Subcellular localization of docosahexaenoic acid and arachidonic acid omega-hydroxylation activity in the brain, liver and colonic adenocarcinoma

A homogenate of rat brain, rat liver or human colonic well differentiated adenocarcinoma was prepared in 250 mM sucrose isoosmolaric buffer (pH 7.6) and fractionated by differential centrifugation at 10(3), 10(4) and 10(5) g. Each precipitate or supernatant was incubated with NADPH and docosahexaenoic acid or arachidonic acid as a substrate for 30 min at 37 degrees C under aerobic conditions. omega-Hydroxydocosahexaenoic acid or omega-hydroxyeicosatetraenoic acid from an incubation mixture was detected by reversed-phase high-performance liquid chromatography-thermospray mass spectrometry with selected-ion monitoring. omega-Hydroxy polyunsaturated fatty acids were characterized by high intensity of the molecular ion (MH(+)) although common hydroxy polyunsaturated fatty acids were characterized by high intensity of the MH(+) - H2O ion. For the rat brain, omega-hydroxylation activity (the amount of omega-hydroxy product produced in 30 min) was concentrated to a 10(3) g precipitate although the specific activity (the activity per 1 mg of protein) in the 10(3) g precipitate did not indicate superiority over other fractions. However, the specific activity of the rat brain increased on addition of a 10(4) or 10(5) g precipitate. For the rat liver, although omega-hydroxylation activity was concentrated to a 10(3) g precipitate, the specific activity was concentrated to a 10(5) g precipitate and the subcellular localization differed from that of rat brain. In the human colonic well differentiated adenocarcinoma, although omega-hydroxylation activity was relatively high in the 10(3) g supernatant, the specific activity was relatively high in the 10(3) and 10(5) g precipitates. These results suggest that there is a difference regarding subcellular localization of the omega-hydroxylation activity depending on the species of the organs.