title: Antigenic determinants and reactive sites of a trypsin/chymotrypsin double-headed inhibitor from horse gram (Dolichos biflorus).
abstract: The major proteinase inhibitor in horse gram (Dolichos biflorus) is a low molecular weight (approximately 8500) Bowman-Birk inhibitor (BBI), HGI-III, that inhibits both trypsin and chymotrypsin simultaneously. Analysis of the reactivity of the polyclonal antibodies raised against native HGI-III, with tryptic, lysylendoproteinase-C and CNBr peptides, in dot-blot assays, revealed the presence of three sequential epitopes (Asp1-Lys14 (I), Leu37-Lys63 (II) and Asp64-Lys71 (III)). Of these, epitope II and III occur consecutively in the sequence of HGI-III. The reactive site peptide bonds were identified by cleavage with catalytic quantities of either trypsin or chymotrypsin at acidic pH. The reactive site peptide bond for trypsin was found to be Lys24-Ser25, whereas for chymotrypsin it was Phe51-Ser52. The highly conserved reactive site loop residues of the Bowman-Birk inhibitors are also conserved in HGI-III. The less immunogenic peptide sequence, Leu37-Lys63, also contains the chymotrypsin reactive site. The recognition of this polypeptide by the immune system provides for a new strategy in the design of ideal, smaller proteinase inhibitors as cancer preventive agents.
authors.affiliation: Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, India.
authors.forename: Y N
authors.initials: YN
authors.lastname: Sreerama
authors.forename: L R
authors.initials: LR
authors.lastname: Gowda
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D017124:Conserved Sequence; D003488:Cyanogen Bromide / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D007887:Fabaceae; D015151:Immunoblotting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology; D010946:Plants, Medicinal; D012639:Seeds / Q000737:chemistry*; D017421:Sequence Analysis; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology
chemical_list: D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D014357:Trypsin; D003488:Cyanogen Bromide