Lawrence J C, Hiken J F, Inkster M, Scott C W, Mumby M C
Proc Natl Acad Sci U S A. 1986 Jun;83(11):3649-53. doi: 10.1073/pnas.83.11.3649.
Incubating 32P-labeled fat cells with insulin increased by as much as 80-fold the amount of 32Pi in a soluble species of apparent Mr 62,000. This species, designated isp62, was specifically immunoprecipitated from cellular extracts with a monoclonal antibody against the type II regulatory subunit (RII) of cAMP-dependent protein kinase. Fat-cell RII, purified from extracts with cAMP-Sepharose or labeled with 8-azido [32P]cAMP, had an apparent Mr 51,000. Peptide mapping indicated that isp62 and adipocyte RII were different proteins. When cells were metabolically labeled with [35S]methionine, insulin stimulated the appearance of 35S-labeled isp62, indicating that the hormonal effect involves generation of the protein. The insulin-induced increase in isp62 could be observed within 1 min, occurred with physiological concentrations of the hormone, and was rapidly reversible. The increase in isp62 was unaffected by cycloheximide, indicating that insulin stimulates the posttranslational processing of a precursor, rather than de novo synthesis of the protein.
用胰岛素孵育32P标记的脂肪细胞，可使表观分子量为62,000的可溶性蛋白中的32Pi含量增加多达80倍。这种蛋白被命名为isp62，用抗环磷酸腺苷（cAMP）依赖性蛋白激酶II型调节亚基（RII）的单克隆抗体从细胞提取物中特异性免疫沉淀得到。从提取物中用cAMP-琼脂糖纯化或用8-叠氮基[32P]cAMP标记的脂肪细胞RII，其表观分子量为51,000。肽图谱分析表明isp62和脂肪细胞RII是不同的蛋白质。当细胞用[35S]甲硫氨酸进行代谢标记时，胰岛素刺激了35S标记的isp62的出现，表明激素的作用涉及该蛋白质的生成。胰岛素诱导的isp62增加在1分钟内即可观察到，在生理浓度的激素作用下发生，且迅速可逆。isp62的增加不受放线菌酮的影响，表明胰岛素刺激前体的翻译后加工，而不是该蛋白质的从头合成。
