Hartzell H C, Glass D B
J Biol Chem. 1984 Dec 25;259(24):15587-96.
C-protein, a component of the thick filament of striated muscles, becomes phosphorylated in response to beta-adrenergic receptor stimulation and dephosphorylated in response to cholinergic receptor stimulation in heart. We have purified C-protein in high yield from cardiac muscle (approximately 50% yield: 0.3 mg of C-protein/g of frozen chicken heart). C-protein has a molecular weight on sodium dodecyl sulfate polyacrylamide gels of 155,000 but the native protein migrates as a globular protein of 209,000 daltons in gel filtration on Sephacryl S-300, suggesting that it is an asymmetric molecule composed of a single 155,000-dalton polypeptide. C-protein from chicken cardiac muscle has an amino acid composition similar to that of C-proteins from other muscles. The purified protein contains approximately 0.2 mol of phosphate/mol of C-protein. The purified C-protein has no endogenous protein phosphatase activity but does exhibit protein kinase activity in the presence of calcium and calmodulin (approximately 160 pmol of phosphate incorporated/min/mg of C-protein). This endogenous kinase catalyzes the incorporation of approximately 1 mol of phosphate/mol of C-protein. C-protein is an excellent substrate for catalytic subunit of cAMP-dependent protein kinase (Km = 4 microM, Vmax = 18.6 mumol/min/mg). Phosphorylation by catalytic subunit of cAMP-dependent protein kinase exhibits a broad pH optimum between pH 8 and 9 and results in the incorporation of up to 3 mol of phosphate/mol of C-protein. Phosphate is incorporated into 3-5 different sites at both phosphothreonine and phosphoserine residues. The phosphorylated C-protein does not differ from unphosphorylated C-protein with regard to Stokes radius, migration on sodium dodecyl sulfate-polyacrylamide gels, or UV spectrum.
C蛋白是横纹肌粗肌丝的一个组成部分，在心脏中，它会因β-肾上腺素能受体刺激而发生磷酸化，因胆碱能受体刺激而发生去磷酸化。我们已从心肌中高产率地纯化出C蛋白（产率约为50%：每克冷冻鸡心可获得0.3毫克C蛋白）。在十二烷基硫酸钠聚丙烯酰胺凝胶上，C蛋白的分子量为155,000，但在Sephacryl S - 300凝胶过滤中，天然蛋白迁移时表现为209,000道尔顿的球状蛋白，这表明它是由一条155,000道尔顿的单多肽链组成的不对称分子。鸡心肌中的C蛋白氨基酸组成与其他肌肉中的C蛋白相似。纯化后的蛋白每摩尔C蛋白约含0.2摩尔磷酸盐。纯化后的C蛋白没有内源性蛋白磷酸酶活性，但在有钙和钙调蛋白存在时确实表现出蛋白激酶活性（每毫克C蛋白每分钟约掺入160皮摩尔磷酸盐）。这种内源性激酶催化每摩尔C蛋白掺入约1摩尔磷酸盐。C蛋白是环磷酸腺苷依赖性蛋白激酶催化亚基的优良底物（米氏常数Km = 4微摩尔，最大反应速度Vmax = 18.6微摩尔/分钟/毫克）。环磷酸腺苷依赖性蛋白激酶催化亚基的磷酸化作用在pH值8至9之间表现出较宽的最佳pH值范围，导致每摩尔C蛋白最多掺入3摩尔磷酸盐。磷酸盐掺入到磷酸苏氨酸和磷酸丝氨酸残基的3至5个不同位点。磷酸化的C蛋白在斯托克斯半径、在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上的迁移或紫外光谱方面与未磷酸化的C蛋白没有差异。
