Substrate specificity and characterization of partially purified rat liver 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase.
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 Km of 6.3 microM and a Vmax 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.
Bronstein
J C
JC
Department of Chemistry, Oakland University, Rochester, Michigan 48309-4401, USA.
Bull
A W
AW
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000645:Ammonium Sulfate; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity
D046911:Macromolecular Substances; D017886:Durapatite; D000429:Alcohol Oxidoreductases; C067701:13-hydroxyoctadecadienoic acid dehydrogenase; D000645:Ammonium Sulfate