Substrate specificity and characterization of partially purified rat liver 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase
Substrate specificity and characterization of partially purified rat liver 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase
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DOI：
10.1006/abbi.1997.0364
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发表时间：
1997-12-01
影响因子：
3.9
通讯作者：
Bull, AW
中科院分区：
文献类型：
--
作者：
Bronstein, JC;Bull, AW
亚油酸的氧化产物，如13-羟基十八碳二烯酸（13-HODE），在许多系统中表现出生物活性。13-HODE的一个主要代谢归宿是通过NAD（+）依赖性脱氢酶（13-HODE脱氢酶）氧化成2，4-二烯酮，13-氧代十八碳二烯酸。本文报道了大鼠肝胞液13-HODE脱氢酶的部分纯化和性质。使用硫酸铵沉淀，以及羟基磷灰石，凝胶渗透和疏水相互作用色谱法的组合纯化酶。通过SDS-聚丙烯酰胺凝胶电泳分析最纯化的制剂表明约55 kDa的两个亚基，表明异二聚体酶的可能性。然而，由于纯化制剂中的聚集，尚未获得天然酶的准确分子量。使用13-HODE作为底物，纯化的酶具有6.3 μ M的Km和5.7 nmol/min/mg的V-max。更重要的是，该酶具有窄的底物特异性，13-HODE是优选的底物。在17种底物中，只有9-HODE（13-HODE活性的53%）和15-羟基二十碳四烯酸（13-HODE活性的64%）具有显著的底物活性。许多其他不饱和羟基脂肪酸，包括几种类二十烷酸，不是底物。该酶所显示的狭窄底物特异性表明，它可能在调节亚油酸氧化衍生物的作用中发挥关键作用。(C)北京：科学出版社.
Oxidation products of linoleic acid, such as 13-hydroxyoctadecadienoic acid (13-HODE), exhibit biological activity in a number of systems. One major metabolic fate of 13-HODE is oxidation to the 2,4-dienone, 13-oxooctadecadienoic acid by an NAD(+)-dependent dehydrogenase (13-HODE dehydrogenase). The present work describes the partial purification and characterization of 13-HODE dehydrogenase from rat liver cytosol. The enzyme was purified using a combination of ammonium sulfate precipitation, as well as hydroxylapatite, gel permeation, and hydrophobic interaction chromatography. Analysis of the most purified preparation by SDS-polyacrylamide gel electrophoresis indicates two subunits of approximately 55 kDa, suggesting the possibility of a heterodimeric enzyme. However, due to aggregation in the purified preparation, an accurate molecular mass for the native enzyme has not yet been obtained. Using 13-HODE as a substrate, the purified enzyme has a K-m of 6.3 mu M and a V-max of 5.7 nmol/min/mg. More importantly, the enzyme has a narrow substrate specificity with 13-HODE being the preferred substrate. From a series of 17 potential substrates, only 9-HODE (53% the activity of 13-HODE) and 15-hydroxyeicosatetraenoic acid (64% the activity of 13-HODE) showed significant activity as substrates. A number of other unsaturated hydroxy fatty acids, including several eicosanoids, are not substrates. The narrow substrate specificity displayed by the enzyme suggests that it could play a key role in modulating the effects of oxidized derivatives of linoleic acid. (C) 1997 Academic Press.