Characterization of phosphohydrolase activity in bovine spleen extracts: identification of a bis(p-nitrophenyl)phosphate-hydrolyzing activity (phosphodiesterase IV) which also acts on adenosine triphosphate.
Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p-nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl)-and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4-methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate).
D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006868:Hydrolysis; D009596:Nitrophenols; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D009596:Nitrophenols; C002887:bis(4-nitrophenyl)phosphate; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D000251:Adenosine Triphosphatases