Purification and characterization of guanosine 3',5'-monophosphate-inhibited low K(m) adenosine 3',5'-monophosphate phosphodiesterase from human placental cytosolic fractions.
We previously characterized human placental cytosolic cAMP phosphodiesterase (PDE) and found that two low K(m) cAMP PDE isoforms that were very sensitive to inhibition by cGMP and cilostamide were activated by insulin. As a first step toward understanding the mechanisms by which insulin activates this enzyme, we purified the cGMP-inhibited low K(m) cAMP PDE (cGI-PDE) from human placentas. The enzyme was purified 11,700-fold from a pool of 100,000 x g supernatant fractions of 10-15 placentas by ammonium sulfate precipitation, diethylaminoethyl-cellulose chromatography, and affinity chromatography, using an isothiocyanate derivative of cilostamide (CIT-agarose). The specific activity of the affinity-purified enzyme was 432 +/- 17 nmol/min.mg (mean +/- SD; n = 4). Gel permeation chromatography of the CIT-agarose eluates revealed one protein peak that coincided with PDE activity at an elution position of 135,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of this protein peak and CIT-agarose eluates revealed the same patterns, indicating that the purified PDE preparations contained multiple proteins with apparent mol wt of 138K, 83K, 72K, 67K, 63K, and 44K. The 138K form appears to be an intact enzyme; an analogous approximately 135K form has recently been identified in rat adipocyte particulate fractions by specific immunoprecipitation or Western immunoblots. In addition, other smaller forms eluted at 135,000 daltons on gel permeation chromatography, suggesting that, although proteolyzed, they must have been associated by either noncovalent interactions or disulfide bonds. All of the protein bands observed on the sodium dodecyl sulfate-polyacrylamide electrophoresis gel reacted with rabbit antibodies raised against human platelet cGI-PDE. Ten peptides from endoproteinase Lys-C-digests of the affinity-purified placental cGI-PDE were isolated and sequenced; sequences of eight peptides were identical to the deduced amino acid sequences in the C-terminal half of a human heart cGI-PDE cDNA, while those of two peptides were not found in the heart enzyme. The sequences of the eight peptides also matched peptide sequences derived from a purified human platelet cGI-PDE. These results provide evidence that the catalytic C-terminal half domain of the placental insulin-sensitive cGI-PDE shares homology with those of human heart and platelet cGI-PDEs. K(m) and maximum velocity values for cAMP and cGMP were 0.57 microM and 862 nmol/min.mg, and 15 microM and 467 nmol/min.mg, respectively. ED50 values for cGMP, cilostamide, and Ro 20-1724 were 0.12, 0.22, and 120 microM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
LeBon
T R
TR
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Kasuya
J
J
Paxton
R J
RJ
Belfrage
P
P
Hockman
S
S
Manganiello
V C
VC
Fujita Yamaguchi
Y
Y
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006152:Cyclic GMP / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010920:Placenta / Q000201:enzymology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011247:Pregnancy; D015363:Quinolones / Q000494:pharmacology
D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D015363:Quinolones; C021294:cilostamide; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP