Antigenic determinant of a monoclonal antibody: extracellular domain at the M3-M4 junction of the alpha-subunit of Na,K-ATPase.
The binding site of a monoclonal antibody, M45-80, against the alpha-subunit of horse Na,K-ATPase was determined. Various sizes of DNA fragments derived from rat Na,K-ATPase alpha 1-subunit cDNA were cloned into pUC19 expression vector and some fragments of horse genomic DNA were cloned into pUC18. Escherichia coli JM83 cells harboring the plasmids were grown and the cell lysates were used as antigens. An enzyme-linked immunosorbent assay revealed that M45-80 recognizes the hexapeptide Glu-Tyr-Thr-Trp-Leu-Glu (which is identical to the rat and horse alpha 1-subunits) at the M3-M4 junction located on the extracellular side. The ouabain-binding site is discussed in relation to the recognition site of M45-80.
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Japan.
I
I
Kano
K
K
Satoh
F
F
Nagai
K
K
Ushiyama
T
T
Nakao
Y
Y
Hara
K
K
Kano
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics; D015698:Genomic Library; D006736:Horses; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010042:Ouabain / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase