title: Proteolytic enzymes in the blood-feeding parasitic copepod, Phrixocephalus cincinnatus.
abstract: To understand digestive and invasive mechanisms employed by blood-feeding parasitic copepods, extracts of Phrixocephalus cincinnatus were assayed for specific proteolytic enzyme activity. Intact parasites were dissected into the 3 major body regions, cephalothorax (CT), genital segment (GS), and eggstrings (ES), and homogenized in ice-cold 1% Triton X-100 (v/v) in water. Protease activity in each body region was assayed using several synthetic fluorogenic peptide substrates. The greatest activity was detected when samples were incubated with carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin (CBZ-phe-arg-NHMec) in the presence of cysteine or reducing agents. Substrate specificity, pH profile, and inhibitor sensitivity indicated that the proteolytic enzyme(s) belonged to the cysteine class of endopeptidases and were most similar to mammalian cathepsins L, B, and H, respectively. Intense protease activity was also detected with carbobenzoxy-glycyl-L-prolyl-L-arginine-7-amido-4-methylcoumarin (CBZ-gly-pro-arg-NHMec), a substrate for the serine proteases, plasmin and thrombin. Substrate gel electrophoresis revealed intense gelatinolytic activity in all body regions; however, the ES extract presented a pattern different from that of the adult body, suggesting that distinct proteolytic enzymes are expressed during development. Gelatinolytic activity was inhibited at low pH and in the presence of serine protease inhibitors but not cysteine protease inhibitors. Collectively, the results indicate the presence of 2 major classes of proteolytic enzymes, cysteine and serine proteases. Differential expression of these proteases may be important for the successful completion of the parasite's life cycle, as well as survival of the adult.
authors.affiliation: Department of Medicine, University of California, San Diego, USA.
authors.forename: P S
authors.initials: PS
authors.lastname: Perkins
authors.forename: D
authors.initials: D
authors.lastname: Haley
authors.forename: R
authors.initials: R
authors.lastname: Rosenblatt
mesh_terms: D000818:Animals; D003445:Crustacea / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015822:Eye Infections, Parasitic / Q000469:parasitology / Q000662:veterinary*; D005393:Fish Diseases / Q000469:parasitology*; D005432:Flounder / Q000469:parasitology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D011480:Protease Inhibitors; D003546:Cysteine Endopeptidases