On the nature of leukotriene C4 synthase in human platelets.

Leukotriene C4 is considered to play a major role in several important pathophysiological conditions, e.g., allergy, asthma, and shock. The present investigation demonstrates the presence in human platelets of a membrane-associated enzyme catalyzing the final step in the biosynthesis of leukotriene C4. This leukotriene C4 synthase was shown to be distinct from previously characterized "microsomal" and soluble glutathione transferases. The latter enzymes did not contribute significantly to the leukotriene A4 conjugating activity in platelets. As determined with leukotriene C4 synthase of a crude membrane fraction from human platelets, the Km value was 7 microM and the V value was 0.56 nmol x min-1 x mg-1 with leukotriene A4 as substrate. The enzyme was 20-fold more efficient with leukotriene A4 than with leukotriene A5 and 30-fold more efficient than with the unphysiological derivative leukotriene A4 methyl ester, as measured by the corresponding V/Km values; 14,15-leukotriene A4 was not a substrate. Platelets should be a useful source for the purification and further characterization of human leukotriene C4 synthase.     

Leukotriene A4 hydrolase in the human lung. Inactivation of the enzyme with leukotriene A4 isomers

Leukotriene A4 hydrolase from the human lung was purified to apparent homogeneity. The molecular weight (68,000-71,000), the amino acid composition, and the N-terminal amino acid sequence were similar to those of the human neutrophil enzyme but different from those of human erythrocyte enzyme. The lung enzyme was inactivated by its substrate, leukotriene A4. To elucidate the substrate and the inactivator specificity of this enzyme, we synthesized various geometric and positional isomers of leukotriene A4. 14,15-Leukotriene A4, leukotriene A4 methyl ester, and geometric isomers of leukotriene A4 could not serve as substrates, but they inactivated the enzyme. On the other hand, styrene oxide and (5S)-trans-5,6-oxide-8,10,14-cis-12-trans-eicosatetraenoic acid neither served as substrates nor inactivated the enzyme. These results indicate that whereas allylic epoxide structures of arachidonic acids are responsible for inactivation of the enzyme, the free carboxylic acid, 5,6-oxide, and the tetraene structure with the 7,9-trans-11,14-cis configuration are required as a substrate for leukotriene A4 hydrolase.     

Racemic LTA4 methyl ester bioconversion into LTC4 methyl ester by various glutathione S-transferases

It has been shown that various glutathione transferases can synthesize leukotriene C4, or its methyl ester, from glutathione and leukotriene A4. We questioned whether the same enzymes could be used to resolve racemic leukotriene A4 methyl ester (more easily prepared than the optically active enantiomer) and to produce leukotriene C4 methyl ester selectively. We present in this paper a study of the enantioselectivity of some rat liver glutathione transferase isozymes and of the glutathione transferase of human placenta for the leukotriene A4 methyl ester isomers. The rat liver 3-4 glutathione transferase exhibited the highest conversion rate but preferentially converted the (5R, 6R) leukotriene A4 methyl ester. The placental enzyme was fairly selective for the natural (5S, 6S) enantiomer but the rate of conversion was low.     

HPLC studies on leukotriene A4 obtained from the hydrolysis of its methyl ester

Alkaline hydrolysis of leukotriene A4 methyl ester to leukotriene A4 was studied in either methanol or acetone. Hydrolysis in acetone yielded larger amounts of leukotriene A4 than similar hydrolysis in methanol. The maximum amount was obtained 60 minutes after the beginning of the hydrolysis. Leukotriene A4, as well as leukotriene B4 methoxy isomers were obtained from hydrolysis of leukotriene A4 methyl ester in methanol. It was found that initial leukotriene A4 methyl ester concentration affected the amount of LTA4 produced during the hydrolysis. The maximum concentration of leukotriene A4 was obtained by hydrolyzing solutions of 0.25 mg/ml leukotriene methyl ester in acetone. Spontaneous degradation of leukotriene A4 occurred when it was diluted with tris buffer. Addition of bovine serum albumin to the tris buffer significantly prolonged the half life of leukotriene A4.     

Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.     

Reverse phase high performance liquid chromatography in the synthesis of leukotrienes A4 and C4

The chromatographic (RP HPLC) behaviour of leukotriene C4, its methyl ester, leukotriene A4 methyl ester and some chemicals involved in their synthesis have been investigated. Optimal conditions of separation were determined for the gradient and isocratic HPLC. Parameters of the interaction of the substances with hydrophobic surface are discussed in terms of solvophobic theory.     

Synthesis of the leukotriene A4 methyl ester via acetylene intermediates

The strategy of acyclic eicosanoid synthesis via polyacetylenic intermediates is examplified by the synthesis of the racemic leukotriene A4 methyl ester. Leukotriene synthons, namely, trideca-1,4,7-triyne and methyl 6-formyl-5,6-trans-epoxyhexanoate, were synthesised using propargylic alcohol (thrice) and 1-heptyne as starting materials. In the course of the synthesis all new carbon-carbon bonds were created through acetylenide anion condensations and (Z)-double bonds are introduced by triple bond hydrogenations. The strategy provides a straightforward and stereospecific synthetic pathway.     

Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid

Arachidonic acid, linolenic acid and 14 different oxygenated fatty acid derivatives were tested as activators of human protein kinase C in vitro using histone as substrate. Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic activated the kinase in the presence of calcium at 30 fold lower concentration (1 microM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C4, certain mono- and dihydroxylated eicosanoids and one lipoxin B isomer had stimulatory effects, albeit at higher concentrations. The substrate specificity of protein kinase C activated by lipoxin A proved to be different from that of the phosphatidylserine or phorbol ester activated kinase. Results of the present study suggest that arachidonic acid derived oxygenation products, in particular lipoxin A, may serve as intracellular activators of protein kinase C.     

Biosynthesis of 20,20,20-trifluoroleukotriene B4 from 20,20,20-trifluoroarachidonic acid: a metabolically stable analog of leukotriene B4 and its application to a study of stimulation of leukotriene B4 synthesis by immunoglobulin G1,2

Leukotriene B4 is rapidly metabolized through omega-oxidation, preventing its detection when it is produced under certain biological conditions. To investigate leukotriene B4 production in various physiological conditions, analogs of arachidonic acid which are converted to metabolically stable analogs of leukotriene B4 would be useful. We have synthesized 20,20,20-trifluoroarachidonic acid by the cis-selective Wittig reaction of the C12-C20 fragment with phosphonium salt. 20,20,20-trifluoroarachidonic acid was transformed into 20,20,20-trifluoroleukotriene B4 when incubated with human neutrophils in the presence of the calcium ionophore A23187. The product was identified by uv absorption spectrophotometry, gas chromatography-mass spectrometry, and coelution on high-performance liquid chromatography with 20,20,20-trifluoroleukotriene B4, which was enantioselectively synthesized by the reaction of the fluorine-containing C11-C20 fragment with the C1-C10 phosphonate. The fluorinated leukotriene B4 demonstrated as much chemotactic activity on human neutrophils as natural leukotriene B4 and was metabolically stable when incubated with human neutrophils, probably by blocking omega-oxidation. Also, enzymes catalyzing the transformation of arachidonic acid (AA) into leukotriene B4 did not discriminate the fluorinated precursors from the natural, nonfluorinated AA, thus 20-F3-AA is a valid analog of AA to be used in the study of AA metabolism. When 50 microM of the fluorinated acid was incubated with neutrophils stimulated with heat-aggregated human immunoglobulin G, a significant amount of fluorinated leukotriene B4 (4.3 ng/10(6) cells/40 min, at most) was formed in a dose-dependent manner while little leukotriene B4 was detected with incubation with 50 microM arachidonic acid, probably due to omega-oxidation of the product, leukotriene B4. 20,20,20-Trifluoroarachidonic acid appears to be a useful tool for studying the capacity of leukotriene B4 synthesis in various biological systems while long-lasting 20,20,20-trifluoroleukotriene B4 would serve as an excellent analog of leukotriene B4 in pharmacological studies to understand functions of leukotrienes B4.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: possible pitfall of using the calcium ionophore A23187 to stimulate 5' lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5' lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5' lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B4 by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC50 1.6 X 10(-4)M) was approximately 100 times less potent than BW755C (IC50 1.7 X 10(-6)M) at inhibiting leukotriene B4 synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 X 10(-6)M). The data obtained using STZ as stimulus are consistent with previous in vivo studies and indicate that benoxaprofen is a relatively selective inhibitor of cyclo-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5' lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.     

Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.     

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.     

Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.     

Metabolism of leukotriene A4 by human erythrocytes. A novel cellular source of leukotriene B4

Human erythrocytes transformed leukotriene A4 into leukotriene B4. Metabolism was proportional to the erythrocyte concentration, even at subphysiological levels (0.08-4 X 10(9) erythrocytes/ml). Comparative metabolic studies excluded the possibility that leukotriene B4 originated from trace amounts of polymorphonuclear leukocytes or platelets present in the purified erythrocyte suspensions. For example, suspensions of isolated platelets (100-500 X 10(6) cells/ml) failed to convert leukotriene A4 into leukotriene B4; and conversion by suspensions of isolated polymorphonuclear neutrophils was insufficient to account for the amounts of leukotriene B4 formed by erythrocytes. Leukotriene B4 formation was maximal within 2 min and substrate concentration dependent. Enzymatic activity originated from a 56 degrees C labile nondialyzable (Mr greater than 30,000) soluble component in the 100,000 X g supernatant obtained from lysed erythrocytes. In contrast to the contemporary view, our results indicate that human erythrocytes are not metabolically inert in terms of eicosanoid biosynthesis. The role of human erythrocytes during inflammatory or pulmonary disorders deserves re-examination in this context.     

Inhibition of human thrombin assessed with different substrates and inhibitors. Characterization of fibrinopeptide binding interaction.

The activity of human thrombin has been assessed with fibrinogen, N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide, N-alpha-benzoyl-arginine-p-nitroanilide, N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and p-nitrophenylacetate: increased rates of hydrolysis were found for N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester and N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide compared to N-alpha-benzoyl-arginine-p-nitroanilide and p-nitrophenylacetate. Phenylmethyl sulfonyl fluoride and N-alpha-tosyl-L-lysine chloromethyl ketone inhibited, to the same degree, the activity toward each substrate. Inclusion of N-alpha-tosyl-arginine methyl ester in the phenylmethyl sulfonyl fluoride reaction mixtures protected the enzyme from inhibition as shown with N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. N-Acetylimidazole inhibited the activity towards fibrinogen, N-alphrosine-p-nitrophenyl ester to varying degrees. Inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide was completely reversible with neutral hydroxylamine, whereas coagulant activity towards fibrinogen was only partially regained. Human fibrinopeptide A inhibited activity toward N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide and N-alpha-carbobenzoxy-tyrosine-p-nitrophenyl ester. The mode of inhibition of N-alpha-benzoyl-phenylalanyl-valyl-arginine-p-nitroanilide by fibrinopeptide A was non=competitive (K1 = 3.02.10(-5) M), whereas N-alpha-toysyl-arginine methyl ester was a competitive inhibitor of this substrate (K1 = 2.6.10(-5) M). These studies demonstrate more than one binding domain for fibrinogen on human thrombin.     

Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase

The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase.     

Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit human neutrophil migration: comparisons between synthetic 15 epimers in chemotaxis and transmigration with microvessel endothelial cells and epithelial cells

Lipoxins (LX) are bioactive eicosanoids that can be formed during cell to cell interactions in human tissues to self limit key responses in host defense and promote resolution. Aspirin treatment initiates biosynthesis of carbon 15 epimeric LXs, and both series of epimers (LX and aspirin-triggered 15-epi-LX) display counter-regulatory actions with neutrophils. In this study, we report that synthetic lipoxin A(4) (LXA(4)) and 15-epi-LXA(4) (i.e., 15(R)-LXA(4) or aspirin-triggered LXA(4)) are essentially equipotent in inhibiting human polymorphonuclear leukocytes (PMN) in vitro chemotaxis in response to leukotriene B(4), with the maximum inhibition ( approximately 50% reduction) obtained at 1 nM LXA(4). At higher concentrations, 15-epi-LXA(4) proved more potent than LXA(4) as its corresponding carboxyl methyl ester. Also, exposure of PMN to LXA(4) and 15-epi-LXA(4) markedly decreased PMN transmigration across both human microvessel endothelial and epithelial cells, where 15-epi-LXA(4) was more active than LXA(4) at "stopping" migration across epithelial cells. Differences in potency existed between LXA(4) and 15-epi-LXA(4) as their carboxyl methyl esters appear to arise from cell type-specific conversion of their respective carboxyl methyl esters to their corresponding carboxylates as monitored by liquid chromatography tandem mass spectrometry. Both synthetic LXA(4) and 15-epi-LXA(4) as free acids activate recombinant human LXA(4) receptor (ALXR) to regulate gene expression, whereas the corresponding methyl ester of LXA(4) proved to be a partial ALXR antagonist and did not effectively regulate gene expression. These results demonstrate the potent stereospecific actions shared by LXA(4) and 15-epi-LXA(4) for activating human ALXR-regulated gene expression and their ability to inhibit human PMN migration during PMN vascular as well as mucosal cell to cell interactions.     

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.     

Specificity of the effect of lipoxygenase metabolites of arachidonic acid on calcium homeostasis in neutrophils. Correlation with functional activity

The ability of the major neutrophil-derived lipoxygenase metabolites of arachidonic acid to increase the rate of 45Ca influx in rabbit neutrophils was examined. The results obtained demonstrate that (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid (leukotriene B4) is the most active of the arachidonic acid metabolites. The activity of leukotriene B4 is highly stereospecific in that its three nonenzymatically derived isomers are essentially inactive. The omega-hydroxylation of leukotriene B4 results in a compound that is nearly as active as leukotriene B4 as far as its ability to stimulate calcium influx and neutrophil aggregation while being a much weaker secretagogue. The further conversion of leukotriene B4 into a dicarboxylic acid removes all detectable biological activity. 5,6-Oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) methyl ester was also found to increase the rate of calcium influx, while the degradation products of native leukotriene A4 were essentially inactive. These results demonstrate that a close correlation exists between the ability of the various lipoxygenase products to alter calcium homeostasis in rabbit neutrophils and their biological activities.