Binding of a monoclonal antibody E12 to Gc globulin (vitamin D-binding protein) is inhibited by actin.
A monoclonal antibody, E12, to human Gc globulin was raised in murine somatic cell using purified Gc. The antibody was subtyped IgG2b kappa and had a kd of 3.0 x 10(-8) M for antigen Gc. Monospecificity for Gc was demonstrated by Western blotting of normal human serum using nondenaturing polyacrylamide gel electrophoresis. As judged by ELISA, actin inhibited binding of E12 to Gc in dose-dependent fashion. Affinity chromatography studies further showed that ternary complexes of actin-Gc-E12 were not formed, and actin displaced Gc from Gc-E12 complexes. Proteolytic digestion of Gc with trypsin showed that the monoclonal antibody E12 reacted with the major 30-kDa tryptic fragment containing the amino terminal fragment of Gc, but actin did not react with this fragment. These results indicate that interaction of actin with Gc causes conformational changes which inhibit binding of E12.
Osawa
M
M
Department of Microbiology, Medical University of South Carolina, Charleston 29425-2230.
Sabbatini
A R
AR
Erukhimov
J
J
Werner
P A
PA
Galbraith
R M
RM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D003429:Cross Reactions; D000939:Epitopes / Q000378:metabolism; D006801:Humans; D006825:Hybridomas; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011485:Protein Binding; D014809:Vitamin D-Binding Protein / Q000378:metabolism*
D000199:Actins; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D014809:Vitamin D-Binding Protein