Maizels E T, Jungmann R A
Endocrinology. 1983 Jun;112(6):1895-902. doi: 10.1210/endo-112-6-1895.
Studies were undertaken to determine if calmodulin-regulated Ca2+-dependent protein kinase system(s) exist in the prepubertal rat ovary. Phosphorylation studies were performed with ovarian cytosol, calmodulin-depleted cytosol, and nuclear extracts. In vitro phosphorylation of endogenous substrate proteins was accomplished by incubation of tissue fractions with [gamma-32P]ATP followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Calmodulin-dependent phosphorylations of a cytosol protein, mol wt, 95,000, and of three nuclear proteins in the mol wt range of 50,000-60,000, were established by demonstrating: Ca2+ requirement; inhibition by the phenothiazine derivative chlorpromazine; and dependence upon addition of exogenous calmodulin to the calmodulin-depleted cytosol, or to the nuclear extract. These findings demonstrate the presence of rat ovarian cytosol and nuclear Ca2+-calmodulin-dependent protein kinase activities capable of recognizing endogenous substrate proteins.
开展了多项研究以确定青春期前大鼠卵巢中是否存在钙调蛋白调节的钙依赖性蛋白激酶系统。使用卵巢胞质溶胶、去除钙调蛋白的胞质溶胶和核提取物进行磷酸化研究。通过将组织部分与[γ-32P]ATP孵育，然后进行电泳分离和磷酸化蛋白的放射自显影显示，实现了内源性底物蛋白的体外磷酸化。通过证明以下几点，确定了一种分子量为95,000的胞质溶胶蛋白以及分子量在50,000 - 60,000范围内的三种核蛋白的钙调蛋白依赖性磷酸化：对钙的需求；吩噻嗪衍生物氯丙嗪的抑制作用；以及依赖于向去除钙调蛋白的胞质溶胶或核提取物中添加外源性钙调蛋白。这些发现证明了大鼠卵巢胞质溶胶和核中存在能够识别内源性底物蛋白的钙 - 钙调蛋白依赖性蛋白激酶活性。
