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The activity of the enzyme dihydropteridine reductase, which catalyses the reduction of quinoid dihydropteridine cofactor in the presence of NADH or NADPH, was measured in a system contajning H,O, and peroxidase. Peroxidase and H202 continually reoxidize the reduced tetrahydropteridine cofactor to the quinoid dihydropteridine cofactor, and thus maintains the concentration of the latter virtually constant. A method for calculation of the actual concentration of dihydropteridine cofactor and for correction of the dihydropteridine reductase activity to allow for variations in the dihydropteridine cofactor concentration have been derived. Even though dihydropteridine reductase can utilize either NADH or NADPH as the reductant, the former is 15 to 45-fold more active than the latter. This specificity for the nucleotide cofactor has been found for dihydropteridine reductases prepared from five different mammalian species.The quinoid dihydropteridine cofactor, which is formed during the enzymatically catalysed hydroxylation of L-phenylalanine to tyrosine [I-31, can be reduced by either NADH or NADPH to the corresponding tetrahydropteridine cofactor. This reduction can occur both spontaneously and by an enzyme catalyzed mechanism [4], the enzyme was named dihydropteridine reductase by Kaufman [5]. The 7,s-dihydropteridine derivative, formed by spontaneous isomerisation from the unstable quinoid dihydropteridine cofactor, is neither reduced spontaneously nor by dihydropteridine reductase catalysis in the presence of NADH or NADPH [l,6]. The turnovers of NADH and the di-and tetrahydropteridine cofactors in a system containing an oxidant is shown in Fig. 1. The oxidant will recycle the reduced pteridine cofactor (reaction c) and also to a certain extend directly oxidize NADH (reaction d). The formulas of the hydropterines in Fig.1

Vesicles consisting of (Ca(2+)+Mg(2+))-dependent ATPase (adenosine triphosphatase), and lipid were prepared from sarcoplasmic reticulum of rabbit skeletal muscle. As with non-ionic detergents [le Maire, Møller & Tanford (1976) Biochemistry15, 2336-2342] the (Ca(2+)+Mg(2+))-dependent ATPase after solubilization by deoxycholate showed a pronounced tendency to form oligomers in gel-chromatographic experiments, when eluted in the presence of deoxycholate and phosphatidylcholine. To evaluate the functional significance of oligomer formation the properties of enzymically active preparations of ATPase, solubilized by deoxycholate, were studied. Such preparations were obtained at a protein concentration of 2.5mg/ml in the presence of a high salt concentration (0.4m-KCl) and sucrose (0.3m) in the solubilization medium. Analytical ultracentrifugation of solubilized ATPase showed one protein boundary moving at the same rate as gel-chromatographically prepared monomeric ATPase (s(20,w)=6.0S). From simultaneous measurements of the diffusion coefficient an apparent molecular weight of 133000 was calculated, consistent with solubilization of ATPase in predominantly monomeric form. The enzymic activity of deoxycholate-solubilized ATPase when measured directly in the solubilization medium at optimal Ca(2+) and MgATP concentrations was about 35-50% of that of vesicular ATPase. The dependence of enzymic activity on MgATP concentration indicated that the solubilized ATPase retained high-affinity binding of MgATP, but the presence of high concentrations of the nucleotide did not stimulate activity further, in contrast with that of vesicular ATPase. The dependence of enzymic activity on the free Ca(2+) concentration was essentially the same for both solubilized and vesicular forms, indicating that interaction of ATPase with more than one molecule of Ca(2+) is required for enzyme activity. Solubilized enzyme at 20 degrees C was phosphorylated to about the same degree as vesicular ATPase. It is concluded that the catalytic activity of monomeric ATPase retains most of the features of vesicular ATPase and that extensive oligomer formation in gel-chromatographic experiments in the presence of deoxycholate probably reflects processes taking place during inactivation and delipidation of the protein.

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