Substrate Specificity and Characterization of Partially Purified Rat Liver 13-Hydroxyoctadecadienoic Acid (13-HODE) Dehydrogenase

Abstract

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 aKmof 6.3 μM and aVmaxof 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.