Monoclonal antibodies recognizing amino-terminal and carboxy-terminal regions of human aldolase A: probes to detect conformational changes of the enzyme.
Three monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution.(ABSTRACT TRUNCATED AT 250 WORDS)
Kitajima
Y
Y
Department of Biochemistry, Saga Medical School.
Matsuhashi
S
S
Nishida
H
H
Takasaki
Y
Y
Takahashi
I
I
Hisatsugu
T
T
Hori
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002678:Chimera; D003488:Cyanogen Bromide; D000939:Epitopes / Q000032:analysis*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D010449:Peptide Mapping; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase; D003488:Cyanogen Bromide