Metabolism of 5-HETE by Different Subcellular Fractions in the Presence and Absence of NAD+ and NADP+
| Fraction (n = 7) . | Cofactor . | 5h Dehydrogenase (pmol/mg protein/min) . |
|---|---|---|
| 1,500g supernatant | NADP+ | 68.7 ± 6.8 |
| 10,000g pellet | NADP+ | 82.7 ± 7.8 |
| 200,000g pellet | None | 2.4 ± 1.1 |
| 200,000g pellet | NAD+ | 41.6 ± 6.3 |
| 200,000g pellet | NADP+ | 192.9 ± 29.3 |
| 200,000g supernatant | NADP+ | 2.4 ± 0.3 |
| Fraction (n = 7) . | Cofactor . | 5h Dehydrogenase (pmol/mg protein/min) . |
|---|---|---|
| 1,500g supernatant | NADP+ | 68.7 ± 6.8 |
| 10,000g pellet | NADP+ | 82.7 ± 7.8 |
| 200,000g pellet | None | 2.4 ± 1.1 |
| 200,000g pellet | NAD+ | 41.6 ± 6.3 |
| 200,000g pellet | NADP+ | 192.9 ± 29.3 |
| 200,000g supernatant | NADP+ | 2.4 ± 0.3 |
Platelets were sonicated and subcellular fractions prepared as described in Materials and Methods. Fractions (1 mL) equivalent to either 20 × 106 (1,500g supernatant and 200,000g pellet) or 50 × 106 (10,000gpellet and 200,000g supernatant) platelets/mL were incubated for 40 minutes at 37°C with 5-HETE (2 μmol/L) in the presence or absence of either NAD+ (1 mmol/L) or NADP+ (1 mmol/L). Eicosanoids were measured by RP-HPLC as shown in Fig 1. 5-hydroxyeicosanoid dehydrogenase activity was calculated from the total amounts of 5-hydroxyeicosanoids (ie, 5-oxo-ETE + 5-oxo-12-HETE + 8-trans-5-oxo-12-HETE) formed. The values are means ± SE (n = 7).
Abbreviation: 5h dehydrogenase, 5-hydroxyeicosanoid dehydrogenase.