Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation.
Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation.
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牛肾上腺皮质胆固醇酯水解酶的纯化和控制以及通过磷酸化激活该酶的证据。
DOI：
10.1111/j.1432-1033.1977.tb11243.x
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发表时间：
1977
期刊：
影响因子：
--
通讯作者：
George S. Boyd
中科院分区：
文献类型：
--
作者：
Geoffrey J. Beckett;George S. Boyd
报道了一种从牛肾上腺皮质105000×g上清液中纯化胆固醇酯水解酶的方法。粗酶提取物与[伽马-32P]-三磷酸腺苷预孵育，然后进行纯化，得到一种磷酸化的胆固醇酯水解酶制剂。磷酸化胆固醇酯水解酶似乎由4个亚基组成，每个亚基的相对分子质量为41000+/-280，其中只有一个亚基可能被磷酸化。粗酶制剂与[α-32P]ATP预孵育，然后进行纯化，未产生磷酸化的胆固醇酯水解酶制剂。纯化的胆固醇酯水解酶的体外激活需要依赖于环磷酸腺苷的蛋白激酶、环磷酸腺苷、三磷酸腺苷和镁离子，激活的时间进程与酶的磷酸化时间进程密切相关。在牛肾上腺皮质上清液中加入三磷酸腺苷、环磷酸腺苷和镁离子，可使胆固醇酯水解酶活性提高2.5倍。如果在加入三磷酸腺苷、环磷酸腺苷和镁离子之前加入蛋白激酶抑制剂，则这种刺激作用被取消。在胆固醇酯水解酶的粗制制剂中加入镁离子或钙离子可抑制酶的活性，而在精制制剂中添加相同的镁离子或钙离子则不能抑制酶的活性。在与镁离子孵育时，胆固醇酯水解酶活性的降低伴随着蛋白质中~(32)P放射性的丧失。将粗制的胆固醇酯水解酶与碱性磷酸酶预孵育，可使胆固醇酯水解酶失活。这表明，牛肾上腺皮质胆固醇酯水解酶是由环-AMP依赖的蛋白激酶催化的磷酸化激活的。胆固醇酯水解酶的失活是由依赖于镁或钙离子的磷酸蛋白磷酸酶催化的去磷酸化完成的。
A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.