Large scale isolation, purification, and partial characterization of the intermediate filament-specific, Ca2+-activated proteinase from porcine kidney and Ehrlich ascites tumor cells: a comparative study.
The Ca2+-activated, neutral thiol proteinase specific for intermediate filament subunit proteins was isolated at large scale from the postribosomal supernatant of a low-ionic-strength extract of porcine kidney and Ehrlich ascites tumor (EAT) cells, respectively. The purification procedure encompassed DEAE-Sephacel ion exchange chromatography of the material precipitating between 23 and 55% (NH4)2SO4 saturation, followed by hydroxylapatite chromatography and activated thiol Sepharose 4B covalent chromatography. On the average, 25 mg of 62% pure enzyme was obtained from 500 g frozen kidney and 55 mg of 51% pure enzyme from 500 g EAT cells within a week. Both enzyme preparations were free of Ca2+-independent proteolytic activities and indistinguishable with respect to their physicochemical and functional properties; their catalytic properties were indistinguishable from those of enzyme purified to homogeneity on arginine methylester Sepharose 4B. Because of this identity, porcine kidney proves to be an inexpensive source for the Ca2+-activated proteinase which had previously been isolated and purified at small scale from EAT cells (W.J. Nelson and P. Traub, (1983) J. Biol. Chem. 257, 5544-5553). Despite a 38% protein contamination, the partially purified enzyme from porcine kidney is useful for the in vitro study of structure-function relationships of intermediate filaments and their subunit proteins. During affinity chromatography of the partially purified proteinase from EAT cells on arginine methylester Sepharose 4B, a 100-kDa protein was purified which has a high affinity for arginine residues. It also occurs in porcine kidney, although at a considerably lower concentration. Its cellular localization and function remain to be determined.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002845:Chromatography; D003546:Cysteine Endopeptidases; D003599:Cytoskeleton / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005590:Fractional Precipitation; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008970:Molecular Weight; D013552:Swine; D014746:Vimentin
D007381:Intermediate Filament Proteins; D014746:Vimentin; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002118:Calcium