Identification of cGMP-stimulated cyclic nucleotide phosphodiesterase in lung tissue with monoclonal antibodies.
Monoclonal antibodies (CGS-1, 2, 3, and 4) and rabbit antisera have been produced against bovine heart cyclic GMP-stimulated cyclic nucleotide phosphodiesterase. The monoclonal antibodies were all of the IgG1 subclass and had relatively high affinities (kd values, 1-5 X 10(-10) M), making them suitable for many sensitive immunological and analytical procedures. Most of the monoclonal antibodies did not inhibit the enzyme, which allowed a rapid and specific assay for this form of phosphodiesterase to be developed. All of the activity could be adsorbed from purified preparations of cGMP-stimulated phosphodiesterase by each of the antibodies indicating antigenic homogeneity of the cGMP-stimulated phosphodiesterase. Neither the monoclonal antibodies nor the antisera inhibited cGMP-binding to the phosphodiesterase. Substrate specificity, Electrophoresis, and cyclic GMP binding studies carried out on immunoadsorbed material indicated that a major part of the cyclic nucleotide phosphodiesterase activity present in bovine lung was due to the cyclic GMP-stimulated form. The presence of this enzyme was difficult to identify by other more common procedures and has not been previously demonstrated in this tissue. These studies also indicated that the cGMP-stimulated phosphodiesterase was immunologically distinct from the other major cGMP-binding proteins present in lung extract and that it could be separated from these binding proteins by chromatography on DEAE-cellulose. Finally, the data suggest that none of the other peaks of cyclic nucleotide phosphodiesterase activity, seen upon DEAE-cellulose fractionation of lung extracts, contain the immunological determinants present on the cGMP-stimulated form which are recognized by the antibodies.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002417:Cattle; D006152:Cyclic GMP / Q000494:pharmacology*; D005779:Immunodiffusion; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP