A monoclonal antibody showing cross-reactivity toward three calmodulin-dependent enzymes.
The spleen cells of a Balb/c mouse immunized with purified bovine calmodulin-dependent cyclic nucleotide phosphodiesterase were fused with nonsecreting mouse myeloma cells (P3-X63-Ag8-653). Antibody producing hybridomas were screened by the enzyme-linked immunosorbent assay using purified phosphodiesterase as the antigen. One monoclonal cell line, CR-B1, was found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with bovine brain calcineurin and rabbit muscle phosphorylase kinase in addition to phosphodiesterase. The antibody was purified and characterized. It was shown to immunoprecipitate the calmodulin (CaM)-dependent phosphodiesterase and phosphorylase kinase activities but not those of CaM itself, CaM-independent phosphodiesterase and the catalytic unit of cAMP-dependent protein kinase. The immunoprecipitation of phosphodiesterase could be inhibited by calcineurin and phosphorylase kinase. These results suggest that the antibody interacts at a common site on these calmodulin-dependent proteins. The antigenic determinant in phosphodiesterase does not appear to reside in the calmodulin-binding domain of the enzyme since the antibody and phosphodiesterase interaction is not inhibited by calmodulin, and the calmodulin activation of phosphodiesterase is not affected by CR-B1 antibody. It is therefore suggested that the structural similarity among the three calmodulin-dependent proteins extends beyond the calmodulin-binding domains.
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000276:immunology / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000276:immunology* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000276:immunology*; D010764:Phosphorylase Kinase / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010764:Phosphorylase Kinase; D010749:Phosphoprotein Phosphatases; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D010727:Phosphoric Diester Hydrolases