Human skin proteases. Separation and characterization of two acid proteases resembling cathepsin B1 and cathepsin D and of an inhibitor of cathepsin B1.
Two acid proteases, one hydrolysing hemoglobin and the other hydrolysing benzoyl arginine naphthyamide (BANA), were separated and partially purified from human skin buffer extract. The acid protease hydrolysing hemoglobin was purified about 190 fold by Sephadex G-100 gel filtration and DEAE-cellulose chromatography. It hydrolysed hemoglobin at pH 3.5, casein at pH 5.8 and skin protein substrate at pH 6.0. It did not markedly hydrolyse synthetic protease substrates. The molecular size of this protease was 38000. The protease was insensitive to common protease modifiers and closely resembles cathepsin D purified from other organs. The BANA-hydrolysing acid protease was purified about 760 fold by Sephadex G-100 gel filtration and affinity chromatography on organomercurial Sepharose 4B gel. It preferentially hydrolysed BAEE, BANA and BAA with an optimum at pH 5.8. The hydrolysis of BAPA, LeuNA and protein substrates was very low. This acid protease was found to be highly dependent on reducing agents, as DTT, and chelating agents, as EDTA, and was inhibited by pCMB and TLCK. The molecular size of the enzyme was 28000. This protease closely resembles cathepsin B1 purified from other organs. Human skin was also shown to contain a low activity of benzoyl arginine amide (BAA) hydrolysing acid protease with a molecular size of about 50000 and resembling cathepsin B2. Human skin contained an inhibitor with a molecular size of about 13000 against human skin cathepsin B1. This inhibitor did not inhibit trypsin, chymotrypsin or skin proteases other than cathepsin B1.
D001587:Benzoylarginine-2-Naphthylamide; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D012867:Skin / Q000201:enzymology*
D006454:Hemoglobins; D001587:Benzoylarginine-2-Naphthylamide; D002403:Cathepsins; D010447:Peptide Hydrolases