Intermediate (10 nm) filament proteins and the Ca2+-activated proteinase specific for vimentin and desmin in the cells from fish to man: an example of evolutionary conservation

Abstract

Cultured cells from representative species of the five main Classes of Chordates (Mammalia, Aves, Reptilia, Amphibia and Pisces) were examined for the presence of intermediate (1o nm) filament proteins. Using a combination of morphological and immunological methods and detailed protein-chemical characterization it is demonstrated that intermediate filament proteins, as shown also for actin and tubulin, are highly conserved during evolution. This conclusion is based on the following observations: (1) intermediate (10 nm) filaments were found in all the cell lines examined from taxonomically distant species; (2) the intermediate filaments were stable in buffers of low ionic strength in the presence of 4 mM-Mg²⁺ and Triton X-100; (3) under these ionic conditions, detergent-resistant residual cell structures were formed in which the intermediate filament morphology was highly preserved; (4) the intermediate filaments could be completely solubilized in the absence of Mg²⁺; (5) all intermediate filament proteins were quantitatively precipitated by (NH₄)₂SO₄ at 25% saturation; (6) the intermediate filament proteins, with the exception of some of the proteins from Xenopus and viper cells, all bound to single-stranded DNA-cellulose, and could be eluted at various NaCl concentrations depending on the individual proteins; (7) the intermediate filament proteins had similar molecular weights (50 000 to 58 000), isoelectric points (pl 5·6–5·3) and isoelectric variants, probably due to different degrees of phosphorylation; (8) the major intermediate filament proteins were degraded by the vimentin-specific, Ca²⁺-activated proteinase and had similar peptide patterns; (9) all the intermediate filament proteins had an antigenic determinant in common and cross-reacted with the monoclonal antibody α-IFA. In addition, the presence of intermediate filament proteins in taxonomically distant species was indirectly supported by the finding that a Ca²⁺-activated proteinase activity could be detected in the cells from fish to man. This proteinase had identical characteristics in all the cells examined. It had a molecular weight of 100000, a pl of 5·2, Ca²⁺ dependence, neutral pH optimum and degraded Ehrlich ascites tumour cell-vimentin with the production of a specific and characteristic peptide pattern. The proteinase was also classified as a thiol-type enzyme. The proteinases from the taxonomically distant species had, therefore, identical properties to the vimentin-specific, Ca²⁺-activated proteinase purified from Ehrlich ascites tumour cells.