Rainer Wiesner

Analysis of the stereochemistry of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid chromatography

A chiral phase HPLC method was developed for the simultaneous determination of the positional and optical isomers of the lipoxygenase-derived hydroxypolyenoic fatty acids. With a Bakerbond chiral phase HPLC column (dinitrobenzoyl phenylglycine as chiral phase) the positional and optical isomers of the reduced dioxygenation products (by triphenylphosphine or borohydride) of linoleic acid and arachidonic acid were separated after methylation of the carboxylic groups. No cumbersome chemical derivatization such as conversion to a diastereomer was necessary. As compared with the methods used up till now chiral phase HPLC proved to be simpler and more sensitive. About 10 pmol of hydroxy fatty acids suffice for an analysis. The chiral phase HPLC can be used for the preparative separation of the optical antipodes of the lipoxygenase products. An optical purity of more than 90% can be reached in one preparative run. The method was applied to the determination of the stereochemistry of the dioxygenation products of polyenoic fatty acids formed by the lipoxygenases from soybeans, reticulocytes, pea seeds (isoenzyme I and II), tomato fruits, by the quasilipoxygenase activity of hemoglobin, and by the methylene blue-mediated photooxidation of arachidonic acid.

The oxygenation of cholesterol esters by the reticulocyte lipoxygenase

The arachidonate 15‐lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV‐spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.

Nitric oxide oxidises a ferrous mammalian lipoxygenase to a pre-activated ferric species

Nitric oxide is known as an inhibitor of soybean lipoxygenase‐1. Investigating the interaction of a mammalian 15‐lipoxygenase with nitric oxide, we found that this enzyme is also inhibited reversibly when incubated with nitric oxide for a short time period (5 s) under anaerobic conditions. This inhibition may be due to the formation of a dissociable lipoxygenase‐nitric oxide complex. With longer incubation periods the ferrous lipoxygenase is oxidised to a ferric form. This oxidation renders the enzyme more susceptible to peroxide activation, causing a time‐dependent shortening of the kinetic lag phase.

Reticulocyte lipoxygenase exhibits both n-6 and n-9 activities

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15‐ and 12‐hydroxyperoxyeicosatetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n‐6 and n‐9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11‐eicosatriynoic and 5,8,11,14‐eicosatetraynoic acids in contrast to soybean lipoxygenase‐1 which inactivated only by 5,8,11,14‐eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n‐6 and n‐9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n‐9 lipoxygenase from thrombocytes appears to be excluded.

Formation of lipoxin B by the pure reticulocyte lipoxygenase

The pure reticulocyte lipoxygenase converts 5,15‐DiHETE via a lipoxygenase reaction to 5,14,15‐trihy‐droxy‐6, 8,10,12‐eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15‐HETE and 15‐HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.

The formation of products containing a conjugated tetraenoic system by pure reticulocyte lipoxygenase

Lipoxygenase product Reticulocyte lipoxygenase Conjugated tetraene Reaction mechanism

Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase

Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.

Reversibility of the inhibition of cytochrome c oxidase by reticulocyte lipoxygenase.

During the maturation of reticulocytes the mitochondria are lyzed and inactivated by a cell-specific lipoxygenase (LOX) [ 11. In addition to oxygenation of mitochondrial phospholipids the LOX action gives rise to an inhibition of the respiratory chain at three sites. Two inhibitory sites are located between the Fe-S centers of complex I and complex II and ubiquinone [2]. The third inhibitory site located at the cytochrome c oxidase differs from the others with respect to the kinetics, dose-dependence and the extent of the inhibition [3,4]. The present paper deals with the mechanism of the inhibitory action of reticulocyte LOX on particulate cytochrome c oxidase. As reported elsewhere, even extensive LOX-treatment of submitochondrial particles (SMP) does not affect the amount and the spectral properties of the redox-active components of the cytochrome c oxidase [ 1,2], which rules out the possibility of co-oxidative destruction of these components. In this paper evidence will be adduced that LOX inhibits cytochrome c oxidase by chemical modification of the membrane phospholipids which are essential for its activity.

Suicidal inactivation of the rabbit 15-lipoxygenase by 15S-HpETE is paralleled by covalent modification of active site peptides.

Lipoxygenases (LOXs) are multifunctional enzymes that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives; they also convert hydroperoxy fatty acids to epoxy leukotrienes and other secondary products. LOXs undergo suicidal inactivation but the mechanism of this process is still unclear. We investigated the mechanism of suicidal inactivation of the rabbit 15-lipoxygenase by [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid (15-HpETE) and observed covalent modification of the enzyme protein. In contrast, nonlipoxygenase proteins (bovine serum albumin and human gamma-globulin) were not significantly modified. Under the conditions of complete enzyme inactivation we found that 1.3 +/- 0.2 moles (n = 10) of inactivator were bound per mole lipoxygenase, and this value did depend neither on the enzyme/inactivator ratio nor on the duration of the inactivation period. Covalent modification required active enzyme protein and proceeded to a similar extent under aerobic and anaerobic conditions. In contrast, [1-(14)C]-(15S,5Z,8Z,11Z,13E)-15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE), which is no substrate for epoxy-leukotriene formation, did not inactivate the enzyme and protein labeling was minimal. Separation of proteolytic cleavage peptides (Lys-C endoproteinase digestion) by tricine SDS-PAGE and isoelectric focusing in connection with N-terminal amino acid sequencing revealed covalent modification of several active site peptides. These data suggest that 15-lipoxygenase-catalyzed conversion of (15S,5Z,8Z,11Z,13E)-15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid to 14,15-epoxy-leukotriene leads to the formation of reactive intermediate(s), which are covalently linked to the active site. Therefore, this protein modification contributes to suicidal inactivation.

Requirement of monohydroperoxy fatty acids for the oxygenation of 15Ls‐HETE by reticulocyte lipoxygenase

The lipoxygenase from reticulocytes oxygenates 15Ls‐HETE to 8‐hydroperoxy‐15‐hydroxy‐5,9,ll,13‐eicosatetraenoic acid and 5‐hydroperoxy‐15‐hydroxy‐6,8,11,13‐eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15LsHETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15Ls‐HETE.