Gotoh Y, Sumimoto H, Minakami S
Department of Biochemistry, Kyushu University School of Medicine, Fukuoka, Japan.
Eur J Biochem. 1989 Feb 1;179(2):315-21. doi: 10.1111/j.1432-1033.1989.tb14557.x.
Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD+ and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD+ and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for omega-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes omega-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1:2. The affinity of the dehydrogenase for 20-OH-LTB4 may be higher than that for 12-hydroxylaurate (Km = 70 microM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase.
白三烯B4（LTB4）被转化为20-羟基-LTB4（20-OH-LTB4），随后20-OH-LTB4被氧化为20-羧基-LTB4（20-COOH-LTB4）。人中性粒细胞将羟基LTB4氧化为羧基LTB4的过程与NAD+的还原相关，且需要胞质和微粒体部分共同参与。我们分离出一种胞质蛋白，它在NAD+和微粒体部分存在的情况下能氧化羟基LTB4。该蛋白在SDS/PAGE上呈现均一性，亚基分子量为37 kDa，可能是一种由两个相同或相似亚基组成的二聚体蛋白，因为通过葡聚糖G-100柱层析估计其分子量约为80 kDa。该蛋白是一种对ω-羟基脂肪酸（如12-羟基月桂酸和16-羟基棕榈酸）具有高亲和力的醇脱氢酶。基于以下发现，我们得出结论：胞质蛋白将20-OH-LTB4氧化为20-氧代-LTB4，微粒体部分将氧代-LTB4氧化为羧基-LTB4，即氧化ω-羟基脂肪酸的活性仅存在于胞质部分，而氧化疏水醛的活性仅存在于微粒体部分，且20-COOH-LTB4形成与NAD+还原的化学计量比为1:2。该脱氢酶对20-OH-LTB4的亲和力可能高于对12-羟基月桂酸的亲和力（Km = 70 microM），因为在12-羟基月桂酸浓度比20-OH-LTB4高10倍时，后者仅抑制前者氧化40%。该酶活性不受毫摩尔浓度的吡唑和4-甲基吡唑影响。这些特性表明该脱氢酶是一种独特类型的醇脱氢酶。
