title: Isolation and characterization of dihydropteridine reductase from human liver.
abstract: Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a three-step chromatographic procedure with the use of (1) a naphthoquinone affinity adsorbent, (2) DEAE-Sephadex and (3) CM-Sephadex. The enzyme was typically purified 1000-fold with a yield of 25%. It gave a single band on non-denaturing and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and showed one spot on two-dimensional gel electrophoresis. The molecular weight of the enzyme was determined to be 50000 by sedimentation-equilibrium analysis and 47500 by gel filtration. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a single subunit with mol.wt. 26000 was observed. A complex of dihydropteridine reductase with NADH was observed on gel electrophoresis. The isoelectric point of the enzyme was estimated to be pH 7.0. Amino acid analysis showed a residue composition similar to that seen for the sheep and bovine liver enzymes. The enzyme showed anomalous migration in polyacrylamide-gel electrophoresis. A Ferguson plot indicated that this behaviour is due to a low net charge/size ratio of the enzyme under the electrophoretic conditions used. The kinetic properties of the enzyme with tetrahydrobiopterin, 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, NADH and NADPH are compared, and the effects of pH, temperature and a number of different compounds on catalytic activity are presented.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004093:Dihydropteridine Reductase / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D009243:NAD; D004093:Dihydropteridine Reductase; D009247:NADH, NADPH Oxidoreductases