Shanker G, Ahrens H, Sharma R K
Proc Natl Acad Sci U S A. 1979 Jan;76(1):66-70. doi: 10.1073/pnas.76.1.66.
We describe the purification to apparent homogeneity of a protein kinase (designated AUT-PK 85) from adrenocortical carcinoma 494, as evidenced by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme binds cyclic AMP (cAMP) and autophosphorylates but does not use histone, casein, or polysomes as substrates in the presence or absence of cAMP. Stoichiometry of phosphate incorporation was 0.71 mol/mol of enzyme. The enzyme was found to have a molecular weight of 85,000 based on gel filtration. The protein was composed of polypeptides having the same molecular weight 42,000, and thus it appears to consist of two subunits of equal size. The enzyme bound two cAMP molecules, indicating that each subunit binds one molecule of cAMP. The homogeneous enzyme did not inhibit the protein kinase activity of the free catalytic subunit of normal adrenal cAMP-dependent protein kinase under conditions such that recombination with the free regulatory subunit occurred. cAMP bound specifically to the enzyme with an apparent dissociation constant (cfKd) of 1.2 X 10(-8) M. Scatchard plot data indicated one type of binding sites for cAMP. The enzyme did not bind adenosine. This novel autophosphorylating, cAMP-binding, protein kinase may be a characteristic of certain adrenal neoplasms.
我们描述了从肾上腺皮质癌494中纯化出一种蛋白激酶（命名为AUT-PK 85）并使其达到表观均一性，这通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳得以证明。该酶能结合环磷酸腺苷（cAMP）并进行自身磷酸化，但在有无cAMP的情况下，均不以组蛋白、酪蛋白或多聚核糖体作为底物。磷酸掺入的化学计量比为每摩尔酶0.71摩尔。基于凝胶过滤法，该酶的分子量为85,000。该蛋白由分子量相同的42,000的多肽组成，因此它似乎由两个大小相等的亚基组成。该酶结合两个cAMP分子，表明每个亚基结合一个cAMP分子。在与游离调节亚基发生重组的条件下，均一化的该酶并不抑制正常肾上腺cAMP依赖性蛋白激酶游离催化亚基的蛋白激酶活性。cAMP以1.2×10⁻⁸ M的表观解离常数（cfKd）特异性结合该酶。Scatchard图数据表明存在一种cAMP结合位点。该酶不结合腺苷。这种新型的自身磷酸化、cAMP结合蛋白激酶可能是某些肾上腺肿瘤的一个特征。
