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

Abstract

A homogenate of rat brain, rat liver or human colonic well differentiated adenocarcinoma was prepared in 250 m M 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°C under aerobic conditions. ω-Hydroxydocosahexaenoic acid or ω-hydroxyeicosatetraenoic acid from an incubation mixture was detected by reversed-phase high-performance liquid chromatography-thermospray mass spectrometry with selected-ion monitoring. ω-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 + - H 2O ion. For the rat brain, ω-hydroxylation activity (the amount of ω-hydroxy product produced in 30 min) was concentrated to a 103 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 ω-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 ω-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 ω-hydroxylation activity depending on the species of the organs.