Alteration of calmodulin-protein interactions by a monoclonal antibody to calmodulin.
The effects of specific anti-calmodulin monoclonal antibodies on the conformation and interaction of calmodulin with two enzymes, the insulin receptor tyrosine kinase and casein kinase II, are examined. Addition of the anti-calmodulin antibody 2D1 in vitro augments phosphorylation of calmodulin by rat hepatocyte insulin receptors 4.9 +/- 0.5-fold (n = 7). Nonimmune immunoglobulin has no effect. Maximal phosphorylation is observed at a molar ratio of calmodulin:antibody of approx. 2:1, with higher concentrations of antibody producing lesser enhancement. Increasing Ca2+ concentrations in the physiological range progressively inhibit phosphorylation both in the absence and presence of antibody 2D1. Phosphate is incorporated predominantly on Tyr-99, which is distant from the antibody binding site. Enhancement of casein kinase II-catalyzed calmodulin phosphorylation is also produced by the antibody 2D1, implying that antibody binding induces a change in calmodulin conformation. In contrast, two other anti-calmodulin monoclonal antibodies, 4F4 and 4G2, decrease phosphorylation of calmodulin by both the insulin receptor kinase and casein kinase II. These data indicate that secondary and tertiary structures are important in enzyme-substrate interactions and suggest that the antibodies may be useful in investigating the mechanism of calmodulin function.
Sacks
D B
DB
Department of Pathology, Brigham and Women's Hospital, Boston, MA.
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000937:Antigen-Antibody Reactions; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000276:immunology / Q000378:metabolism*; D047388:Casein Kinases; D004305:Dose-Response Relationship, Drug; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013917:Thrombin
D000911:Antibodies, Monoclonal; D002147:Calmodulin; D011494:Protein Kinases; D011972:Receptor, Insulin; D047388:Casein Kinases; D013917:Thrombin; D002118:Calcium