Purification and further characterization of the Ca2+-activated proteinase specific for the intermediate filament proteins vimentin and desmin.
A calcium (Ca2+)-activated, neutral proteinase has been purified from Ehrlich ascites tumor cells. The protocol used has resulted in a 3,600-fold purification of the enzyme in a yield of 21% from the Ehrlich ascites tumor cell postnuclear supernatant. The purified proteinase has a high substrate specificity for the intermediate filament subunit proteins, vimentin and desmin, and showed no activity towards other intermediate filament proteins except a 60,000-dalton protein of the cytokeratins. Also, there was no degradation of actin, tubulin, the major constituent proteins of myofibrils and several standard proteins. Characterization of the purified proteinase has shown that it is activated by Ca2+ (10 to 100 microM), is probably calmodulin-independent and irreversibly loses activity when incubated in the presence of Ca2+ without substrate. The enzyme has a Km of 1.7 x 10(-8) M for vimentin and 5.2 x 10(-7) M for desmin. The proteinase has a major subunit of 72,000 daltons which has the catalytic center and a minor component of 29,000 daltons; by gel permeation chromatography it has an apparent molecular weight of 100,000. It requires a reduced sulfhydryl group for activity and can be inhibited by sulfhydryl-blocking reagents. The high substrate specificity of the proteinase indicates that it is involved in the regulation of the distribution and turnover of vimentin- and desmin-containing intermediate filaments.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D003893:Desmin; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D014746:Vimentin
D003893:Desmin; D009124:Muscle Proteins; D014746:Vimentin; D010447:Peptide Hydrolases; D002118:Calcium