Wooge C H, Conn P M
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242.
Mol Cell Endocrinol. 1988 Mar;56(1-2):41-51. doi: 10.1016/0303-7207(88)90007-x.
We have used an 125I-calmodulin gel overlayer assay to identify calmodulin-binding component in the rat pituitary. Tissue-specific and Ca2+ -dependent patterns of 125I-calmodulin binding were observed, with five major Ca2+-dependent 125I-calmodulin-labeled components of subunit Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. Ca2+-dependent binding was defined as that which was abolished in the presence of 1 mM EGTA. Calmodulin binding was inhibited by calmodulin antagonists such as penfluridol (1 microM) or pimozide (1 microM). Some Ca2+-independent binding was observed and appears to be due to (nonspecific) hydrophobic interaction of calmodulin with acid-soluble proteins, principally histones. Subcellular fractionation revealed that the Ca2+-dependent calmodulin-binding components are localized primarily in the cytosolic fraction. Separation of dispersed anterior pituitary cells by a linear metrizamide gradient yielded gonadotrope-enriched fractions; these contained all five 125I-calmodulin-binding components corresponding to the major bands in the pituitary homogenate. Studies with ovariectomized and steroid-replaced animals indicated that the tissue content of calmodulin-binding components, like those of calmodulin itself, did not appear to be differentially regulated by steroids. A comparison of rat and bovine pituitary tissue homogenates revealed that binding components migrating at the same apparent Mrs were found for four of the components (the largest component is lacking in the bovine system).
我们使用了一种¹²⁵I-钙调蛋白凝胶覆盖分析法来鉴定大鼠垂体中的钙调蛋白结合成分。观察到了¹²⁵I-钙调蛋白结合的组织特异性和Ca²⁺依赖性模式，有五个主要的Ca²⁺依赖性¹²⁵I-钙调蛋白标记成分，其亚基Mr分别大于205,000、200,000、135,000、60,000和52,000。Ca²⁺依赖性结合被定义为在1 mM乙二醇双四乙酸（EGTA）存在下被消除的结合。钙调蛋白结合受到钙调蛋白拮抗剂如五氟利多（1 μM）或匹莫齐特（1 μM）的抑制。观察到一些Ca²⁺非依赖性结合，这似乎是由于钙调蛋白与酸溶性蛋白（主要是组蛋白）的（非特异性）疏水相互作用所致。亚细胞分级分离显示，Ca²⁺依赖性钙调蛋白结合成分主要定位于胞质部分。通过线性甲泛影酰胺梯度分离分散的垂体前叶细胞产生了富含促性腺激素细胞的部分；这些部分包含了与垂体匀浆中的主要条带相对应的所有五个¹²⁵I-钙调蛋白结合成分。对去卵巢并进行类固醇替代的动物的研究表明，钙调蛋白结合成分的组织含量，与钙调蛋白自身的含量一样，似乎不受类固醇的差异调节。大鼠和牛垂体组织匀浆的比较显示，在四个成分中发现了迁移至相同表观Mr的结合成分（牛系统中缺少最大的成分）。
