title: Proteolysis of sialoglycoprotein by Pasteurella haemolytica cytotoxic culture supernatant.
abstract: Proteolytic enzyme activity releasing sialo glycopeptides from 3H-labeled human erythrocyte ghosts was detected in cytotoxic (leukotoxic) culture supernatants from 9 of 12 Pasteurella haemolytica serotypes. Microcrystalline cellulose thin-layer chromatograms of radioactive water-soluble products showed the following two radioactive peaks: a high-mobility minor peak (Rf, 0.54 to 0.74), identified as sialic acid, and a low-mobility major peak (Rf, 0.18 to 0.21), partially characterized as a trichloroacetic acid-soluble, sialic acid-rich fragment with a molecular weight of greater than 3,500, not extractable by chloroform. The sialic acid content of this fragment after treatment with Clostridium perfringens neuraminidase was estimated to be 7.2 X 10(-2) mumol mg-1. The presence of neuraminidase as a separate activity in some culture supernatants was confirmed. It is considered to be responsible for the observed release of free sialic acid. Preliminary studies with the crude enzyme showed that it has a broad pH optimum around pH 7.0 and that activity is not affected by inhibitors of trypsin, chymotrypsin, thermolysin, thio and serine enzymes, nor by an inhibitor of neuraminidase, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. Activity was, however, inhibited by o-phenanthroline at a high concentration after prolonged treatment. The enzyme hydrolyzed glycophorin at a rate four times higher than the rate for casein. Free glycophorin inhibited the enzyme-induced release of radioactive products from 3H-labeled ghosts. It is speculated that the novel enzyme is a neutral protease, probably metal-dependent, with specificity for sialoglycopeptides. The possible relationship of this protease to the previously reported host species-specific leukotoxicity of P. haemolytica and its potential role in virulence is discussed.
mesh_terms: D002364:Caseins / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006021:Glycophorins / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008970:Molecular Weight; D009439:Neuraminidase / Q000378:metabolism; D010325:Pasteurella / Q000145:classification / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010618:Phenanthrolines / Q000494:pharmacology; D012703:Serotyping; D012794:Sialic Acids / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000097:blood*
chemical_list: D002364:Caseins; D006021:Glycophorins; D007328:Insulin; D010618:Phenanthrolines; D012794:Sialic Acids; D012795:Sialoglycoproteins; C001635:ferroin; D009439:Neuraminidase; D010447:Peptide Hydrolases