A simple procedure for purification of NADH-specific dihydropteridine reductase from mammalian liver.
A simple and convenient purification method which can yield a homogeneous preparation from even a small amount of starting material was devised for NADH-specific dihydropteridine reductase from rat liver. The procedure is essentially composed of two steps, i.e., affinity chromatography on Matrex gel blue A and hydrophobic chromatography on Phenyl-Sepharose. Prior to the Matrex gel blue A chromatography, the crude extract of rat liver was oxidized to dissociate NADH, bound in the enzyme-NADH complex, from the enzyme. Low molecular weight substances in the extract were removed by Sephadex G-25 gel filtration; then the enzyme was purified by successive chromatographies on Matrex gel blue A and Phenyl-Sepharose columns. Thus about 0.1 mg of purified enzyme was obtained from 3 g of rat liver with 40% recovery. The preparation showed a single protein band on polyacrylamide and SDS-gel electrophoresis. Using NADH and tetrahydro-6-methylpterin, the maximal velocity of the enzyme was determined to be 64.2 mumol quinonoid-dihydro-6-methylpterin reduced/min/mg. Km values of the enzyme were 0.85 microM and 3.4 microM for NADH and quinonoid-dihydro-6-methylpterin, respectively. This simple purification method was also applicable to livers from other mammalian sources such as human and bovine.
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002845:Chromatography / Q000379:methods; D004093:Dihydropteridine Reductase / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D051381:Rats
D009243:NAD; D004093:Dihydropteridine Reductase; D009247:NADH, NADPH Oxidoreductases