IDENTIFICATION OF CARDIAC OXIDOREDUCTASE(S) INVOLVED IN THE METABOLISM OF THE LIPID PEROXIDATION-DERIVED ALDEHYDE-4-HYDROXYNONENAL

The aim of this study was to identify the cardiac oxidoreductases invo lved in the metabolism of 4-hydroxy-2-trans-nonenal (HNE), an alpha,be ta unsaturated aldehyde generated during the peroxidation of omega-6 p olyunsaturated fatty acids. In homogenates of bovine, human and rat ve ntricles the primary pyridine coenzyme-linked metabolism of HNE was as sociated with NADPH oxidation. The NADPH-dependent enzyme catalysing H NE reduction was purified to homogeneity from bovine heart. The purifi ed enzyme displayed kinetic and immunological properties identical wit h the polyol pathway enzyme aldose reductase (AR), and catalysed the r eduction of HNE to its alcohol 1,4-dihydroxynonene (DHN), with a K-m o f 7 +/- 2 mu M. In the presence of NADP the enzyme did not catalyse th e oxidation of DHN. During catalysis, HNE did not cause inactivation o f AR. Nevertheless when the apoenzyme was incubated with HNE a dissoci able complex was formed between the enzyme and HNE, followed by irreve rsible loss of activity. Inactivation of the enzyme by HNE was prevent ed by NADP. Partial modification of the enzyme with HNE led to a 17-fo ld increase in the K-m(HNE) and K-m(glyceraldehyde), and the HNE-modif ied enzyme had a 500-fold higher IC50 for sorbinil than for the reduce d enzyme, whereas the IC50 for tolrestat increased 25-fold. Incubation of the enzyme with radiolabelled HNE resulted in the incorporation of 2 mol of the aldehyde per mol of the enzyme. Sequence analysis of the radiolabelled peptides revealed modification of Cys-298 and Cys-187. The amino acid sequence of the HNE-modified peptides confirmed that th e HNE-reducing cardiac enzyme is AR and not a related protein such as the fibroblast-growth-factor-regulated protein FR-1 or the mouse vas d eferens protein MVDP. These results indicate that AR represents the on ly major oxidoreductase in the heart capable of utilizing HNE. The hig h affinity of the enzyme for HNE, the lack of inactivation during cata lysis, and the lack of significant alcohol dehydrogenase activity of t he protein suggests that AR-mediated catalysis of HNE is unlikely to b e limited by substrate/product inhibition. Thus AR might constitute an against endogenous and exogenous cytotoxic aldehydes and against oxid ative stress.