Functional conformations of calmodulin: I. Preparation and characterization of a conformational specific anti-bovine calmodulin monoclonal antibody.
Calmodulin, similarly to many other Ca(2+)-activated proteins, undergoes considerable conformational changes in the presence of Ca2+ ions. These changes were followed using specific monoclonal antibodies against calmodulin. Since calmodulin is a poor immunogen due to its high phylogenetic conservancy, glutaraldehyde-crosslinked bovine brain extract, which contains a considerable amount of functionally active calmodulin complexed with its target proteins, was used as an antigen. Out of nine anti-calmodulin mAbs isolated, three (namely, CAM1, CAM2 and CAM4) were purified and characterized. MAb CAM1 was identified as an IgG1 while mAbs CAM2 and CAM4 belong to IgM class. Additivity ELISA showed that mAb CAM1 binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize close epitopes. MAb CAM1 was found to be especially sensitive to the conformational state of calmodulin in the presence of Ca2+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca2+ removal. In addition mAb CAM1 failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM1 and CAM4 share common epitopes with the above molecules.
Wolf
T
T
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Israel.
Fleminger
G
G
Solomon
B
B
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D011487:Protein Conformation; D018724:Spinacia oleracea
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D000939:Epitopes