Cobalt-dependent protein phosphatases from human cord blood erythrocytes. I. Submolecular structure and regulation of activity of E3 casein phosphatase.
We have identified three phosphoprotein phosphatases in the cytosol of human cord blood erythrocytes by sequential anion-exchange chromatography and gel filtration. The most abundant was E3 protein phosphatase. After rechromatography on a column of Ultrogel AcA-44 the enzyme had a molecular weight of 95,000 daltons. According to the data obtained by SDS/PAGE, the 95,000-dalton form was composed of non-identical subunits with a molecular mass of 23,000 and 16,000 daltons. Since ethanol decreased the molecular mass of the 95,000-dalton enzyme to 25,000 daltons, we suggest that the protein of 23,000-25,000 daltons represents the catalytic subunit. The decrease in the molecular weight is followed by a 2-fold increase in the Vmax value and by a change in kinetics: the negatively cooperative 95,000-dalton enzyme (h = 0.45) transforms into Michaelis-Menten kinetics (h = 1.0) in the 25,000-dalton form. Both molecular forms, 95,000 and 25,000 daltons, only dephosphorylated casein but not phosvitine and histones. Both forms were activated by CoCl2 and inhibited by organic, and most potently, by inorganic pyrophosphates to approximately the same degree. As opposed to the inorganic pyrophosphate, which affects the catalytic properties of the enzyme molecule, CoCl2 did not affect the catalytic properties of the enzymes, but it probably did affect the rate of 'E-S' complex formation. CoCl2 protected the 95,000-dalton enzyme from pyrophosphate inhibition. The data indicate that CoCl2 and pyrophosphate may take part in the regulation of the activity of both forms of E3 phosphatase.
Japundzić
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I
Institute of Biochemistry, Faculty of Medicine, Belgrade, Yugoslavia.
Levi
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Japundzić
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D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003035:Cobalt / Q000494:pharmacology*; D003600:Cytosol / Q000201:enzymology; D011756:Diphosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D005312:Fetal Blood / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000097:blood*; D010766:Phosphorylation
D011756:Diphosphates; D003035:Cobalt; D010749:Phosphoprotein Phosphatases