Human skin proteases: separation and characterization of two alkaline proteases, one splitting trypsin and the other chymotrypsin substrates.
Two alkaline proteases, one splitting preferentially the substrates of chymotrypsin (ATEE) and the other one those of trypsin (BAEE), were separated and partially purified by chromatographic means from human skin extract made in a buffer containing 1.07 mol/1 KC1. The proteins soluble in dilute buffer were removed by a prior extraction. The enzymes could be separated effectively only in the presence of KC1 at a high conc-ntration since large molecular size aggregates or polymers were formed in solutions of low ionic strength. In the presence of 2 mol/1 KC1 the molecular size of the BAEE-hydrolysing enzyme was 120000 and that of the ATEE-hydrolysing enzyme 30000. The ATEE-hydrolysing enzyme was purified by Sephadex G-100 gel filtration and DEAE-cellulose chromatography about 250 fold. It also hydrolysed esters of tryptophane and phenylalanine as well as casein with optimum pH 7.8--8.2. The enzyme was inhibited effectively by LBTI, SBTI and partially by trasylol, TPCK and TLCK, but not by E-600 and SH-modifers. The hydrolysis of ATEE was doubled in the presence of 1 mol/lKCl, NaCl, KBr or NaBr but that of casein was inhibited to some extent. Human serum and alpha-1-antitrypsin inhibited this enzyme but not C1-inactivator. alpha-2-Macroglobulin did not protect if from inhibition by SBTI. The BAEE-hydrolysing enzyme was purified by Sephadex G-100 gel filtration and hydroxylapatite chromatography about 30 fold. It also split other esters of substituted basic amino acids as well as BAPA and histone proteins with optimum pH 7.5--8.2. It was inhibited by Trasylol and TLCK, but not by LBTI, SBTI, OMTI, TPCK, E-600, SH-modifiers, human serum, C1-inactivator or alpha-1-antitrypsin. Neither of these enzymes is exactly similar to any one of the enzymes so far separated from human tissues or fluids.
D000085:Acetates; D001120:Arginine; D001565:Benzoates; D001586:Benzoylarginine Nitroanilide; D002364:Caseins; D011232:Chemical Precipitation; D002712:Chlorides / Q000494:pharmacology; D006657:Histones; D007700:Kinetics; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000032:analysis* / Q000302:isolation & purification; D012867:Skin / Q000201:enzymology*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014443:Tyrosine
D000085:Acetates; D001565:Benzoates; D002364:Caseins; D002712:Chlorides; D006657:Histones; D014361:Trypsin Inhibitors; D014443:Tyrosine; D001586:Benzoylarginine Nitroanilide; D001120:Arginine; D010447:Peptide Hydrolases