Cobalt-dependent protein phosphatases from human cord blood erythrocytes. II. Further characterization of E2 casein phosphatase.
Of three casein phosphatases isolated from the cytosol of human cord blood erythrocytes two were cobalt-dependent, E2 and E3. In the presence of CoCl2, E2 activity was the most prominent. In addition to casein, E2 dephosphorylated phosvitin and p-nitrophenyl phosphate (p-NPP) with pH optima at 6.8-7.2 for proteins and 9.0 for p-NPP. The native enzyme had a molecular weight of 104,000 daltons after AcA-44 Ultrogel filtration. According to SDS/PAGE it consisted of two subunits, 78,000 and 15,000 daltons. The 104,000-dalton form exhibited Michaelis-Menten kinetics and had the greatest affinity for casein between protein substrates tested. Ethanol denaturated the enzyme by 80%. Optimal activation of E2 phosphatase was achieved with 5 mmol/l CoCl2 which did not affect the catalytic properties of the enzyme but did affect the rate of 'E-S' complex formation. Inorganic pyrophosphate was not inhibitory for the 104,000-dalton enzyme. Judging by all these properties the natural substrate for E2 casein phosphatase could be P-pyruvate kinase.
Japundzić
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Institute of Biochemistry, Faculty of Medicine, Belgrade, Yugoslavia.
Levi
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Japundzić
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D002364:Caseins / Q000378:metabolism; D003035:Cobalt / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D000431:Ethanol / Q000494:pharmacology; D005312:Fetal Blood / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000097:blood*; D010774:Phosvitin / Q000378:metabolism; D011489:Protein Denaturation; D013379:Substrate Specificity
D002364:Caseins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D003035:Cobalt; D000431:Ethanol; D010774:Phosvitin; D010749:Phosphoprotein Phosphatases