Properties of a Ca²⁺‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells

Abstract

A Ca2 +-activated neutral protease is described which, when tested against various native proteins, appears to be specific for vimentin, the 58000-M, subunit protein of intermediate-sized (7-11 nm) filaments in Ehrlichascites-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 detergentresistant 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 CaZf concentration required for activation is 10 pM. The pH optimum is between pH 7.5 and 8.0, and the KCI 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-ascitestumour cells

Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins.

“…The proteinase appears to be coconserved during evolution with the substrate vimentin (20) and is present in reduced amounts in cells in which little or no vimentin is synthesized (36). Together with the fact that the Ca2+-activated proteinase does not significantly degrade other proteins (18,19), these results indicate that the proteinase plays an important role in the regulation of the function of vimentin and desmin. To investigate this, we have analyzed in detail the effect of the proteinase on vimentin and desmin.…”

“…The proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins (18,19) proceeds in two stages and is in the form of a limited proteolysis. At first, the rate of reaction is very fast with the cleavage, in a stepwise manner, of a peptide from the N-terminal of the vimentin and desmin molecule with the same molecular weight of ca.…”

“…The optimum conditions for the degradation of vimentin and desmin by the Ca21+-activated proteinase have been described previously (18,19). Briefly, the standard reaction mixture contained (in 275 ,ul) 100 p.1 of vimentin or desmin solution…”

“…This is due to the activity of a highly specific, Ca2'-activated, neutral proteinase for which we have recently described the purification and characterization (18,19). The proteinase appears to be coconserved during evolution with the substrate vimentin (20) and is present in reduced amounts in cells in which little or no vimentin is synthesized (36).…”

Molecular Interactions in Intermediate‐Sized Filaments Revealed by Chemical Cross‐Linking

“…All steps and subsequent experiments were carried out at 4 "C unless otherwise stated. 5 mM EDTA was included in all extraction buffers to prevent proteolysis [51]. Radioactively labelled vimentin filaments were obtained from RVF-SMC cultures using the described extraction procedure after labelling with [35S]methionine (specific radioactivity about 1000 Ci/nimol; New England Nuclear, Boston, USA).…”