Brooks C L, Landt M
Arch Biochem Biophys. 1985 Aug 1;240(2):663-73. doi: 10.1016/0003-9861(85)90074-8.
Acini isolated from lactating rat mammary tissue were used as the starting material to determine the subcellular location and characteristics of a calcium and calmodulin-dependent protein kinase. The kinase activity phosphorylated a 53,600-Da endogenous protein, required Mg2+, and was stimulated only by the simultaneous presence of calcium and calmodulin. Fractionation by differential and sucrose gradient centrifugation demonstrated the enzyme activity in acinar homogenates to be largely particulate; yet the activity did not co-fractionate with markers for nuclei, secretory vesicles, endoplasmic reticulum, mitochondria, lysozymes, Golgi or plasma membranes. The addition of dephosphorylated K-casein to these preparations resulted in a calcium and calmodulin-dependent phosphorylation of the exogenous substrate. A combination of differential centrifugation and equilibrium sucrose density gradient centrifugation purified the kinase 15-fold and revealed a density for the kinase activity between 1.33 and 1.27 g/cm3, suggesting that the kinase was associated with a particle composed largely or entirely of protein. Gel chromatography on Sephacryl S-1000 also purified the activity significantly, and provided a molecular weight of approximately 10(6). In both procedures, the enzymatic activity and principal endogenous protein substrate were enriched indicating that the kinase was associated with the 53,600-Da substrate. Sodium dodecyl sulfate-gel electrophoresis of the fractions enriched in kinase activity by either gel-exclusion chromatography or equilibrium density gradient centrifugation revealed a discrete set of proteins common to both preparations. These included proteins with molecular weights of approximately 32, 35, 54, 70, 94, 100 and 103 K. The calmodulin-dependent protein kinase of mammary acini may be associated in a large complex with these protein species or may represent a polymer of one or several of the proteins. Despite no apparent association with the common phospholipid membranous organelles, the kinase activity was solubilized by treatment with a mixture of phospholipases C and D. After phospholipase treatment and chromatography on Sephacryl S-1000, calcium and calmodulin-dependent phosphorylation was no longer detectable, indicating separation of enzyme and endogenous substrate. Phospholipase treatment of the kinase preparation may be useful in future studies as a method to solubilize the activity.
从泌乳大鼠乳腺组织分离出的腺泡用作起始材料，以确定钙和钙调蛋白依赖性蛋白激酶的亚细胞定位及特性。该激酶活性可磷酸化一种53,600道尔顿的内源性蛋白，需要Mg2+，并且仅在钙和钙调蛋白同时存在时受到刺激。通过差速离心和蔗糖梯度离心分级分离表明，腺泡匀浆中的酶活性主要存在于颗粒部分；然而，该活性并未与细胞核、分泌小泡、内质网、线粒体、溶菌酶、高尔基体或质膜的标志物共分级。向这些制剂中加入去磷酸化的κ-酪蛋白会导致外源底物发生钙和钙调蛋白依赖性磷酸化。差速离心和平衡蔗糖密度梯度离心相结合使激酶纯化了15倍，并显示激酶活性的密度在1.33至1.27 g/cm3之间，表明该激酶与一种主要或完全由蛋白质组成的颗粒相关。在Sephacryl S - 1000上进行凝胶过滤也显著纯化了该活性，并得出分子量约为10(6)。在这两种方法中，酶活性和主要内源性蛋白底物都得到了富集，表明该激酶与53,600道尔顿的底物相关。通过凝胶排阻色谱或平衡密度梯度离心富集激酶活性的组分进行十二烷基硫酸钠 - 凝胶电泳，揭示了两种制剂共有的一组离散蛋白质。这些蛋白质包括分子量约为32、35、54、70、94、100和103 K的蛋白质。乳腺腺泡的钙调蛋白依赖性蛋白激酶可能与这些蛋白质种类形成大的复合物相关，或者可能代表其中一种或几种蛋白质的聚合物。尽管与常见的磷脂膜细胞器没有明显关联，但该激酶活性可通过用磷脂酶C和D的混合物处理而溶解。经过磷脂酶处理并在Sephacryl S - 1000上进行色谱分离后，不再能检测到钙和钙调蛋白依赖性磷酸化，表明酶与内源性底物已分离。激酶制剂的磷脂酶处理作为一种溶解该活性的方法，可能在未来的研究中有用。
