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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.

Dihydropteridine reductase was found in extracts of Crithidia fasciculata and was demonstrated by the fact that the enzyme required both quinonoid-dihydropterin and NADH as substrates. 7,8-Dihydropterin and dihydrofolate failed to serve as substrates; tetrahydropterin was formed as the reaction product. The molecular weight of the enzyme was estimated to be about 55 000 by Sephadex G-100 gel filtration. NADH was more effective than NADPH as substrate for the enzyme. Tetrahydropterin (1.35 nmol tetrahydrobiopterin equivalents/g cells) was also detected in C. fasciculata.

NADH-specific dihydropteridine reductase [EC 1.6.99.7] was purified from mouse mastocytoma P-815 cells. Km values for NADH and NADPH were determined to be 1.4 microM and 32 microM, respectively, using tetrahydro-6-methylpterin. Molecular weight was 50,000, and subunit molecular weight was 25,000. The enzymes from P-815 and liver of host mouse (DBA/2) showed similar electrophoretic mobility on polyacrylamide gel. The P-815 enzyme reacted with antiserum against bovine liver NADH-specific dihydropteridine reductase, forming a single precipitin line.