title: Purification and some properties of two kinds of keratin-hydrolyzing enzymes of cow snout epithelium.
abstract: Two kinds of keratin-hydrolyzing enzymes (KHEs) from cow snout epithelium were highly purified by affinity chromatography using soybean trypsin inhibitor-bound Sepharose. On gel filtration chromatography, the KHEs were eluted at a volume corresponding to a relative molecular mass (Mr) of 21,000. They were separated from each other by ion exchange chromatography. One of the enzymes had the same characteristics as urea extracted alkaline proteinase, of which optimal pH was at 8.5 to 9.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single band with Mr of 21,500 in the presence or absence of a reducing agent. The other enzyme was a neutral proteinase, with an optimal pH of 7.5. Both enzymes were inhibited by phenylmethylsulfonyl fluoride and soybean trypsin inhibitor. Among the fluorogenic peptides that were hydrolyzed most effectively by the alkaline proteinase were peptidyl MCAs (4-methyl-coumaryl-7-amides) with extended sequences, Boc-Leu-Ser-Thr-Arg-MCA, and then Boc-Val-Pro-ARg-MCA. The neutral proteinase hydrolyzed the latter most effectively. They hydrolyzed preferentially high Mr keratins of cow snout and of newborn mouse epidermis, and showed a limited proteolysis toward 68,000 polypeptide, giving rise to distinct products. The high substrate specificity and extended subsites of the KHEs suggest their role on the metabolism of the high Mr keratins.
mesh_terms: D000818:Animals; D002417:Cattle; D004817:Epidermis / Q000201:enzymology*; D004848:Epithelium / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007633:Keratins / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D011480:Protease Inhibitors; D007633:Keratins; D012697:Serine Endopeptidases