Oxygenation of biological membranes by the pure reticulocyte lipoxygenase.
We find that the reticulocyte lipoxygenase can oxygenate rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes (inside-out and right side-out ghosts) without prior action of a phospholipase. After alkaline hydrolysis of the ester lipids, the main products were identified as 15S-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetr aenoic acid, 17S-hydro(pero)xy-4Z,7Z,10Z,13Z,15E, 19Z,-docosahexaenoic acid, 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid, 9(S/R)-hydro(pero)xy-10E,12Z-octadecadienoic acid as well as the two all-E hydro(pero)xy octadecadienoic acid isomers. At low membrane concentrations (1 mg of protein/ml), the enzyme maintains a high stereospecificity for the S-configuration, but at higher concentrations (20 mg/ml), the products were virtually racemic. Addition of the antioxidant 2,6-ditert-butyl-p-cresol counteracted this tendency to lose stereospecificity. During these enzyme-catalyzed reactions, substantially more oxygen is consumed than can be accounted for as the hydro(pero)xy products. This discrepancy is due to secondary reactions which lead to the decomposition of the primary oxygenation products, the hydroperoxy lipids, and to oxidative modifications of membrane proteins. These data indicate that the reticulocyte lipoxygenase can oxygenate polyenoic fatty acids in various types of biological membrane and that the oxidative modifications are not restricted to the membrane lipids. The results are discussed in terms of the proposed role of the enzyme in the breakdown of mitochondria and other intracellular organelles during the maturation of red blood cells.
Kuhn
H
H
Institute of Biochemistry, School of Medicine (Charité), Humboldt University, Berlin, East Germany.
Belkner
J
J
Wiesner
R
R
Brash
A R
AR
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006880:Hydroxy Acids / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D008084:Lipoxygenase / Q000378:metabolism*; D013058:Mass Spectrometry; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D012156:Reticulocytes / Q000201:enzymology*; D013367:Submitochondrial Particles / Q000378:metabolism
D005231:Fatty Acids, Unsaturated; D006880:Hydroxy Acids; D008563:Membrane Lipids; D008084:Lipoxygenase; D010100:Oxygen