title: Binding of trypsin to fibrillar amyloid beta-protein.
abstract: We have recently reported that fibrillar amyloid beta-protein (Abeta) inhibits the proteolytic activity of trypsin and high molecular weight bovine brain protease. We report here that trypsin binds to fibrillar Abeta (fAbeta) and the resulting complex of trypsin/fAbeta is sodium dodecyl sulfate (SDS)-stable. Electron microscopic analysis confirmed the binding of trypsin on the fibrils of both Abeta 1-40 and Abeta 1-42. SDS-polyacrylamide gel electrophoresis (PAGE) of fAbeta sample incubated in the presence of trypsin showed that major amount of trypsin was associated with fAbeta that did not enter the gel. The presence of trypsin in this protein complex was confirmed by Western blotting after its elution from the gel. Kinetic studies showed that the binding of trypsin to fibrillar Abeta was dependent on the degree of Abeta fibrillization and on the concentration of fAbeta. However, the trypsin binding to Abeta oligomers did not affect the fibril growth. The maximum binding (B(max)) of trypsin to fAbeta 1-40 and fAbeta 1-42 was 36 pmol and 40 pmol, and dissociation constant (K(d)) was 18.31 microM and 20 microM respectively. Similar to fAbeta, trypsin could also bind to fibrillar amylin. This binding was dependent on the concentration of fibrillar amylin. Under similar conditions, bovine serum albumin did not bind to fibrillar Abeta. These results suggest that fAbeta and fibrillar amylin have strong affinities for trypsin, and chelation of proteases by abnormal aggregated proteins may be a general mechanism for inflicting pathological conditions in various diseases.
authors.affiliation: NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA.
authors.forename: Harish
authors.initials: H
authors.lastname: Chander
authors.forename: Abha
authors.initials: A
authors.lastname: Chauhan
authors.forename: Jerzy
authors.initials: J
authors.lastname: Wegiel
authors.forename: Mazhar
authors.initials: M
authors.lastname: Malik
authors.forename: Ashfaq
authors.initials: A
authors.lastname: Sheikh
authors.forename: Ved
authors.initials: V
authors.lastname: Chauhan
mesh_terms: D000682:Amyloid / Q000494:pharmacology; D016229:Amyloid beta-Peptides / Q000378:metabolism* / Q000493:pharmacokinetics / Q000648:ultrastructure; D015153:Blotting, Western / Q000379:methods; D003720:Densitometry / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D066298:In Vitro Techniques; D058228:Islet Amyloid Polypeptide; D046911:Macromolecular Substances; D008854:Microscopy, Electron / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics / Q000648:ultrastructure; D011485:Protein Binding / Q000187:drug effects; D014357:Trypsin / Q000378:metabolism*
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D058228:Islet Amyloid Polypeptide; D046911:Macromolecular Substances; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D014357:Trypsin