-dimensional maps of the tryptic peptides confirmed that the polypeptide chains were very homologous although apparently not identical. In contrast, the dissociated regulatory subunits differed substantially. The calculated molecular weight of the native type II subunit was 109,000 as opposed to 92,500 for the type I regulatory subunit. Molecular weights based on sodium dodecyl sulfate-gel electrophoresis were 55,000 and 47,000, respectively. Significant ionic differences were reflected in the elution of both subunits from DEAE-cellulose, with the type II representing a more acidic protein. This acidic property was reflected in the amino acid compositions of both subunits which were different but both unusually rich in glutamic and aspartic acid residues. Following gel electrophoresis in Tris/glycine buffer, the type II regulatory subunit migrated as a doublet which appeared to be attributable to the phospho and dephospho forms of the protein. A similar doublet was not observed for the type I regulatory subunit. In addition, maps of the tryptic peptides indicated substantial differences in primary structure. In marked contrast to the catalytic subunits, very few, if any, peptides were superimposable, suggesting that, at least in terms of primary structure, the 2 molecules did not share a common highly homologous core. Clearly, the type I regulatory subunit cannot be explained as a proteolytic fragment derived from the type II subunit. The regulatory subunit was also shown to be labile to endogenous proteolysis which yielded a M, = 37,000, monomeric, CAMP-binding protein.