Isolation and characterization of dihydropteridine reductase from human liver

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Abstract
Dihydropteridine reductase (EC 1.6.99.7) was purified from human liver obtained at autopsy by a 3-step chromatographic procedure with the use of a naphthoquinone affinity adsorbent, DEAE-Sephadex and 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 sulfate/polyacrylamide-gel electrophoresis, and showed 1 spot on 2-dimensional gel electrophoresis. The MW of the enzyme was determined to be 50,000 by sedimentation-equilibrium analysis and 47,500 by gel filtration. On sodium dodecyl sulfate/polyacrylamide-gel electrophoresis a single subunit with MW 26,000 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 behavior 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.