Adachi Y, Kobayashi N, Murachi T, Hatanaka M
Biochem Biophys Res Commun. 1986 May 14;136(3):1090-6. doi: 10.1016/0006-291x(86)90445-6.
To clarify phosphorylation of calpains I and II in vivo, we purified both calpains concurrently from the [32P] metabolic-labeled human chronic myelogenous leukemia cell line K-562. By Ultragel AcA34 column chromatography, enzymatic activity of calpain I was separated from [32P] radioactivity. Whereas calpain II activity was closely associated with [32P] radioactivity on Ultragel AcA34 and Blue Sepharose CL-6B column chromatographies. By the above purification procedures, calpain I was purified 1300-fold from the crude extract and calpain II was 920-fold from the original sample, respectively. Autoradiographies of purified calpains I and II from [32P] labeled K-562 cells revealed that both calpains were not specifically phosphorylated in vivo. The autophosphorylation in vitro on calpains and modulation of their proteolytic activities reported recently thus may not occur within cells.
为了阐明体内钙蛋白酶I和II的磷酸化情况，我们从经[32P]代谢标记的人慢性髓性白血病细胞系K-562中同时纯化了这两种钙蛋白酶。通过Ultragel AcA34柱层析，钙蛋白酶I的酶活性与[32P]放射性分离。而在Ultragel AcA34和Blue Sepharose CL-6B柱层析中，钙蛋白酶II的活性与[32P]放射性紧密相关。通过上述纯化程序，钙蛋白酶I从粗提物中纯化了1300倍，钙蛋白酶II从原始样品中纯化了920倍。对来自[32P]标记的K-562细胞的纯化钙蛋白酶I和II进行放射自显影显示，两种钙蛋白酶在体内均未发生特异性磷酸化。因此，最近报道的钙蛋白酶体外自磷酸化及其蛋白水解活性的调节可能不会在细胞内发生。
