Kozma S C, Lane H A, Ferrari S, Luther H, Siegmann M, Thomas G
Friedrich Miescher-Institut, Basel, Switzerland.
EMBO J. 1989 Dec 20;8(13):4125-32. doi: 10.1002/j.1460-2075.1989.tb08597.x.
A number of approaches were tested for their ability to induce S6 phosphorylation and S6 kinase activation in rat liver, including i.p. injection of insulin, sodium orthovanadate or cycloheximide, as well as refeeding starved animals. All treatments led to increased S6 phosphorylation and activation of the apparent same enzyme. The most potent activator of the S6 kinase in liver extracts was cycloheximide. Maximum activation was achieved in 20 min at 1 mg cycloheximide/100 g body weight, with half-maximal activation in 10 min. Based on these findings a large-scale kinase purification procedure was established involving seven steps of chromatography. Following the final step a major protein band of Mr 70,000 was revealed. The protein was purified 20,000-fold, had a sp. act. of 640 nmol/min/mg of protein towards S6, autophosphorylated and was inactivated by phosphatase 2A. Peptide maps of autophosphorylated material were identical to those derived from the mitogen-activated kinase of 3T3 cells.
人们测试了多种方法在大鼠肝脏中诱导S6磷酸化和S6激酶激活的能力，包括腹腔注射胰岛素、原钒酸钠或环己酰亚胺，以及对饥饿动物重新喂食。所有处理均导致S6磷酸化增加和同一种酶的激活。肝脏提取物中S6激酶最有效的激活剂是环己酰亚胺。在1 mg环己酰亚胺/100 g体重的情况下，20分钟可实现最大激活，10分钟时达到半最大激活。基于这些发现，建立了一个包括七个色谱步骤的大规模激酶纯化程序。在最后一步之后，发现了一条分子量为70,000的主要蛋白带。该蛋白被纯化了20,000倍，对S6的比活性为640 nmol/分钟/毫克蛋白，可自动磷酸化，并被2A磷酸酶灭活。自动磷酸化物质的肽图与来自3T3细胞的丝裂原活化激酶的肽图相同。
