Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.
A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.
Ogawa
T
T
Department of Nutrition, School of Medicine, University of Tokushima, Japan.
Tsuji
H
H
Kimoto
M
M
Sasaoka
K
K
D000364:Agaricus / Q000201:enzymology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D000939:Epitopes / Q000276:immunology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006825:Hybridomas / Q000276:immunology; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D051379:Mice; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010449:Peptide Mapping; D012697:Serine Endopeptidases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C050092:4-aminobenzoate hydroxylase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase