TABLE 3. Activity of NADPH:cytochrome P-450 reductase, NADH:cytochrome b5 reductase, and xanthine dehydrogenase/oxidase in parental CHO cells and transfectants expressing human NQO1.
Values are mean ± standard deviations of at least four independent determinations. NADPH:cytochrome P-450 reductase and NADH:cytochrome b5 reductase were measured in whole-cell lysates by monitoring the reduction of cytochrome c in the presence of NADPH or NADH, respectively. Xanthine dehydrogenase/oxidase activity was measured by monitoring the conversion of xanthine to uric acid in the presence and absence of NAD+. Assays were done on whole-cell lysates for NADPH:cytochrome P-450 reductase and NADH:cytochrome b5 reductase and the supernatant after centrifugation at 15,000 × g for xanthine dehydrogenase/oxidase. Lysate from ~106 cells was used for each assay, and total protein was determined in the samples using the BCA total protein assay.
| Cell line | P-450Ra | b5Rb | XDH/XOc |
|---|---|---|---|
| CHO | 3.3 ± 1.2 | 33.7 ± 12.0 | 5.7 ± 2.3 |
| DTD1 | 5.0 ± 2.1 | ||
| DTD812 | 4.5 ± 1.1 | 47.9 ± 4.0 | 3.4 ± 1.4 |
| DTD815 | 9.6 ± 4.0 | 55.4 ± 9.0 | 1.3 ± 1.2 |
| DTD818 | 7.4 ± 1.7 | 51.0 ± 6.2 | 4.9 ± 1.0 |
NADPH:cytochrome P-450 reductase activity is expressed in nmol of cytochrome c reduced/min/mg of protein.
NADH:cytochrome b5 reductase activity is expressed in nmol of cytochrome c reduced/min/mg of protein.
Xanthine dehydrogenase/oxidase activity is expressed in nmol of uric acid formed/min/mg of protein. All XDH/XO activity was NAD+ dependent.