leukotriene-a4 and 5-6-dihydroxy-7-9-11-14-eicosatetraenoic-acid

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37 degrees C and approximately 40 s at 4 degrees C. Regardless of this aqueous instability, LTA(4) is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA(4) to approximately 20 min at 4 degrees C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA(4) at 4 degrees C to approximately 20 min with protein present. Stabilization of LTA(4) was examined when arachidonic acid was present to compete with LTA(4) for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA(4) and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA(4). Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B(4), were found to have no effect on the stability of LTA(4) induced by E-FABP even when present at concentrations 3-fold higher than LTA(4).

Topics: Animals; Arachidonic Acid; Binding Sites; Binding, Competitive; Biochemical Phenomena; Biochemistry; Carrier Proteins; Cell Line; Dose-Response Relationship, Drug; Fatty Acid-Binding Proteins; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Lipoxygenase; Mass Spectrometry; Models, Biological; Protein Binding; Rats; Temperature; Time Factors

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the proinflammatory mediator leukotriene B4. In previous studies with site-directed mutagenesis on mouse leukotriene A4 hydrolase, we have identified Tyr-383 as a catalytic amino acid involved in the peptidase reaction. Further characterization of the mutants in position 383 revealed that [Y383H], [Y383F], and [Y383Q] leukotriene A4 hydrolases catalyzed hydrolysis of leukotriene A4 into a novel enzymatic metabolite. From analysis by high performance liquid chromatography, gas chromatography/mass spectrometry of material generated in the presence of H216O or H218O, steric analysis of the hydroxyl groups, treatment with soybean lipoxygenase, and comparison with a synthetic standard, the novel metabolite was assigned the structure 5S, 6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE). The kinetic parameters for the formation of 5S,6S-DHETE and leukotriene B4 were found to be similar. Also, both activities were susceptible to suicide inactivation and were equally sensitive to inhibition by bestatin. Moreover, from the stereochemical configuration of the vicinal diol, it could be inferred that 5S, 6S-DHETE is formed via an SN1 mechanism involving a carbocation intermediate, which in turn indicates that enzymatic hydrolysis of leukotriene A4 into leukotriene B4 follows the same mechanism. Inasmuch as soluble epoxide hydrolase utilizes leukotriene A4 as substrate to produce 5S,6R-DHETE, our results also suggest a functional relationship between leukotriene A4 hydrolase and xenobiotic epoxide hydrolases.

Topics: Aminopeptidases; Animals; Chromatography, High Pressure Liquid; Epoxide Hydrolases; Hydroxyeicosatetraenoic Acids; Leucine; Leukotriene A4; Leukotriene B4; Mass Spectrometry; Metalloproteins; Mice; Models, Chemical; Multienzyme Complexes; Mutagenesis, Site-Directed; Spectrophotometry, Ultraviolet; Tyrosine; Zinc

Topics: Animals; Cell Fractionation; Chromatography; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cytosol; Durapatite; Epoxide Hydrolases; Gas Chromatography-Mass Spectrometry; Guinea Pigs; Hydrolysis; Hydroxyapatites; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene A4; Leukotriene B4; Leukotrienes; Liver; Mice; Microsomes, Liver; Substrate Specificity; Ultracentrifugation; Ultrafiltration

The enzymatic conversion of leukotriene A4 into 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid, catalyzed by mouse liver cytosolic epoxide hydrolase (EC 3.3.2.3), was recently described (Haeggström, J., Meijer, J. and Rådmark, O. (1986) J. Biol. Chem. 261, 6332-6337). In the present study, we report analytical data confirming the stereochemistry of this novel enzymatic metabolite of leukotriene A4. By steric analysis of the vicinal diol and comparison with synthetic material, the structure was established as (5S,6R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid. Apparent kinetic constants of this reaction were determined and found to be 5 microM and 550 nmol.mg-1.min-1, for Km and Vmax, respectively. Also, a semipurified preparation of human liver cytosolic epoxide hydrolase avidly catalyzed the same hydrolysis of leukotriene A4 (apparent Km was 8 microM). The enzyme was not inactivated by leukotriene A4, as judged by time-course experiments with a second substrate addition.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Cytosol; Epoxide Hydrolases; Gas Chromatography-Mass Spectrometry; Humans; Hydrolysis; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene A4; Liver; Mice; Molecular Conformation; Spectrophotometry, Ultraviolet

The transformation of leukotriene A4 into dihydroxyeicosatetraenoic acids and sulfidopeptide leukotrienes was determined in homogenates of rat tissues supplied with glutathione and albumin. The highest production of leukotriene B4 was found in spleen, lung and small intestine, while leukotriene C4 dominated in liver and lung. 5(S),6(R)-Dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5,6-DHETE) was formed in all tissues, most prominently in kidney, heart and brain. We also found another isomer of 5,6-dihydroxyeicosatetraenoic acid produced in the kidney. This compound was derived from 5,6-DHETE by isomerization, probably of the 11-cis double bond to 11-trans, and the process appeared to be catalyzed by a membrane-bound factor.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Leukotriene A4; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Spectrophotometry, Ultraviolet; SRS-A

Topics: Animals; Arachidonic Acids; Cytosol; Epoxide Hydrolases; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene A4; Leukotriene B4; Liver; Mice

Homogenates from rat and pig kidney converted leukotriene A4 to 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid as well as leukotriene B4. Both hydrolyses were enzymatic as judged by the effects of heat treatment and proteolytic digestion. Upon subcellular fractionation, conversion of leukotriene A4 to 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid occurred both in the 105,000xg supernatant and the 20,000xg pellet from rat kidney, whereas conversion to leukotriene B4 was confined to the 105,000xg supernatant. We also found production of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene B4 in isolated rat renal epithelial cells, either from exogenous leukotriene A4 or from this substrate supplied by human leukocytes.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Kidney; Kidney Cortex; Kidney Medulla; Leukotriene A4; Leukotriene B4; Rats; Swine

Mouse liver homogenates transformed leukotriene A4 into a 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid. This novel enzymatic metabolite of leukotriene A4 was characterized by physical means including ultraviolet spectroscopy, high performance liquid chromatography, and gas chromatography-mass spectrometry. After subcellular fractionation, the enzymatic activity was mostly recovered in the 105,000 X g supernatant and 20,000 X g pellet. Heat treatment (80 degrees C, 10 min) or digestion with a proteolytic enzyme abolished the enzymatic activity in the high speed supernatant. A purified cytosolic epoxide hydrolase from mouse liver also transformed leukotriene A4 into a 5,6-dihydroxyeicosatetraenoic acid with the same physico-chemical characteristics as the compound formed in crude cytosol, but not into leukotriene B4, a compound previously reported to be formed in liver cytosol (Haeggström, J., Rådmark, O., and Fitzpatrick, F.A. (1985) Biochim. Biophys. Acta 835, 378-384). These findings suggest a role for leukotriene A4 as an endogenous substrate for cytosolic epoxide hydrolase, an enzyme earlier characterized by xenobiotic substrates. Furthermore, they indicate that leukotriene A4 hydrolase in liver cytosol is a distinct enzyme, separate from previously described forms of epoxide hydrolases in liver.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Cytosol; Epoxide Hydrolases; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Liver; Mice; Stereoisomerism