Characterization of the serine protease and serine protease inhibitor from the tissue-penetrating nematode Anisakis simplex.
A serine protease and a serine protease inhibitor were purified from infective larvae of the parasitic nematode Anisakis simplex. The serine protease was found to be trypsin-like and preferentially cleaved substrates with the basic amino acid arginine at the P1 position (Z-Gly-Pro-Arg-AMC (where Z is benzyloxycarbonyl), Km = 0.019 mM, and Z-Phe-Pro-Arg-AMC, Km = 0.013 mM) at rates similar to those determined for trypsin (0.002 mM and 0.006 mM, respectively). However, the presence of a bulky hydrophobic residue at the P2 position (Z-Phe-Arg-AMC, Km = 13.3 mM, and Z-Ile-Leu-Val-Arg-AMC, Km = 24.7 mM) greatly decreased the rate of substrate hydrolysis. Internal amino acid sequence information was obtained from three endo Lys-C digestion fragments of the purified enzyme. These sequences were > 89% (33:37) identical with that of porcine trypsin. A second serine protease 85% (11:13) identical with that of a secreted tissue-destructive serine protease from the pathogenic bacterium Dichelobacter nodosus was also identified. The serine protease inhibitor was found to inhibit trypsin, elastase, and the Anisakis serine protease stoichiometrically, but did not inhibit chymotrypsin. The amino acid sequence of the amino terminus as well as two internal endo Lys-C fragments were determined. Approximately 96% (47:49) of the residues were identical with soybean trypsin inhibitor, indicating that this inhibitor belongs to the Kunitz-type family of inhibitors.
University of California, San Francisco 94121.
S R
SR
Morris
J A
JA
Sakanari
D000595:Amino Acid Sequence; D000818:Animals; D017163:Anisakis / Q000201:enzymology*; D015801:Helminth Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D013379:Substrate Specificity; D013552:Swine
D015801:Helminth Proteins; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases