Purification and characterization of vitamin B6-phosphate phosphatase from human erythrocytes.
Human erythrocytes rapidly convert vitamin B6 to pyridoxal-P and contain soluble phosphatase activity which dephosphorylates pyridoxal-P at a pH optimum of 6-6.5. This phosphatase was purified 51,000-fold with a yield of 39% by ammonium sulfate precipitation and chromatography on DEAE-Sepharose, Sephacryl S-200, hydroxylapatite, and reactive yellow 86-agarose. Sephacryl S-200 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was a dimer with a molecular mass of approximately 64 kDa. The phosphatase required Mg2+ for activity. It specifically catalyzed the removal of phosphate from pyridoxal-P, pyridoxine-P, pyridoxamine-P, 4-pyridoxic acid-P, and 4-deoxypyridoxine-P at pH 7.4. Nucleotide phosphates, phosphoamino acids, and other phosphorylated compounds were not hydrolyzed significantly nor were they effective inhibitors of the enzyme. The phosphatase showed Michaelis-Menten kinetics with its substrates. It had a Km of 1.5 microM and a Vmax of 3.2 mumol/min/mg with pyridoxal-P. The Vmax/Km was greatest with pyridoxal-P greater than 4-pyridoxic acid-P greater than pyridoxine-P greater than pyridoxamine-P. The phosphatase was competitively inhibited by the product, inorganic phosphate, with a Ki of 0.8 mM, and weakly inhibited by pyridoxal. It was also inhibited by Zn2+, fluoride, molybdate, and EDTA, but was not inhibited by levamisole, L-phenylalanine, or L(+)-tartrate. These properties of the purified enzyme suggest that it is a unique acid phosphatase that specifically dephosphorylates vitamin B6-phosphates.
Fonda
M L
ML
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000097:blood* / Q000302:isolation & purification; D011730:Pyridoxal / Q000031:analogs & derivatives / Q000494:pharmacology; D013379:Substrate Specificity
D006882:Hydroxyapatites; D011730:Pyridoxal; D017886:Durapatite; C027138:pyridoxine phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases