Yamane M, Abe A, Shimizu S, Nakajima M
Department of Biochemistry, Tokyo Medical College, Japan.
J Chromatogr A. 1996 Apr 12;730(1-2):91-8. doi: 10.1016/0021-9673(95)00829-2.
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) 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.
将大鼠脑、大鼠肝脏或人结肠高分化腺癌制成匀浆，置于250 mM蔗糖等渗缓冲液（pH 7.6）中，通过在10³、10⁴和10⁵ g下进行差速离心进行分级分离。将各沉淀或上清液与NADPH以及二十二碳六烯酸或花生四烯酸作为底物，在有氧条件下于37℃孵育30分钟。通过反相高效液相色谱 - 热喷雾质谱联用选择离子监测法检测孵育混合物中的ω-羟基二十二碳六烯酸或ω-羟基二十碳四烯酸。ω-羟基多不饱和脂肪酸以分子离子（MH⁺）的高强度为特征，而常见的羟基多不饱和脂肪酸以MH⁺ - H₂O离子的高强度为特征。对于大鼠脑，ω-羟基化活性（30分钟内产生的ω-羟基产物量）集中在10³ g沉淀中，尽管10³ g沉淀中的比活性（每1 mg蛋白质的活性）并不比其他组分高。然而，加入10⁴或10⁵ g沉淀后，大鼠脑的比活性增加。对于大鼠肝脏，尽管ω-羟基化活性集中在10³ g沉淀中，但比活性集中在10⁵ g沉淀中，亚细胞定位与大鼠脑不同。在人结肠高分化腺癌中，尽管ω-羟基化活性在10³ g上清液中相对较高，但比活性在10³ g沉淀中相对较高。这些结果表明，根据器官种类不同，ω-羟基化活性的亚细胞定位存在差异。
