A procedure for nondenaturing immunopurification of bovine calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) is described that utilizes chromatography on a confirmation-specific monolonal antibody column. Hybridomas derived from spleen cells of mice immunized with Ca2+/calmodulin/phosphodiesterase were screened for antiphosphodiesterase antibody production. A stable cell line was established that secrets a monoclonal antibody that binds to the Ca2+/calmodulin/enzyme complex with an approximate K(d) of 10-9 M. The dissociation constant was increased by two orders of magnitude when calmodulin interaction with the enzyme was inhibited by Ca2+ chelation. This differential reactivity was utilized for affinity chromatography of heart and brain phosphodiesterases on monoclonal antibody columns. Highly purified phosphodiesterases were eluted in good yield with buffer containing EGTA. The immunopurified enzymes from heart and brain exhibited specific activities of ≃300 units/mg when assayed at millimolar concentrations of cGMP or cAMP. Calmodulin stimulated both enzymes 10- to 15-fold over basal activity under these conditions. However analysis of the two preparations by NaDodSO4/polyacrylamide gel electrophoresis revealed an apparent subunit of M(r)61,000 for the brain enzyme, in contrast to the M(r)59,000 cardiac subunit. The observed difference was not an artifact of tissue homogenization because both forms were detected after purification from mixed-tissue homogenates. These results suggest that mild, biospecific elution from a conformation-specific monoclonal antibody column may be a general technique applicable to the rapid isolation of proteins whose antigenic determinants can be altered with specific ligands.
CITATION STYLE
Hansen, R. S., & Beavo, J. A. (1982). Purification of two calcium/calmodulin-dependent forms of cyclic nucleotide phosphodiesterase by using conformation-specific monoclonal antibody chromatography. Proceedings of the National Academy of Sciences of the United States of America, 79(9 I), 2788–2792. https://doi.org/10.1073/pnas.79.9.2788
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