L-Arginine 7-amido-4-methylcoumarin dihydrochloride

The excretory-secretory product (ESP) derived from Cyathostominea in vitro was assessed, in terms of subunit composition, and proteolytic activity using as substrates azocasein and two synthetic fluorogenic peptides. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits. DTT also enhanced azocaseinolysis, and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NHMec) and carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec). At the optimum pH of 5.5, hydrolysis of Z-Phe-Arg-NHMec was three-fold greater than that of Z-Arg-Arg-NHMec suggesting that the proteolytic specificities of the ESP are more like those of papain or cathepsin L, rather than cathepsin B. In SDS-PAGE gelatin gels, DTT was a requirement for proteolysis by the ESP. Optimum resolution was at pH=5.5, resolving six bands ranging from 114-20kDa. Cysteine proteinase inhibitors abolished all gelatinolytic activity at the pH values tested. Such data indicate the presence of cysteine-class proteinases in the ESP of Cyathostominea.

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37 degrees C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K(m) values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 microM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.