Mizukami Y, Sumimoto H, Isobe R, Minakami S, Takeshige K
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Eur J Biochem. 1994 Sep 15;224(3):959-65. doi: 10.1111/j.1432-1033.1994.00959.x.
Lipoxin B4 (LXB4) is metabolized to 20-hydroxy-LXB4 by rat liver microsomes. The omega-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, indicating involvement of a cytochrome P-450 (P-450). This is supported by inhibition of the reaction by antibodies raised against NADPH-P-450 reductase. The P-450 appears to be the one responsible for leukotriene B4 omega-hydroxylation, because leukotriene B4 inhibits the formation of 20-hydroxy-LXB4 and LXB4 blocks the leukotriene B4 omega-hydroxylase activity in microsomes. Incubation of 20-hydroxy-LXB4 with both rat liver cytosol and NAD+ leads to formation of a more polar metabolite on high-performance liquid chromatography. The metabolite is identified as 20-carboxy-LXB4, a novel metabolite of LXB4, based on analyses by ultraviolet spectrometry and by gas chromatography/mass spectrometry. The 20-carboxy-LXB4-forming activity is localized in cytosol, with an optimal pH of 8.5. The activity is dependent on NAD+, but NADP+ can not replace NAD+. The reaction is inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by substrates of the enzyme such as ethanol and 20-hydroxy-leukotriene B4. Disulfiram, an inhibitor of aldehyde dehydrogenase, also blocks the 20-carboxy-LXB4 formation. These observations suggest that both alcohol dehydrogenase and aldehyde dehydrogenase participate in the oxidation of 20-hydroxy-LXB4 to 20-carboxy-LXB4.
脂氧素B4（LXB4）被大鼠肝脏微粒体代谢为20-羟基-LXB4。ω-羟基化反应既需要分子氧也需要NADPH，并且会被一氧化碳抑制，这表明有细胞色素P-450（P-450）参与。针对NADPH-P-450还原酶产生的抗体对该反应的抑制作用也支持了这一点。这种P-450似乎是负责白三烯B4 ω-羟基化的酶，因为白三烯B4会抑制20-羟基-LXB4的形成，而LXB4会阻断微粒体中的白三烯B4 ω-羟化酶活性。将20-羟基-LXB4与大鼠肝脏胞质溶胶和NAD⁺一起孵育，在高效液相色谱上会形成一种极性更强的代谢产物。基于紫外光谱分析和气相色谱/质谱分析，该代谢产物被鉴定为20-羧基-LXB4，这是LXB4的一种新代谢产物。形成20-羧基-LXB4的活性定位于胞质溶胶中，最适pH为8.5。该活性依赖于NAD⁺，但NADP⁺不能替代NAD⁺。该反应会被吡唑和4-甲基吡唑（乙醇脱氢酶抑制剂）以及该酶的底物（如乙醇和20-羟基白三烯B4）抑制。双硫仑（一种乙醛脱氢酶抑制剂）也会阻断20-羧基-LXB4的形成。这些观察结果表明，乙醇脱氢酶和乙醛脱氢酶都参与了将20-羟基-LXB4氧化为20-羧基-LXB4的过程。
