Purification of two calcium/calmodulin-dependent forms of cyclic nucleotide phosphodiesterase by using conformation-specific monoclonal antibody chromatography

Abstract

A procedure for nondenaturing immunopurification of bovine calmodulin-dependent 3'',5''-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17) is described that utilizes chromatography on a conformation-specific monoclonal antibody column. Hybridomas derived from fusion of P3-NSI/1-Ag4-1 myeloma cells with spleen cells of mice immunized with Ca2+/calmodulin/phosphodiesterase were screened for antiphosphodiesterase antibody production. A stable cell line was established that secretes a monoclonal antibody that binds to the Ca2+/calmodulin/enzyme complex with an approximate Kd of 10-9 M. The Kd was increased by 2 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 [ethylene glycol bis(.beta.-aminoethyl ether) N,N,N'',N''-tetraacetic acid]. The immunopurifed enzymes from heart and brain exhibited specific activities of .apprxeq. 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 2 preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed an apparent subunit of MW 61,000 for the brain enzyme, in contrast to the MW 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 genral technique applicable to the rapid isolation of proteins whose antigenic determinants can be altered with specific ligands.