Figure 6.

Assays of NDC1 with Naphthoquinones as Electron Acceptors.

(A) Coupled assays with demethylnaphthoquinone methyltransferase. Assays contained 0.52 μM demethylphylloquinone, 370 μM NADPH, 370 μM AdoMet, 89 μg of NDC1, and 10 μg of MenG and were performed for 3 h at 30°C. Overnight preincubation of NDC1 with FAD prior to the assay stimulated phylloquinone formation ∼25-fold (upper trace) compared with no preincubation (lower trace). Inset shows the formation of phylloquinone as a function of the concentration of NDC1 preincubated with FAD. Note that the point at 0 μg NDC1 also contained FAD. HPLC traces have been offset for clarity. DPhQ, demethylphylloquinone; PhQ, phylloquinone.

(B) NDC1-catalyzed oxidation of NADPH as a function of menadione concentration.

(C) Inhibition of NDC1 by dicumarol. Menadione concentration was 20 μM.

(D) ndbB-catalyzed oxidation of NADPH as a function of menadione concentration.

(E) Inhibition of ndbB by dicumarol.

The menadione concentration was 2.5 μM. The concentration of NADPH was 100 μM, and that of the FAD cofactor was 0.6 μM in (B) and (C) and 1.12 μM in (D) and (E). Data are means ± se of three replicates. Error bars smaller than the points are not visible.