Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity.
Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity.
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DOI：
10.1021/bi00450a006
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发表时间：
1989-11
期刊：
影响因子：
2.9
通讯作者：
M. Orlowski;C. Michaud
中科院分区：
文献类型：
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作者：
M. Orlowski;C. Michaud
从牛垂体中提取的700 kDa多催化蛋白酶复合物在解离和还原条件下经聚丙烯酰胺凝胶电泳分离为11个组分，分子量在21 ~ 32 kDa之间。没有检测到更高的分子质量成分。针对该复合物的兔多克隆抗体可识别五种免疫活性成分。如前所述，该复合物表现出三种不同的蛋白水解活性，即胰凝乳蛋白酶样、胰蛋白酶样和肽酰谷氨酰肽水解活性。这三种活性都能被一种通用的丝氨酸蛋白酶抑制剂3,4-二氯异香豆素迅速失活，然而，这三种成分的失活伪一级速率常数差异很大，其中凝乳胰蛋白酶样活性对抑制最敏感。低浓度的十二烷基硫酸钠和脂肪酸极大地激活了肽基谷氨酰肽的水解活性，似乎构成了蛋白质底物降解的主要成分。除了裂解谷氨酰基残基羧基侧的键外，这种活性还裂解疏水残基羧基侧的键，尽管速率较慢；然而，该成分的二级特异性明显不同于凝乳胰蛋白酶样活性。肝素选择性地激活凝乳胰蛋白酶样活性。在低浓度十二烷基硫酸钠的作用下，该复合物能迅速地裂解天然和去磷酸化的β -酪蛋白。蛋白水解产物的性质以及酸溶性茚三酮反应产物的形成速率对于β -酪蛋白的磷酸化和去磷酸化形式是不同的，这表明磷酸化的程度影响蛋白水解的速率和模式。（摘要删节250字）
The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)