[Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico-chemical properties].
The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS-polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis
D013438:Sulfhydryl Compounds; D010749:Phosphoprotein Phosphatases
Fosfoproteinfosfataza kletochnykh iader selezenki byka: fiziko-khimicheskie svoĭstva.