Sakuma S, Fujimoto Y, Doi K, Nagamatsu S, Nishida H, Fujita T
Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, Japan.
Biochem Biophys Res Commun. 1994 Jul 29;202(2):1054-9. doi: 10.1006/bbrc.1994.2035.
We examined whether arachidonoyl CoA (AA-CoA) can be a possible supplier of arachidonic acid (AA) for prostaglandin (PG) synthesis in rabbit kidney medulla. AA-CoA was preincubated with or without the 105,000 g supernatant (cytosol) fraction from the kidney medulla for 5 min at 37 degrees C followed by the incubation with the microsomal fraction (0.5 mg protein) (a rich source of PG synthesizing enzymes) in the presence of reduced glutathione and hydroquinone for 5 min at 37 degrees C, and the formed PGE2, F2 alpha and D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of AA-CoA (20 nmol) alone changed the total PG formation (the sum of PGE2, F2 alpha and D2) from 0.14 to 1.55 ng. When the cytosolic fraction (10-100 microliters) was added together with 20 nmol-AA-CoA in the preincubation mixture, total PG formation was further increased 2- to 3-fold compared to AA-CoA alone. Experiments utilizing AA, instead of AA-CoA and boiled cytosolic fraction revealed that the stimulatory action of the cytosolic fraction on the AA-CoA-induced PG synthesis may not be due to the effect on cyclooxygenase activity and due to long-chain acyl CoA hydrolase. These results show that long-chain acyl CoA hydrolase which hydrolyzes AA-CoA to AA is present in the cytosol of rabbit kidney medulla and suggest that this enzyme activity is a potential mediator of supply of AA for PG synthesis in this region.
我们研究了花生四烯酰辅酶A（AA-CoA）是否可能作为花生四烯酸（AA）的供应源，用于兔肾髓质中前列腺素（PG）的合成。将AA-CoA与来自肾髓质的105,000 g上清液（胞质溶胶）部分一起或不一起在37℃下预孵育5分钟，然后在还原型谷胱甘肽和对苯二酚存在下，与微粒体部分（0.5 mg蛋白质）（PG合成酶的丰富来源）在37℃下孵育5分钟，使用9-蒽重氮甲烷进行衍生化，通过高压液相色谱法测定形成的PGE2、F2α和D2。单独添加AA-CoA（20 nmol）可使总PG形成量（PGE2、F2α和D2的总和）从0.14 ng变为1.55 ng。当在预孵育混合物中加入胞质溶胶部分（10 - 100微升）和20 nmol - AA-CoA时，与单独使用AA-CoA相比，总PG形成量进一步增加2至3倍。利用AA替代AA-CoA并使用煮沸的胞质溶胶部分进行的实验表明，胞质溶胶部分对AA-CoA诱导的PG合成的刺激作用可能不是由于对环氧化酶活性的影响，而是由于长链酰基辅酶A水解酶。这些结果表明，兔肾髓质胞质溶胶中存在将AA-CoA水解为AA的长链酰基辅酶A水解酶，并表明该酶活性是该区域PG合成中AA供应的潜在调节因子。
