title: Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment.
abstract: A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.
authors.affiliation: Laboratorium voor Biochemie, Katholieke Universiteit Leuven, Celestijnenlaan 200 G, B-3001, Leuven-Heverlee, Belgium.
authors.forename: Y
authors.initials: Y
authors.lastname: Yigzaw
authors.forename: C
authors.initials: C
authors.lastname: Gielens
authors.forename: G
authors.initials: G
authors.lastname: Préaux
mesh_terms: D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006372:Helix, Snails / Q000737:chemistry*; D006433:Hemocyanins / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry; D006801:Humans; D008744:Methylamines / Q000494:pharmacology; D011487:Protein Conformation; D013045:Species Specificity; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D002364:Caseins; D008744:Methylamines; D000511:alpha-Macroglobulins; D006433:Hemocyanins; D001586:Benzoylarginine Nitroanilide; C027451:methylamine; D014357:Trypsin