Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization.
1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.
Panara
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Istituto di Biologia Cellulare, Facoltá di Scienze MM.FF.NN., Universitá di Perugia, Italy.
D009597:4-Nitrophenylphosphatase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002645:Chickens; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism*
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010710:Phosphates; C008644:nitrophenylphosphate; D010744:Phosphoric Monoester Hydrolases; D009597:4-Nitrophenylphosphatase; D015032:Zinc