Properties of Ca2+-activated protease specific for the intermediate-sized filament protein vimentin in Ehrlich-ascites-tumour cells.

A Ca2+-activated neutral protease is described which, when tested against various native proteins, appears to be specific for vimentin, the 58,000-Mr subunit protein of intermediate-sized (7--11 nm) filaments in Ehrlich-ascites-tumour cells. The protein subunits of other classes of intermediate-sized filaments have been tested; neurofilament protein and glial fibrillary acidic protein are not degraded, however skeletin, the subunit protein of intermediate-sized filaments in smooth muscle, is degraded. The protease is found associated with the detergent-resistant cytoskeleton of Ehrlich-ascites-tumour cells; proteins, other than vimentin, present in this structure are not degraded. The protease is activated by Ca2+ and Sr2+ but not by other divalent cations tested: the Ca2+ concentration required for activation is 10 microM. The pH optimum is between pH 7.5 and 8.0, and the KCl concentration required for optimal activity is 100 mM. The protease is inhibited by 1-chloro-3-tosylamido-7-amino-L-2-heptanone hydrochloride and L-1-tosylamido-2-phenylethyl chloromethyl ketone but not by soybean trypsin inhibitor; inhibition by phenylmethylsulphonyl fluoride is moderate. The high substrate specificity of the protease suggests it may play a role in vimentin intermediate-sized filament protein turnover in Ehrlich-ascites-tumour cells.