CHARACTERIZATION OF A HORMONE-INDUCIBLE, HIGH-AFFINITY ADENOSINE 3'-5'-CYCLIC MONOPHOSPHATE PHOSPHODIESTERASE FROM THE RAT SERTOLI-CELL

In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps. The major purified form of cAMP-PDE had a specific activity of 1-2 mu mol/(min . mg of protein). Polyacrylamide gel electrophoresis and silver staining analysis showed that a 67-68 kDa polypeptide comigrated with the major peak of cAMP hydrolytic activity. The molecular weight of the crude or purified enzyme determined by gel filtration and sucrose density gradients was 150000. Suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a K-m of 1.97 +/- 0.26 mu M. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 mu M. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The activity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxanthine > cilostamide = milrinone. Because mRNAs derived from two distinct PDE4B and PDE4D genes are present in the Sertoli cell, selective and nonselective PDE antibodies were used to determine the origin of the inducible PDE protein. The partially purified PDE was quantitatively precipitated with nonselective or PDE4D-specific antibodies. Conversely, only negligible activities were immunoprecipitated with the PDE4B-selective antibody. Immunoblot analyses using crude preparations or preparations at different stages of purification demonstrate the presence of a predominant 67 kDa band. The nonselective and the PDE4D-selective antibody, but not the PDE4B-selective antibody, recognized this polypeptide. The accumulation of the 67 kDa polypeptide was induced by FSH, confirming that this is the major hormone-regulated form. This 67 kDa PDE recovered from the Sertoli cell had a molecular weight different from the form with similar immunological properties expressed in brain. These data indicate that the major PDE form regulated by hormones and cAMP is a high affinity, cGMP insensitive 67 kDa cAMP-PDE protein variant product of the PDE4D gene.