Hippuryl-L-arginine

Given the importance of the complement anaphylatoxins in cellular recruitment during infection, the ability of secreted products from larval stages of Brugia malayi and Trichinella spiralis to influence C5a-mediated chemotaxis of human peripheral blood granulocytes in vitro was examined. Secreted products from B. malayi microfilariae almost completely abolished chemotaxis. This inhibition was blocked by phenylmethylsulphonyl fluoride, indicating the presence of a serine protease, which was subsequently shown to cleave C5a. In contrast, secreted products from T. spiralis infective larvae showed modest inhibition of C5a-mediated granulocyte chemotaxis, and this was blocked by potato carboxypeptidase inhibitor, an inhibitor of several metallocarboxypeptidases. Adult and larval stages of both parasites were demonstrated to secrete carboxypeptidases which cleaved hippuryl-L-lysine and hippuryl-L-arginine, and the T. spiralis enzyme was partially characterised. The data are discussed with reference to inflammation in parasitic nematode infection.

The influence of the chemical structure of the amino acid (or amino acid analogue) moiety of a number of synthetic cholyl amidates on deconjugation by cholylglycine hydrolase from Clostridium perfringens was studied in vitro at pH 5.4. Conjugates with alkyl homologues of glycine were hydrolyzed more slowly as the number of methylene units increased (cholylglycine greater than cholyl-beta-alanine greater than cholyl-gamma-aminobutyrate). In contrast, for conjugates with the alkyl homologues of taurine, cholylaminopropane sulfonate was hydrolyzed slightly faster than cholyltaurine, whereas cholylaminomethane sulfonate was hydrolyzed much more slowly. When glycine was replaced by other neutral alpha-amino acids, rates of hydrolysis decreased with increasing steric hindrance near the amide bond (cholyl-L-alpha-alanine much much greater than cholyl-L-leucine much greater than cholyl-L-valine greater than cholyl-L-tyrosine much greater than cholyl-D-valine). Conjugation with acidic or basic amino acids also greatly reduced the rates of hydrolysis, as cholyl-L-aspartate, cholyl-L-cysteate, cholyl-L-lysine, and cholyl-L-histidine were all hydrolyzed at a rate less than one-tenth that of cholylglycine. Methyl esterification of the carboxylic group of the amino acid moiety reduced the hydrolysis, but such substrates (cholylglycine methyl ester and cholyl-beta-alanine methyl ester) were completely hydrolyzed after overnight incubation with excess of enzyme. In contrast, cholyl-cholamine was not hydrolyzed at all, suggesting that a negative charge at the end of the side chain is required for optimal hydrolysis. Despite the lack of specificity for the amino acid moiety, a bile salt moiety was required, as the cholylglycine hydrolase did not display general carboxypeptidase activity for other non-bile acid substrates containing a terminal amide bond: hippuryl-L-phenylalanine and hippuryl-L-arginine, as well as oleyltaurine and oleylglycine, were not hydrolyzed. Fecal bacterial cultures from healthy volunteers also hydrolyzed cholyl-L-valine and cholyl-D-valine more slowly than cholylglycine, suggesting that cholylglycine hydrolase from Clostridium perfringens has a substrate specificity similar to that of the deconjugating enzymes of the fecal flora. The results indicate that modification of the position of the amide bond, introduction of steric hindrance near the amide bond, or loss of a negative charge on the terminal group of the amino acid moiety of the bile acid conjugate greatly reduces the rate of bacterial deconjugation in vitro when compared to that of the naturally occurring glycine and taurine conjugates.

A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogeneous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18,500-19,000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N-benzoyl-L-arginine ethyl ester, N-alpha-toluenesulfonyl-L-arginine methyl ester, N-alpha-benzoyl-DL-arginine-p-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeSO2F), chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgCl2, NaCl and KCl, and loses its activity in 5 min at 67 degrees C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2Cl, and alpha 1-antitrypsin. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported.