Search

In a study of the transformation of polyunsaturated fatty acids in rabbit polymorphonuclear leukocytes in 1976 it was discovered that arachidonic acid is oxygenated at C-5 (1). Subsequently a number of derivatives, including leukotriene B 4 (LTB 4 ), were identified (2-4). Extension of these studies led in 1979 to the discovery of the pivot epoxide intermediate, LTA 4 (5), and the elucidation of the structure of SRS-A (slow-reacting substance of anaphylaxis) as a group of cysteinyl-leukotrienes, namely LTC 4 , LTD 4 , and LTE 4 (6-10). Earlier work had shown that the prostaglandins are formed by oxygenation and further transformation of arachidonic acid and other polyunsaturated fatty acids (11). The first intermediate in the formation of prostaglandins, the endoperoxide PGG 2 , was isolated and identified in 1974 (12). At the same time we introduced the name cyclooxygenase for the enzyme catalyzing the transformation of arachidonic acid into PGG 2 (12). However, the endoperoxides, PGG 2 and PGH 2 , had some biological effects that could not be explained by their conversion to the known prostaglandins (12). This finding eventually led to the discovery of thromboxane A 2 , an unstable platelet-aggregating and vasoconstrictor substance (13). Subsequent work showed that the endoperoxide can also be converted into a derivative, prostacyclin (PGI 2 ), with opposite biological effects (14). Prostaglandins E 2 and I 2 have strong proinflammatory effects. Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the enzyme (cyclooxygenase) responsible for conversion of arachidonic acid into prostaglandins and thromboxanes (15). An induced form of the cyclooxygenase (COX-2) seems to play an important role in inflammation, thus opening the possibility of developing antiinflammatory NSAIDs that lack the side effects of the previous generation of such drugs (16, 17). In 1975 antiinflammatory steroids were proposed to inhibit prostaglandin formation by blocking of the release of the precursor acid from phospholipid stores (18). Since steroids and NSAIDs have significantly different antiinflammatory effects it seemed conceivable that some of these differences might be explained by the formation of proinflammatory derivatives of arachidonic acid by cyclooxygenase-independent reactions. Our studies of the metabolism of arachidonic acid in leukocytes unraveled a new metabolic pathway and led to the discovery of the leukotrienes (8). These compounds play a role in allergy and asthma and have pronounced proinflammatory effects (19, 20).

5-Lipoxygenase.- LTC4 synthase: A key enzyme in cysteinyl leukotriene formation.- Leukotriene A4 hydrolase: A key enzyme in chemotactic leukotriene formation.- Metabolism of leukotrienes and formation of new leukotriene structures.- Receptors for cysteinyl-leukotrienes: Targets for new drugs.- Targeted disruption of 5-lipoxygenase.- Transcellular biosynthesis of leukotrienes: A unique mode of cell communication.- Physiological and pathophysiological activities of leukotrienes.- Entry rate and metabolic handling of LTC4 in the human circulation.- Genetic polymorphisms of 5-LO.- Leukotrienes and aspirin-intolerant asthma.- Leukotrienes in nocturnal asthma.- Leukotrienes and allergic asthma.- Inhibitors of leukotrienes: An overview.- The development of zafirlukast (Accolate(R)) and the Zeneca series of peptidyl-leukotriene receptor antagonists.- The development of zileuton (ZYFLO(R)) and the N-hydroxyurea class of 5-lipoxygenase inhibitors.- The development of BAY X 1005 and the Bayer series of leukotriene biosynthesis inhibitors.- The development of iralukast: A member of the Novartis series of cysteinyl leukotriene antagonists.- LTB4 receptor antagonists.- SmithKline Beecham pharmaceuticals' cysteinyl leukotriene receptor antagonists: Pranlukast (SB 205312 Ono-1078 Onon), Pobilukast (SK&F 104353) and SK&F 106203.

5-lipoxygenase (5-LO) is expressed in myeloid cells like granulocytes, monocytes macrophages and in B-lymphocytes. In addition, 5-LO expression was reported for neuronal tissues and keratinocytes. In myeloid cells, expression of the 5-LO pathway (5-LO) is upregulated during cell maturation. It has been shown by various authors that differentiation of HL-60 cells is accompanied by an upregulation of 5-LO mRNA, protein, and activity (Bennett et al., 1993; Kargman and Rouzer, 1989). Depending on the differentiation inducer, 3–6-fold differences in 5-LO mRNA levels were observed for HL-60 cells (Bennett et al., 1993; Steinhilber et al., 1993a). The combination of 1,25-dihydroxyvitamin D3 (calcitriol) and transforming growth factor-s (TGFs), either alone or in combination with dimethylsulfoxide, was the most prominent inducer of the 5-LO pathway, leading to cellular protein expression and activity comparable to (or higher than) what is found for normal human granulocytes (Brungs et al., 1994; Steinhilber et al., 1993b).

Some Recent Advances in Leukotriene Research B. Samuelsson. Practical Aspects of Prostaglandin E1 before and after Solid Organ Transplantation H. Muller et al. Prostaglandins in Liver Transplantation R.M. Merion. Prostaglandins in Heart Transplantation F. Iberer et al. Eicosanoids in Peridontal Diseases: Potential for Systemic Involvement S.M. Damareet al. Mitogen-Activated Protein Kinases and Endothelial Prostacyclin Secretion J.H. Grose et al. Crosstalk between Elevation of [Ca2+I, Reactive Oxygen Species Generation and Phospholipase A2 Stimulation in Human Keratinocyte Cell Line R. Goldman et al. Cloning Tissue-Specific Expression and Regulation of the Bovine Thromboxane A2 Receptor S. Muck, K. Schror. Weak Inhibitors of Cyclooxygenases May Exert their Antinociceptive Effect by Modulation of Transcription Factors N. Scheuren et al. New Group of Lipid Mediators Containing )-Hydroxyarachidonic Acid (20-HETE) K. Mao et al. Biosynthesis of Novel Divinyl Ether Oxylipins by Enzyme from Garlic (Allium Sativum L.) Bulbs A.N. Grechkin, M. Hamberg. Catalytic and Spectroscopic Properties of Linoleate Diol Synthase of the Fungus Gaumannomyces Graminis E.H. Oliw, C. Su. 87 Additional Articles. Index.

Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a soluble monomeric protein with a molecular mass of 69 kDa that converts the unstable epoxide intermediate LTA4 into the proinflammatory compound LTB4, 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosa-tetraenoic acid (For reviews see Refs. 1 and 2). Sequence comparisons of LTA4 hydrolase with certain zinc containing proteases and peptidases led to the discovery of a zinc binding motif in the primary structure of the enzyme3. Further studies verified that LTA4 hydrolase indeed contained one zinc atom per enzyme molecule4,5. These findings also led to the discovery of a previously unknown peptidase/amidase activity5,6 which was specifically stimulated by monovalent anions, e. g., chloride ions7, and also by albumin8. Studies involving site directed mutagenesis in combination with metal determination of the purified mutated proteins demonstrated that the zinc atom was bound to His-295, His-299, and Glu-318, in accordance with previous predictions, and that replacements of either of these three residues led to enzymatically inactive proteins which did not contain zinc9.

The leukotrienes are formed by transformation of arachidonic acid into an unstable epoxide intermediate, leukotriene A4 (LTA4), which can be converted enzymatically by hydration to LTB4, and by addition of glutathione to LTC4. This last compound is metabolized to LTD4 and LTE4 by successive elimination of a γ-glutanyl residue and glycine1, 2.

Leukotriene A4 hydrolase is a zinc-containing enzyme which catalyzes the hydrolysis of LTA4 to LTB4, a proinflammatory mediator. The enzyme also exhibits an aminopeptidase activity. Due to its biological importance, it is of considerable interest to develop selective inhibitors of this enzyme. The design and synthesis of a number of potent beta-amino hydroxylamine and amino hydroxamic acid inhibitors are described here. It was found that having a free amine was essential for high activity. Hydroxylamines were found to be about an order of magnitude less potent than their analogous hydroxamic acids. Our investigation of amino hydroxamic acids as inhibitors of leukotriene A4 hydrolase has led to the development of hydroxamates 16 and 17, which are among the most potent inhibitors found to date. These, compounds were found to be competitive inhibitors with Ki values of 1.6 nM and 3.4 nM respectively, against the peptidase activity. Inhibitor 16 has an IC50 value of < or = 0.15 microM against the epoxide hydrolase activity and is also potent against the production of LTB4 by isolated polymorphonuclear leukocytes (PMNL) activated with ionophore A23187 (IC50 approximately 0.3 microM).

In an effort to better understand the favorable binding interactions between the reversible picomolar inhibitor 3-(4-benzyloxyphenyl)-2-(R)-amino-1- propanethiol (1) and leukotriene A4 (LTA4) hydrolase (EC 3.3.2.6), we prepared a number of derivatives of 1-L and other related structures, and assayed their inhibition of LTA4 hydrolase-catalyzed hydrolysis of L-alanine-p-nitroanilide. The inhibition data was analyzed using a weighted non-linear least-squares curve fitting computer program developed for this purpose to fit data derived under the non-Michaelis-Menten condition of [I]t < [E]t. The free thiol is necessary for sub-micromolar binding and the enzyme prefers the R enantiomer over the S enantiomer, in contrast to the stereoselectivity displayed towards bestatin, an inhibitor of somewhat similar structure. Substitution of acid moieties around the periphery of the benzyloxyphenyl portion of 1-L leads to substantially decreased binding, suggesting that this group resides within a large hydrophobic pocket when bound to the enzyme. Possible LTA4 binding modes in the active site of LTA4 hydrolase, including a possible direct role for the carboxylic acid of LTA4 in the enzyme-catalyzed hydrolysis of leukotriene A4, are discussed.

The non-heme iron centre in human 5-lipoxygenase was studied. Recombinant enzyme was expressed in Escherichia coli, purified and assayed for iron content and enzyme activity. For non-mutated enzyme, the iron content was 1.01 +/- 0.19 mol/mol. Deletion of the C-terminal Ile673 resulted in an iron content of 0.03 +/- 0.07 mol/mol and undetectable lipoxygenase activity. Mutations at His367, Glu376 and Asn554 led to drastically decreased enzyme activity (< 2% of non-mutated control) but iron was still present. In addition to Glu376, eight other conserved acidic residues (Asp/Glu) in 5-lipoxygenase were replaced, none of which was crucial for enzyme activity. We conclude that Ile673 is an iron ligand in 5-lipoxygenase, while our results do not support that Glu376 or Asn554 have this function. The possible role of His367 as a replaceable iron ligand is discussed.

Human 5-lipoxygenase (5LO) becomes very unstable after purification. Commonly used methods for protein stabilization could not prevent this inactivation. However, addition of small amounts of glutathione peroxidase (0.15 micrograms/ml) and superoxide dismutase (1 microgram/ml) to the solution of purified 5LO (300-500 micrograms/ml) stabilized the enzyme during storage. The protected 5LO maintained full activity for at least 12 days at 25 degrees C, while 50% of the activity was lost within 10 h without protection. Glutathione peroxidase alone also preserved the activity of 5-lipoxygenase; however, the effect declined rapidly in the absence of superoxide dismutase. 2-Mercaptoethanol was the most efficient hydrogen donor substrate for glutathione peroxidase in the protection of 5LO. Catalase was less effective as a stabilizing agent, and ebselen, a synthetic glutathione peroxidase-mimicking compound, did not protect 5LO. Since many metal ion binding proteins are susceptible to H2O2 inactivation, this method could be useful also for the stabilization of other proteins.

5-Lipoxygenase activity in DMSO-differentiated HL60 cells is regulated by human serum. The serum effect depended on the differentiation state of the cells. For a stimulatory effect to occur, it was required that the cells had been treated with DMSO before addition of serum. After this regimen, the HL60 cells acquired the same high 5-LO activity as found for human neutrophils isolated from peripheral blood (about 9-times higher than for HL60 cells treated only with DMSO). On the other hand, when serum was added together with DMSO and present during the entire differentiation period (seven days), or withdrawn after the first four days, the 5-LO activity did not increase. 5-LO activity of HL60 cells covaried with the expression of the CD14 molecule, a marker for myeloid cell maturation which was recently identified as a receptor for the complex of LPS and LPS-binding protein. These serum effects on 5-LO activity were only observed for intact cells. The prominent increase in 5-LO activity induced by serum was not concomitant with similar changes in the expression of 5-LO or 5-LO-activating protein (FLAP), as judged from analyses of immunoreactive protein and mRNA. Also, the high 5-LO activity induced by serum was rather insensitive to the drug MK886 under our standard assay conditions, which included addition of exogenous arachidonic acid (40 μM). The results indicate that additional cellular components of importance for 5-LO activity in HL60 cells become operative after serum treatment, and that mere expression of 5-LO and FLAP is insufficient for high 5-LO activity in intact cells.

Recombinant human 5-lipoxygenase was expressed in Escherichia coli and purified to more than 95% homogeneity by ammonium sulfate precipitation and agarose-ATP column chromatography. The specific activity of the purified enzyme was 21-28 mumol/mg, as assessed by the generation of 5-hydro(pero)xyeicosatetraenoic acid. The iron content was analyzed by graphite furnace atomic absorption spectrophotometry for six preparations of the enzyme. The average value of the iron content was 0.86 mol/mol (iron/protein) with a range of 0.74-1.15 mol/mol. All lipoxygenases that have been sequenced contain 6 conserved histidine residues. Mutants of 5-lipoxygenase, with substitutions of these 6 conserved histidines, were purified and analyzed. Mutants H372Q and H550Q had no detectable enzyme activity and were also practically devoid of iron. Three mutants regarding His367 (H367Q, H367N, and H367S) were all inactive but had partial iron contents (0.5, 0.2, and 0.5 mol/mol, respectively). Finally, the mutated proteins H362Q, H390Q, and H399Q displayed reduced enzyme activity but contained similar amounts of iron as non-mutated 5-lipoxygenase. We conclude that histidines 372 and 550 constitute two of the iron ligands in 5-lipoxygenase. Also His367 is necessary for the enzyme activity, but this residue is not crucial for binding of iron.

Leukotriene A4 hydrolase is a zinc-containing enzyme which exhibits both epoxide hydrolase and aminopeptidase activities. Since the enzyme product leukotriene B4 is an inflammatory mediator, it is of interest to develop selective inhibitors of leukotriene A4 hydrolase as potential antiinflammatory agents and as mechanistic probes. A systematic study on the enzyme specificity and the inhibition of its amidase activity with more than 30 synthetic inhibitors has led to the development of an alpha-keto-beta-amino ester (26) and a thioamine (27) as tight-binding, competitive type transition-state analog inhibitors of the aminopeptidase activity, with Ki values of 46 and 18 nM, respectively. Both compounds also inhibit the epoxide hydrolase activity, with the IC50 values of 1 microM and 0.1 microM for 26 and 27, respectively.

X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus sequences for catalytic and structural zinc binding sites (1). Such reference structures have served to predict zinc binding ligands among members of related zinc enzyme families and also, more noticeable, in apparently unrelated proteins. Thus, comparison of the amino acid sequence of LTA4 hydrolase with the corresponding primary structures of certain aminopeptidases and neutral proteases, typified by thermolysin, revealed the presence of a putative catalytic zinc site (1, 2). Consequently, LTA4 hydrolase was found, not only to contain one zinc atom per molecule, essential for the catalytic activity, but also to possess a zinc-dependent peptidase activity towards synthetic amides (3–5).

Leukotriene (LT)A4 is formed from arachidonic acid in two consecutive reactions, both catalyzed by 5-lipoxygenase (1). This unstable epoxide intermediate is hydrolyzed into the proinflamrnatory compound LTB4, by the enzyme LTA4 hydrolase (review in 2). From sequence comparison with certain zinc containing aminopeptidases and proteases, typified by thermolysin, a potential zinc binding site was found in the sequence of LTA4 hydrolase (3, 4). This finding prompted us to investigate the possible zinc content and peptidase activity of LTA4 hydrolase.

The expression of the 5-lipoxygenase and the 5-lipoxygenase activating protein (FLAP) genes in human tonsillar B cells and in a lymphoblastoid B cell line was demonstrated at the transcriptional level by the polymerase chain reaction (PCR) technique. Intact B cells produced very low amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid in comparison to those formed by sonicates of these cells. Incubation of intact lymphoblastoid B cells with the glutathione depleting agent, azodicarboxylic acid bis[dimethylamidel] (diamide), prior to the addition of the calcium ionophore A23187 and arachidonic acid, led to the formation of similar amounts of LTB4 as those produced by sonicated cells. These results indicate that the glutathione status is of importance for the activity of 5-lipoxygenase in B lymphocytes.

Two human 12-lipoxygenase enzyme (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31)-related genes were characterized from 13 distinct clones isolated from three genomic bacteriophage and cosmid libraries. A complete gene (12-lipoxygenase gene 1) spanning approximately 17 kilobases and consisting of 14 exons with sequence matching the cloned platelet/human erythroleukemia (HEL) cell cDNA sequence was identified. Several consensus sites for transcription factors and two potential transcription initiation sites within the 5' flanking region, encompassing the putative promoter region, were identified. A segment of a second, probable pseudogene (12-lipoxygenase gene 2), which displays approximately 85% identity to gene 1 within exon sequences, was also characterized. The presence of two 12-lipoxygenase genes was also substantiated by Southern blot analysis of total human genomic DNA. Exon-intron boundaries for the 12-lipoxygenase genes were located in the identical corresponding positions to the previously cloned human 5-lipoxygenase and rabbit 15-lipoxygenase genes, indicating a highly related gene family. Three lipoxygenase genes (12-lipoxygenase genes 1 and 2, 15-lipoxygenase) were localized to human chromosome 17, whereas the most unrelated lipoxygenase (5-lipoxygenase) was mapped to chromosome 10 by PCR analysis of a human-hamster somatic hybrid DNA panel. 12-Lipoxygenase gene 1 expression could be detected in human erythroleukemia cells, platelets, and human umbilical vein endothelial cells with certainty by reverse transcription-PCR analysis. There was no detectable 12-lipoxygenase gene 2 expression in several tissues and cell lines.

The expression of arachidonate 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) and the 5-lipoxygenase-activating protein (FLAP) genes in human tonsillar B cells and lymphoblastoid B-cell lines was demonstrated at the transcriptional level by reverse transcription-PCR analysis. Also, five lymphoblastoid T-cell lines were investigated and found to express the FLAP gene but not the 5-lipoxygenase gene, suggesting that the transcriptional regulation of these two genes is different. Western blot analysis of the cytosolic proteins from a lymphoblastoid B-cell line with an antiserum raised against purified human leukocyte 5-lipoxygenase revealed an immunoreactive band that comigrated with recombinant human 5-lipoxygenase. Intact B cells produced very low amounts of leukotriene B4 and 5-hydroxyeicosatetraenoic acid upon stimulation with the calcium ionophore A23187 and arachidonic acid, in comparison to the amounts formed by sonicates of these cells. However, preincubation of intact lymphoblastoid B cells with the glutathione-depleting agents azodicarboxylic acid bis(dimethylamide) or 1-chloro-2,4-dinitrobenzene prior to the addition of the calcium ionophore A23187 and arachidonic acid led to similar amounts of leukotriene B4 as were formed by sonicated cells. In contrast, the glutathione synthesis inhibitor buthionine sulfoximine diminished the cellular level of glutathione by greater than 90% but did not influence the production of leukotriene B4 or 5-hydroxyeicosatetraenoic acid in intact cells. These results demonstrate that certain drugs affecting the redox status can stimulate the cryptic 5-lipoxygenase activity in intact lymphoblastoid B cells but that the mechanism of this activation is unclear and appears not to be directly related to intracellular glutathione levels.

Recombinant human 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) was expressed in Escherichia coli. In incubations of E. coli supernatants with arachidonic acid, 5-hydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene A4 were formed, while incubation with 8,11,14-eicosatrienoic acid gave 8-hydroxy-9,11,14-eicosatrienoic acid. Six conserved histidine residues in 5-lipoxygenase were subjected to site-directed mutagenesis. Exchanges of His-367, -372, or -551 gave mutants for which no enzyme activities were detectable. On the other hand, exchanges of His-362, -390, or -399 gave mutants that were enzymatically active, but less so than the nonmutated control. For two of these (exchanges of His-390 or -399), the activities of the mutants were dependent on the expression temperature. Thus, the histidines in the first group (His-367, -372, -551) were crucial for 5-lipoxygenase activity, possibly because of a function of these residues as metal ligands. Mutagenesis aimed at two other conserved elements in 5-lipoxygenase, Gln-558 and the C terminus, gave mutated proteins with only a small residual activity (substitution of Gln-558), or with no detectable activity (deletion of six C-terminal amino acids), indicating that these regions are important for the function of 5-lipoxygenase.

Background: The anthracyclin adriamycin (ADR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. However, this antitumor agent appears not to be successful in triggering apoptotic cell death in prostate cancer cell lines with high metastatic potential. In this study we investigated the effect of ADR on the 5-lipoxygenase (5-LO) pathway and examined whether the activation of 5-LO, the key determinant in biosynthesis of proinflammatory leukotrienes (LTs), is involved in development of prostate cancer cell resistance. Effects of ADR on the activation of the 5-LO pathway were also studied in regard to p53 status and expression levels of pro- and anti-apoptotic members of the Bcl-2 family in two human, prostate cancer cell lines, DU145 and PC3 (both derived from metastases). Methods: Enzymatic activity and modulations of the 5-LO product profile were analyzed by high pressure liquid chromatography. The relationship between enzymes of the 5-LO pathway and members of Bcl-2 family, before and upon the ADR treatment, were investigated by Western blot analysis and Q-PCR. Cytotoxic effect of ADR was assessed by MTT, while apoptosis was determined by caspase activation. Results: In DU145 and PC3 cells, clinically relevant concentrations of ADR were not found to induce apoptosis. Interestingly, after 24h of ADR treatment the expression of 5-LO was strongly up-regulated at the mRNA and protein levels in both cell lines, independently of their p53 status. This consequently resulted in a high ionophore-induced formation of 5-hydroxyeicosatetraenoic acid (5-HETE). The production of LTB4 was also increased, particularly in DU145 cells, although ADR treatment did not alter expression of LTA4 hydrolase. In DU145 cells ADR furthermore induced overexpression of coactosin-like protein (CLP), a small cytoplasmic 5-LO chaperone, which functions as a scaffold during Ca2+-induced 5-LO activation and up-regulates LTA4 formation in in vitro assays. In PC3 cells, ADR did not alter relatively high constitutive expression of CLP, which appeared to be critical for LTB4 production in this cell line, since CLP knock-down by short hairpin RNA completely abolished it. Increased formation of the 5-LO products observed after 24h treatment with ADR resulted in resistance to apoptosis. No changes in Bax and Bcl-2 expression levels were observed upon genotoxic stress induction. Pre-incubation of the cells with the 5-LO inhibitors, BW-4-AC and particularly Hyperforin (a natural compound isolated from the plant Hypericum perforatum (St John's wort) in clinical use) led to inhibition of 5-LO products' formation and resulted in an extensive programmed cell death engaging caspase-3 activation. Conclusions: Together, these results support a novel role of the 5-LO products in cancer cell chemo-resistance, and provide a rationale for the therapeutic use of 5-LO inhibitors to enhance cancer chemosensitivity. This may lead to development of novel pharmacological approaches for combating prostate cancer. Citation Information: Cancer Res 2009;69(23 Suppl):A49.

Two different proteins with arachidonate 15-lipoxygenase activity have been purified to near homogeneity from human leukocytes. Both have the same molecular mass (74 kDa) on SDS/PAGE and appear to be equally active with three different fatty acid substrates. The N-terminal amino acid sequences of both forms were identical to the sequence of human reticulocyte 15-lipoxygenase [Sigal, E., Criak, C. S., Highland, E., Grunberger, D., Costello, L. L., Dixon, R. A. F. & Nadel, J. A. (1988) Biochem. Biophys. Res. Commun. 157 457–464]. The two forms of 15-lipoxygenase could be clearly separated by cation-exchange chromatography. Of particular interest, the relative amounts of the two forms differed markedly between leukocytes obtained from donors and leukocyltes from an individual with eosinophilia.

Recombinant mouse leukotriene A 4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants ( K m and V max ) for the conversion of leukotriene A 4 to leukotriene B 4 (at 0°C, pH 8) were 5 μM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. K m and V max for this reaction (at 37°C, pH 8) were 680 μM and 365 nmol/mg per min, respectively. Apo-leukotriene A 4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A 4 resulted in a dose-dependent inactivation of both enzyme activities.

Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained significant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed in E. coli. The LTA4 hydrolase activities of the total soluble proteins produced in these expressions were less than 10% of that obtained for bacteria harboring nonmutated cDNA. In agreement with earlier predictions, our experimental data demonstrate that His-295, His-299, and Glu-318 constitute the three ligands of the intrinsic zinc atom in LTA4 hydrolase. Additionally, the combined loss of enzyme activities and zinc content in the purified mutated mouse proteins, emphasizes the critical role of the zinc atom for catalysis, whereas the virtually identical chromatographic behaviors of the mutated and nonmutated mouse LTA4 hydrolase proteins suggest that the metal is of limited importance for the maintenance of the enzyme tertiary structure.

Purified leukotriene A4 hydrolase from human leukocytes is shown to exhibit peptidase activity towards the synthetic substrates alanine-4-nitroanilide and leucine-4-nitroanilide. The enzymatic activity is abolished after heat treatment (70 degrees C, 30 min). At 37 degrees C these substrates are hydrolyzed at a rate of 380 and 130 nmol/mg/min, respectively, and there is no enzyme inhibition during catalysis. Apo-leukotriene A4 hydrolase, obtained by removal of the intrinsic zinc atom, exhibits only a low peptidase activity which can be restored by the addition of stoichiometric amounts of zinc. Reconstitution of the apoenzyme with cobalt results in a peptidase activity which exceeds that of enzyme reactivated with zinc. Preincubation of the native enzyme with leukotriene A4 reduces the peptidase activity. Semipurified preparations of bovine intestinal aminopeptidase and porcine kidney aminopeptidase do not hydrolyze leukotriene A4 into leukotriene B4.

Publisher Summary The chapter presents a study on cloning of leukotriene A 4 hydrolase cDNA. Leukotriene A 4 hydrolase (EC 3.3.2.6) is a soluble epoxide hydrolase, which catalyzes the enzymatic hydrolysis of leukotriene A4 (LTA4) to the chemotactic agent leukotriene B 4 . After initial purification and characterization of the enzyme, it was determined to be different from other epoxide hydrolases, and further studies were initiated. The cloning of LTA 4 hydrolase was carried out successfully using two different strategies, they are, screening of λgt11 cDNA libraries (which express recombinant protein) with polyclonal antiserum, and screening of a λgtl0 cDNA library with a single isomer oligonucleotide probe (48-mer) whose structure was based on mammalian codon usage frequencies. Prior to this, screening of plasmid (pBR322) cDNA libraries with mixed shorter oligonucleotide probes (16-mers) was unsuccessful. Thus, the use of better quality cDNA synthesizes, according to the RNase H method in λgt10/11 vectors, as well as the use of more efficient probes were beneficial. In the procedure for screening with 48-mer, a 48-mer oligonucleotide was designed from the amino acid sequence of a cyanogen bromide fragment of LTA hydrolase, according to the mammalian codon usage frequencies.

Publisher Summary The chapter presents a study on isolation and characterization of leukocyte arachidonate 5-lipoxygenase. The chapter discusses the method of assay stating that there are a number of issues that complicate the assay of leukocyte arachidonate 5-1ipoxygenase. The purification procedure described in the chapter was developed for the 5- lipoxygenase from human peripheral blood leukocytes. Unlike the 5-1ipoxygenase of rat and pig, purification of the human leukocyte 5-1ipoxygenase has revealed a number of cellular fractions that stimulate 5-1ipoxygenase product formation in the routine high-performance liquid chromatography (HPLC) assay. In regard to the properties of 5-lipoxygenase, because of limited availability and poor stability, very few detailed kinetic studies have been performed with purified leukocyte 5-1ipoxygenase. Investigations performed with partially purified samples suggest a deviation from Michaelis-Menten behavior, with substrate inhibition observed. As has been shown for the lipoxygenase from soybeans, leukocyte 5- lipoxygenase displays a kinetic lag phase that is eliminated by fatty acyl hydroperoxides, and exacerbated by reducing agents, such as sulfhydryl compounds. This suggests that, like the soybean enzyme, leukocyte 5-1ipoxygenase may undergo a product-mediated oxidative activation step; however, the chemical mechanism of such a step remains to be elucidated.

Publisher Summary The chapter describes the purification and characterization of potato lipoxygenase, and details procedures for the syntheses of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5( S ), 12( S )-diHETE. Synthesis of biologically active leukotrienes (LTs) is initiated by 5-lipoxygenation of arachidonic acid to yield 5( S )-hydroperoxy-6- trans -8, 11, 14- cis -eicosatetraenoic acid (5-HPETE), which is further transformed by dehydration to LTA 4 , a key intermediate in the formation of LTs. It is mentioned that enzyme activity varies significantly from potato to potato. Some lots of potatoes have no detectable enzyme activity, presumably because of storage conditions (duration and temperature, radiation, and so on.) rather than to species differences. Before starting purification of the enzyme, or preparation of 5-HPETE, first the enzyme activity should be checked using the ammonium sulfate fraction. The use of fresh potatoes obtained directly from farms provides the most successful results. Like other lipoxygenases, the potato lipoxygenase activity can be determined by the following four assay methods: (1) Spectrophotometry, (2) oxygen monitoring, (3) thin-layer chromatography (TLC), and (4) high-performance liquid chromatography (HPLC). The chapter summarizes the characteristics of each method.

Publisher Summary The chapter presents a study on molecular biology and cloning of archidonate 5-lipoxygenase. Arachidonate 5-1ipoxygenase (Ecl. 13.11.34) is a complex enzyme requiring calcium, ATP, and various unknown cellular stimulatory factors or maximal conversion of arachidonic acid (20:4) to 5-hydroperoxy-6,8,10,14-eicosatetraenoic acid (5-HPETE) and its subsequent metabolite 5, 6-oxido-7,9,11,14-eicosatetraenoic acid [leukotriene A4 (LTA4)]. The enzyme has been isolated from human leukocytes and has been extensively characterized. To further enhance the knowledge of this enzyme and the regulation of its synthesis, this chapter describes the isolation and characterization of 5-1ipoxygenase cDNA clones and a method for overexpressing the enzyme in a baculovirus-insect cell system. The chapter outlines the strategy for obtaining 5-1ipoxygenase cDNA clones, which can essentially be used for cloning the cDNA for any protein that lends itself to relatively straightforward purification. 5-Lipoxygenase was purified from human leukocytes as described. The chapter mentions a protocol identical to that used for isolation of LTA4 hydrolase cDNA clones. High-level expression of many foreign genes has been achieved using a baculovirus/insect cell system. The chapter discusses the antibody selection assay and DNA sequence analysis in the process of clone characterization.

Inhibitors of leukotriene A4 hydrolase are disclosed, as are processes for making and using the same. A contemplated inhibitor has a structure corresponding to formula (I), wherein the depicted -NH2 group is in the (S) configuration; -W is -CH2SH, -CH2NH2 or C(=Z)-Y, wherein =Z is =O, or -H and -OH; and -Y is selected from the group consisting of (a) phenyl, (b) trifluoromethylphenyl, (c) carboxyphenyl, (d) benzyl, (e) C1-C6 alkylenecarboxyl, (f) C1-C6 alkyl, (g) C2-C6 alkenyl, (h) C1-C6 alkylenephenyl and (i) -C(=O)-X-R1 wherein X is O or NH and, R1 is selected from the group consisting of C?1?-C6 alkyl, C1-C6 alkylenecarboxyl, and benzyl, with the proviso that -Y is other than (i) when =Z is -H and -OH; and R?2? is hydrogen, benzyloxy or 2-naphthylmethyloxy, and a pharmaceutically acceptable acid addition salt thereof.

Maximal activity of human leukocyte 5-lipoxygenase requires Ca2+, ATP, a microsomal membrane preparation, and two cytosolic stimulatory factors. We report here some effects of Ca2+ on the physical properties of the 5-lipoxygenase. When leukocytes were homogenized in the presence of 2 mM EDTA, 5-lipoxygenase was found to be a soluble enzyme. However, when Ca2+ was added to homogenization buffers at 0-1 mM in excess of EDTA, increasing quantities of the enzyme were recovered in the microsomal membrane fraction (100,000 X g pellet). The membrane-associated enzyme was resolubilized by washing pellet preparations in buffers containing 2 mM EDTA and was partially purified by anion-exchange chromatography. Studies of the stimulatory-factor requirements of the membrane-associated, resolubilized, and partially purified enzyme indicated that one of the cytosolic 5-lipoxygenase stimulatory factors exhibited a reversible, Ca2+-dependent membrane association, similar to that of the enzyme itself. Ca2+ also caused a destabilization of the 5-lipoxygenase. Homogenates prepared in the presence of Ca2+ contained lower total enzyme activity, and retention of activity in these samples over time was also diminished.

The metabolism of arachidonic acid by washed human platelets was investigated. [1-14C]Arachidonic acid was extensively converted to [1-14C]12-hydroxyeicosatetraenoic acid. In addition, several minor labeled products were formed with a considerably lower specific activity, indicating their preferential formation from endogenous substrate. These were dihydroxy metabolites of arachidonic acid with conjugated triene structures and were identified as 14,15-dihydroxyeicosatetraenoic acid (three isomers) and 8,15-dihydroxyeicosatetraenoic acid (three isomers). The identification was based on comparison with reference compounds with respect to chromatographic properties, characteristic UV spectra, and mass spectrometry of several derivatives. Bradykinin (10(-8)-10(-5) M) was found to enhance the formation of all these compounds. In addition, the monohydroxy acid fraction was found to contain 15-hydroxyeicosatetraenoic acid. The present investigation thus demonstrates the occurrence of a 15-lipoxygenase in human platelets in addition to the previously known 12-lipoxygenase.

A nomenclature for thromboxanes is proposed in which the parent unsubstituted compound is thrombane and the corresponding carboxylic acid is thrombanoic acid.

The synthesis of tetradeuterated 15-methyl-PGF2α and 17-phenyl-18, 19, 20-trinor-PGF2α, is described. Gas chromatographic -mass spectrometric methods, using the deuterated compounds as internal standards and carriers, for quantitation of corresponding unlabeled prostaglandin analogues is described. With acceptable accuracy and precision the sensitivity is in the subpicomole range.

and adhesion to post- capillary venules [3]. In [4-61 LTB., was suggested to be a weak stimulator of polymorphonuclear leuko- cyte (PMNL) degranulation. However, it is now clear that previous purification procedures for biosyntheti- tally prepared LTB4 were inadequate. The newly discovered LTB4 isomer, SQ,12Q-DHETE, co-chro- matographs with LTB4 in the reverse phase high- pressure liquid chromatography (HPLC) systems commonly used [7]. As a consequence, earlier studies probably tested preparations which contained a mix- ture of both isomers with the non-leukotriene product predominating. Therefore, we have investigated the role of LTB4, SQ,12(S)-DHETE and their o-oxida- tion products (fig.1) [8] in leukocyte degranulation in vitro. In addition, the activity of the non-enzymati- tally formed LTB4 isomers was investigated. Cytochalasin B, phenolphthalein glucuronic acid, and dried

The following labeled compounds were isolated and identified after incubation of 8,11,14-eicosatrien [1-14C] oic acid with human platelets: 12-L-hydroxy-8,10,14-eicosatrienoic acid, 8,11,12-trihydroxy-9,14-eicosadienoic acid, 8,9,12-trihydroxy-10,14-eicosadienoic acid, 12-L-hydroxy-8,10-heptadecadienoic acid, prostaglandin E1, prostaglandin D1, and 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-10-heptadecenoic acid (thromboxane B1).

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)- S -glutathionyl-7,9,11- trans -14- cis -eicosatetraenoic acid (11- trans leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.

Leukotriene C, a “Slow Reacting Substance” (SRS) from mouse mast cell tumors, was earlier shown to be a derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid with a cysteine containing substituent in thioether linkage at C-6 (Murphy, R.C., Hammarstrom, S., Samuelsson, B.: Proc. Natl. Acad. Sci. USA, 76 , 4275–4279 (1979)). The substituent has now been identified as γ-glutamylcysteinylglycine (glutathione).

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 acid) activated the kinase in the presence of calcium at 30 fold lower concentration (1 μM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B 4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C 4 , 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.

A preparation of human leukocytes was incubated with arachidonic acid. Two new dihydroxy acids with conjugated triene structures, were isolated and characterized as 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-leukotriene B4) and 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene B4).

The prostaglandin endoperoxide prostaglandin H 2 (15-hydroxy-9α,11α-peroxidoprosta-5,13-dienoic acid) inhibits basal and hormone-stimulated adenylate cyclase in fat cell ghosts. This inhibition by prostaglandin H 2 has been found to be antagonized by GTP and Gpp(NH)p. Dose response studies have shown GTP and Gpp(NH)p to be maximally effective at 3.3 μM, the lowest concentration tested. Although the system is exceedingly sensitive to modulation by GTP or Gpp(NH)p, UTP, CTP, GMP, and cyclic GMP did not antagonize the antihormone activity of prostaglandin H 2 . Kinetic studies indicate that the GTP or Gpp(NH)p antagonism of prostaglandin H 2 is observable on initial rates of cyclic AMP synthesis, and persists throughout the adenylate cyclase measurements. Preincubation of fat cell ghosts with GTP followed by washing and resuspension results in a prostaglandin H 2 -sensitive adenylate cyclase system. However, the same preincubation experiment with Gpp(NH)p produces an irreversible antagonism of the prostaglandin H 2 inhibition of hormone-stimulated adenylate cyclase. It is suggested that prostaglandin H 2 stabilizes that fat cell adenylate cyclase system in a state that is resistant to hormone stimulation, and GTP or Gpp(NH)p overcome this stabilization.

Binding sites for [3H]LTC4 were observed in crude membrane preparations of rat central nervous system tissue. Equilibrium binding studies indicated one high affinity [3H]LTC4 binding site with a KD of 31.4 +/- 3.4 nM for whole brain preparations. The binding was highly specific for [3H]LTC4 and could be inhibited by the SRS-A antagonist FPL 55172. Specific binding was increased with both mono- and di-valent ions. Regional distribution studies revealed a three-fold difference in binding capacity within different regions of the brain with the highest binding capacity in the brainstem (94.1 +/- 6.9 fmol/mg of protein) and the lowest in the hypothalamus (29.6 +/- 12.8 fmol/mg of protein). In addition, weak low capacity binding was observed for [3H]LTB4 and [3H]LTE4, while no saturable binding was observed for [3H]LTD4. The order of selectivity in inhibiting [3H]LTC4 binding was LTC4 much greater than LTD4 = LTE4 greater than LTB4.

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B4(LTB4). 6- trans -LTB4, 12- epi -6- trans -LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB4 but not 5-HETE formation was impaired. (1-14C) Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate. (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.

The human leukocyte 5-lipoxygenase is a unique enzyme in its class because of its involvement in the synthesis of the biologically active leukotrienes. Furthermore, unlike most other lipoxygenases, this enzyme requires multiple stimulatory factors for maximal activity. These include Ca2+, ATP, and three non-dialyzable cellular components, two cytosolic, and one membrane-associated. The mechanism of action of these factors is not yet well understood; however, a Ca2+-dependent association of the enzyme and one of the cytosolic factors with the membrane has been demonstrated. These findings suggest that stimulation of the leukocyte, resulting in an increased intracellular Ca2+ concentration, may result in the translocation of the enzyme and the factor to a membrane site, thereby facilitating the interaction of these two proteins with other enzymes involved in the 20:4 metabolic cascade. The development of a better understanding of these processes should not only help to define the biochemical basis for the regulation of leukotriene formation, but should also yield valuable information concerning the more general aspects of stimulus-response coupling in the leukocyte.