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 100,000g 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 mumol/(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 150,000, suggesting that the native enzyme is an oligomeric structure. This PDE hydrolyzed cAMP with a Km of 1.97 +/- 0.26 microM. The hydrolysis of cAMP was neither inhibited nor stimulated by cGMP concentrations lower than 50 microM. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Conti
M
M
Department of Gynecology and Obstetrics, Stanford University Medical Center, California 94305, USA.
Iona
S
S
Cuomo
M
M
Swinnen
J V
JV
Odeh
J
J
Svoboda
M E
ME
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006868:Hydrolysis; D007163:Immunosorbent Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000201:enzymology*
D046911:Macromolecular Substances; D003994:Bucladesine; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium