Localization of a highly immunogenic region of carboxypeptidase A recognized by three different monoclonal antibodies and their use in the detection of subtle conformational alterations in this enzyme region.
Three murine monoclonal antibodies (mAb 100, 104, and 121) elicited against carboxypeptidase A (CPA) were prepared and characterized. All three mAbs recognize the same or partially overlapping sites of CPA. This is corroborated by the lack of antibody additivity in the ELISA assay carried out in the presence of pairs of mAbs, the similarity in molecular weight of the immunocomplex formed between CPA and one of the mAbs in the presence of another, and also a competition experiment in which one of the mAbs was labeled enzymatically. The three mAbs do not affect the enzymatic activity of CPA. Even at high concentrations, they do not recognize carboxypeptidase B (CPB) in spite of the similar tertiary structure and the 50% homology in amino acid sequence with CPA. This antigenic determinant is located on one of the four cyanogen bromide fragments of CPA. On the basis of the known sequences of the two enzymes, criteria which predict high antigenicity, and experimental data using synthetic peptides, such a determinant was found to be located within the amino acid sequence from residues 209 to 218 of the CPA molecule. The mAbs prepared detect conformational alterations in the above enzyme epitope when the enzyme is exposed to various conditions. The binding of the mAbs to CPA adsorbed onto a polystyrene plate is characterized by apparent binding constants higher by 1 or 2 orders of magnitude than those characterizing the interaction of the mAbs with CPA in solution. The mAbs also readily detect both conformational alterations of CPA on treatment with urea and subtle, reversible conformational alterations on removal of zinc from the active site of the enzyme.
Solomon
B
B
Department of Biotechnology, Tel-Aviv University, Ramat Aviv, Israel.
Koppel
R
R
Kenett
D
D
Fleminger
G
G
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D002268:Carboxypeptidases / Q000276:immunology / Q000378:metabolism*; D043422:Carboxypeptidases A; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000939:Epitopes; D002268:Carboxypeptidases; D043422:Carboxypeptidases A