Calcium Stimulates the Membrane Oxygenase Activity of the Purified Rabbit 15-Lipoxygenase
| Sample . | Lipid Extracts* Oxygen Uptake (nmol O2) . | SMP Oxygen Uptake (nmol O2/mg protein) . | Disappearance of PUFA (nmol/mg protein) . | Formation of Oxygenated PUFA (nmol/mg protein) . |
|---|---|---|---|---|
| SMP | 77.5 | 13.0 | 1.8 | 0.7 |
| SMP + 1 mmol/L EDTA | 73.8 | 7.3 | 1.5 | 0.6 |
| SMP + 1 mmol/L Ca2+ | 76.8 | 81.0 | 12 | 8.2 |
| Sample . | Lipid Extracts* Oxygen Uptake (nmol O2) . | SMP Oxygen Uptake (nmol O2/mg protein) . | Disappearance of PUFA (nmol/mg protein) . | Formation of Oxygenated PUFA (nmol/mg protein) . |
|---|---|---|---|---|
| SMP | 77.5 | 13.0 | 1.8 | 0.7 |
| SMP + 1 mmol/L EDTA | 73.8 | 7.3 | 1.5 | 0.6 |
| SMP + 1 mmol/L Ca2+ | 76.8 | 81.0 | 12 | 8.2 |
SMP were washed in PBS containing 1 mmol/L EDTA. After resuspending the membranes in PBS, aliquots (0.05 mL) of this membrane suspension were added as substrate stock to 1.8 mL assay mixture used as substrate. Oxygen uptake was measured with a Clark-type oxygen electrode. Disappearance of PUFA and formation of oxygenated PUFAs was quantified by RP-HPLC (see Materials and Methods). Similar results were obtained with three different enzyme preparations. A representative experiment is shown.
Abbreviations: SMP, submitochondrial particles; PUFA, polyenoic fatty acid.
For these experiments, the lipids were extracted from SMP and used as LOX substrates. The final concentration of phospholipids in this assay was the same as in the incubation with SMP.