A new approach to monitoring proteolysis phenomena using antibodies specifically directed against the enzyme cleavage site on its substrate.
Proteolysis is a generic biochemical process that is central in all biological activities. A new strategy for monitoring this biochemical process is proposed here. This approach is based on the production of rabbit polyclonal anti-peptide antibodies directly against the cleavage site on the substrate of the enzyme responsible for proteolysis. So long as the molecule's cleavage site is intact, the antibody will bind to the protein. However, after cleavage of the peptide bond by the protease, the antibody will no longer be able to recognize the substrate. Thus, the development of an ELISA that uses this specific antibody allows hydrolysis of the substrate protein to be monitored. Hydrolysis of beta-casein by plasmin, the main indigenous protease of milk, during the ripening of Swiss-type cheese, has been chosen as a model for this study.
Dupont
Didier
D
INRA, Unité de Recherches en Technologie et Analyses Laitières, B.P. 89 F-39801 Poligny Cedex, France. dupont@poligny.inra.fr
Rolet-Repecaud
Odile
O
Senocq
Daniel
D
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites / Q000276:immunology; D015153:Blotting, Western; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002611:Cheese / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005341:Fibrinolysin / Q000378:metabolism; D006868:Hydrolysis; D008892:Milk / Q000737:chemistry / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013379:Substrate Specificity / Q000276:immunology; D013997:Time Factors
D000906:Antibodies; D002364:Caseins; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D005341:Fibrinolysin