Figure 7.

Membrane oxygenase activity of rabbit ALOX15. Purified rabbit ALOX15 was incubated in 0.5 mL PBS with submitochondrial particles (1.4 mg membrane protein/mL). Reaction products were reduced by the addition of solid borohydride, the sample was acidified, the total lipids were extracted [38], the lipid extracts were hydrolyzed under alkaline conditions, and the hydrolysates were analyzed by RP-HPLC (see Materials and Methods) recording the absorbances at 235 nm (OH-PUFAs, panel (A)) and at 210 nm (non-oxygenated PUFAs, panel (B)). The OH-PUFA/PUFA ratio was calculated as measure for the oxidation degree of the membrane lipids. Calibration curves (6-point measurements) for LA, AA, and 13-HODE were established to convert peak area units into nmoles of the analytes. * UV spectrum of the peak 13-HODE/15-HETE.