Purification and characterization of keratin hydrolase in psoriatic epidermis: application of keratin-agarose plate and keratin-polyacrylamide enzymography methods.
Keratin-agarose plate and keratin-polyacrylamide enzymography methods were developed to demonstrate proteolytic digestion of epidermal keratin. By applying these methods, keratin hydrolase was purified from Tris-buffered saline extract of psoriatic scales by 50% ammonium sulfate precipitation, passage through a lysine-Sepharose column, DEAE-Sepharose, Sephacryl S-200, high-performance cation-exchange chromatography on Mono S, and aprotinin-Sepharose affinity chromatography. The final preparation demonstrated a single protein band at molecular weight 30,000 judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, in keratin-polyacrylamide slab gels, the purified enzyme preparation showed a translucent band at molecular weight 30,000, indicating keratin digestion. Keratin hydrolase digested reassembled epidermal keratin as well, whereas it had no effect on guinea pig hair keratin. The enzyme demonstrated a high level of hydrolytic activity on Ile-Pro-Arg-p-nitroanilide and other peptidyl arginine substrates, while it showed a low level of activity on Val-Leu-Lys-p-nitroanilide, and no activity on Arg-Pro-Tyr-p-nitroanilide, Glu-Pro-Val-p-nitroanilide, or Ala-Ala-Ala-p-nitroanilide. The keratin hydrolase was a serine proteinase, inactivated by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, tosyl-lysyl-chloromethyl ketone, antipain, leupeptin, soybean trypsin inhibitor, aprotinin, and p-aminobenzamidine. The keratinolytic activity was not detected in normal epidermal extract.
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D000328:Adult; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011565:Psoriasis / Q000201:enzymology*; D012685:Sepharose; D012867:Skin / Q000201:enzymology*
D011480:Protease Inhibitors; D007633:Keratins; D012685:Sepharose; D010447:Peptide Hydrolases; C035855:keratinase