Chaudhry P S, Nanez R, Casillas E R
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Arch Biochem Biophys. 1991 Aug 1;288(2):337-42. doi: 10.1016/0003-9861(91)90204-v.
Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.
通过用磷酸纤维素反复层析和用Sephacryl S - 200进行凝胶过滤，将来自牛附睾精子的酪蛋白激酶II纯化至表观均一性。通过凝胶过滤，纯化后的酶显示分子量为130 kDa，通过SDS - 聚丙烯酰胺凝胶电泳显示出分子量分别为26、33和36 kDa的三条多肽带。针对小牛胸腺酪蛋白激酶II产生的抗体与这三种精子多肽发生交叉反应。全酶与[γ - 32P]ATP或[γ - 32P]GTP孵育导致26 kDa亚基的磷酸化。以酪蛋白为底物时，酶活性受到纳摩尔浓度肝素的强烈抑制，并受到微摩尔浓度精胺的极大刺激。以酪蛋白为底物时，纯酶的比活性（0.5 μmol/分钟/毫克蛋白）与来自其他来源的酪蛋白激酶II相当。通过在限制其他蛋白激酶表达的条件下，将精子胞质提取物与[γ - 32P]GTP孵育，并通过SDS - PAGE和放射自显影分析产物，证明了该激酶的内源性底物。当将适当稀释以最小化内源性酪蛋白激酶II的精子提取物与[γ - 32P]GTP和纯精子酪蛋白激酶II的等分试样孵育时，获得了类似的结果。低浓度（50 μM）的精胺强烈增强了92 kDa和106 kDa胞质蛋白的磷酸化。我们的结果清楚地表明，酪蛋白激酶II存在于精子中，并且它具有许多来自其他来源的该酶的特性。此外，它们表明该酶在介导精子蛋白的磷酸化状态中起作用。
