Ca2+-dependent cysteine proteinase, calpains I and II are not phosphorylated in vivo.

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.