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 agonist s regulate the levels of mRNA and increase the activity of a high affi nity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cel l in culture. To identify and characterize the hormone-inducible form( s), the cAMP-PDE activity of the Sertoli cell was partially purified a nd its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100000g supernatant of Ser toli cell extracts was purified more than 2000-fold by four HPLC chrom atographic steps. The major purified form of cAMP-PDE had a specific a ctivity of 1-2 mu mol/(min . mg of protein). Polyacrylamide gel electr ophoresis and silver staining analysis showed that a 67-68 kDa polypep tide comigrated with the major peak of cAMP hydrolytic activity. The m olecular weight of the crude or purified enzyme determined by gel filt ration and sucrose density gradients was 150000. Suggesting that the n ative 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 inhib ited nor stimulated by cGMP concentrations lower than 50 mu M. Cyclic nucleotide catalysis required Mg2+, but was insensitive to Ca2+. The a ctivity of this form was competitively inhibited by several inhibitors with the following potency: rolipram > RO 20-1724 > methylisobutylxan thine > cilostamide = milrinone. Because mRNAs derived from two distin ct PDE4B and PDE4D genes are present in the Sertoli cell, selective an d 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. Conversel y, only negligible activities were immunoprecipitated with the PDE4B-s elective antibody. Immunoblot analyses using crude preparations or pre parations at different stages of purification demonstrate the presence of a predominant 67 kDa band. The nonselective and the PDE4D-selectiv e antibody, but not the PDE4B-selective antibody, recognized this poly peptide. The accumulation of the 67 kDa polypeptide was induced by FSH , confirming that this is the major hormone-regulated form. This 67 kD a PDE recovered from the Sertoli cell had a molecular weight different from the form with similar immunological properties expressed in brai n. These data indicate that the major PDE form regulated by hormones a nd cAMP is a high affinity, cGMP insensitive 67 kDa cAMP-PDE protein v ariant product of the PDE4D gene.