Purification of two calcium/calmodulin-dependent forms of cyclic nucleotide phosphodiesterase by using conformation-specific monoclonal antibody chromatography.
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 conformation-specific monoclonal antibody column. Hybridomas derived from spleen cells of mice immunized with Ca(2+)/calmodulin/phosphodiesterase were screened for antiphosphodiesterase antibody production. A stable cell line was established that secretes a monoclonal antibody that binds to the Ca(2+)/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 Ca(2+) 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 approximately 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 NaDodSO(4)/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.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D009206:Myocardium / Q000201:enzymology; D011234:Precipitins; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D011234:Precipitins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D002118:Calcium