Purification and characterization of human erythrocyte pyridoxine kinase.
A 6000-fold purification of pyridoxine kinase from human erythrocytes has been achieved by a combination of DEAE-cellulose chromatography, ammonium sulfate fractionation, gel filtration and preparative disc polyacrylamide gel electrophoresis. Analytic disc polyacrylamide gel electrophoresis at pH 8.7 reveals two protein bands with similar mobility in the purified enzyme, but only one of these has catalytic activity. The enzyme is found to have a pI of 5.5 and a molecular weight of 65000 by gel filtration, and a broad pH optimum of 8.5. The enzyme is labile at acidic pH. The order of activation of divalent metal ions on the enzyme is Co-2+ greater than Mn-2+ greater than Mg-2+ greater than Zn-2+ greater than Cu-2+ greater than Ni-2+ greater than Fe-2+. Of the monovalent cations studied, K+ is the most effective activator, NH+4 is slightly less effective while Na+ is inhibitory. ATP is the specific phosphate donor for the enzyme. Sulfhydryl reagents did not significantly inhibit the enzyme. The Km for pyridoxine is 5.7 with 10-minus 6 M. Pyridoxal, pyridoxamine and 4-deoxypyridoxine inhibit pyridoxine kinase. Inhibition by pyridoxal is competitive and pyridoxal is observed to be phosphorylated effectively by the enzyme. Young human red cells have a higher activity of pyridoxine kinase than old red cells.
D002413:Cations, Divalent; D002414:Cations, Monovalent; D004589:Electrophoresis, Disc; D004905:Erythrocyte Aging; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D011730:Pyridoxal / Q000378:metabolism* / Q000494:pharmacology; D011733:Pyridoxamine / Q000494:pharmacology; D011736:Pyridoxine / Q000378:metabolism*; D013696:Temperature
D002413:Cations, Divalent; D002414:Cations, Monovalent; D011730:Pyridoxal; D011733:Pyridoxamine; D010770:Phosphotransferases; D011736:Pyridoxine