Purification, properties and polyclonal antibodies for the particulate, low Km cAMP phosphodiesterase from bovine adipose tissue.
The particulate, cGMP- and cilostamide-inhibited "low Km" cAMP phosphodiesterase was purified greater than 100,000-fold in good yield (approximately 20%) from bovine omental fat by a procedure similar to that utilized for purification of an analogous enzyme from rat epididymal adipose tissue; ten-fold more enzyme protein (20-30 micrograms) could be prepared from bovine omentum (25 kg) than from rats (approximately 900 fat pads). Kinetic parameters (all similar to those for the rat enzyme) were: for cAMP, Km and Vmax = 0.3 microM and 2.5 mumol/min/mg, respectively; for cGMP, 0.8 microM and 1.6 mumol/min/mg. For inhibition of cAMP hydrolysis, IC50 values were: for cGMP = 0.6 microM and for OPC 3911, milrinone, CI 930, 0.1-2.0 microM. The purified enzyme, the activity of which eluted from Sephadex G-200 with Mr(app) = 110,000 and was associated with a single protein band during non-denaturing electrophoresis, exhibited on SDS-PAGE silverstained bands of 62 (probably a 61-63 doublet) and 77 kDa, perhaps due to proteolytic nicking. On Western blots, each of the polypeptides cross-reacted with a monoclonal antibody toward the bovine cardiac low Km cAMP and polyclonal rabbit antibody generated toward the purified bovine omental phosphodiesterase. Rabbit anti-phosphodiesterase IgG, which inhibited bovine and rat phosphodiesterase enzymatic activity, did not cross-react with purified bovine retina cGMP-binding or bovine cGMP-stimulated phosphodiesterase.
Degerman
E
E
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Manganiello
V C
VC
Newman
A H
AH
Rice
K C
KC
Belfrage
P
P
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D000273:Adipose Tissue / Q000201:enzymology* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011233:Precipitin Tests
D000906:Antibodies; D015105:3',5'-Cyclic-AMP Phosphodiesterases