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B Samuelsson

Publications and source records attributed to B Samuelsson.

At least 91 records · Page 5Linked to original sources
PubMed1993

Molecular cloning of a 12-lipoxygenase cDNA from rat brain.

A cDNA encoding an arachidonate 12-lipoxygenase from rat brain was obtained by polymerase chain reaction cloning. Primers specific for porcine leukocyte 12-lipoxygenase cDNA were used to isolate the initial polymerase-chain-reaction product (395 bp). The final sequence of the rat 12-lipoxygenase cDNA coding region (1989 bp) was verified by analysis of several separate polymerase-chain-reaction products. The open reading frame corresponded to a protein of 662 amino acid residues, with a calculated molecular mass of 75,305 Da. Also the rat 12-lipoxygenase contained the six conserved histidines, characteristic for all cloned lipoxygenases. It displayed the highest degree of identity to porcine leukocyte 12-lipoxygenase (71%) and to human 15-lipoxygenase (75%), with less resemblance to human platelet 12-lipoxygenase (59%) or rat leukocyte 5-lipoxygenase (41%). The recombinant enzyme was expressed in Escherichia coli and incubated with arachidonic acid. Primarily 12-lipoxygenase (but also some 15-lipoxygenase) enzyme activity was obtained. A part of the brain 12-lipoxygenase cDNA was used as probe in Northern blots. A 2.7-kb mRNA was more abundant in RNA from rat leukocytes, lung, and aorta, than in RNA from rat brain. Sequencing of parts of the corresponding cDNAs (from leukocytes and lung), and comparison to the brain 12-lipoxygenase sequence, indicated that these mRNAs from the different rat tissues were identical.

Amino Acid Sequence↗
PubMed1993

Iron content of human 5-lipoxygenase, effects of mutations regarding conserved histidine residues.

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.

Arachidonate 5-Lipoxygenase↗
PubMed1993

Development of selective tight-binding inhibitors of leukotriene A4 hydrolase.

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.

Amino Acids↗
PubMed1992

Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296.

The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be virtually inactive (less than or equal to 0.2% of unmutated enzyme). To serve as a positive control, we also replaced Ser-298 with an alanine residue, which resulted in a protein ([Ala298]LTA4 hydrolase) with catalytic properties almost indistinguishable from the wild-type enzyme. Substitution of Glu-296 by glutamine or alanine was also carried out with human LTA4 hydrolase, and the mutated human enzymes displayed specific activities similar to the corresponding mouse proteins. Zinc analyses of the purified mouse and human proteins confirmed that the mutations did not significantly influence their zinc content. In conclusion, the results of the present study indicate a direct catalytic role for Glu-296 in the peptidase reaction of LTA4 hydrolase, where it presumably acts as a base to polarize water, whereas its function, if any, is apparently not essential in the epoxide hydrolase reaction.

Amino Acid Sequence↗
PubMed1992

Characterization of human 12-lipoxygenase genes.

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.

Amino Acid Sequence↗
PubMed1992

On the expression and regulation of 5-lipoxygenase in human lymphocytes.

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.

5-Lipoxygenase-Activating Proteins↗
PubMed1992

Mutagenesis of some conserved residues in human 5-lipoxygenase: effects on enzyme activity.

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.

Arachidonate 5-Lipoxygenase↗
PubMed1991

Purification of two forms of arachidonate 15-lipoxygenase from human leukocytes.

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., Craik, 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 normal donors and leukocytes from an individual with eosinophilia.

Amino Acid Sequence↗
PubMed1991

Recombinant mouse leukotriene A4 hydrolase: a zinc metalloenzyme with dual enzymatic activities.

Recombinant mouse leukotriene A4 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 (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 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 A4 resulted in a dose-dependent inactivation of both enzyme activities.

Animals↗
PubMed1991

Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.

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.

Amino Acid Sequence↗
PubMed1991

Glycosylation of extracellular superoxide dismutase studied by high-performance liquid chromatography and mass spectrometry.

Extracellular superoxide dismutase, EC-SOD, the main superoxide dismutase in biological fluids, is known from its lectin binding to be a glycoprotein. We have characterized the glycosylation of recombinant EC-SOD. A tryptic digest of the protein contained only one glycosylated peptide. This peptide was specifically bound to lectins and stained by periodic acid-Schiff stain. Although appearing very large on size-exclusion chromatography, it was shown to be glycosylated at only one site, asparagine-89, by specific cleavage with glycanases followed by mass spectrometry of the resulting peptide. Based on the binding properties of the peptide to concanavalin A and lentil lectin and the elution profile of N-glycanase-treated glycopeptide on ion-exchange chromatography, the carbohydrate appears to be the complex biantennary type with a core fucose.

Amino Acids↗
PubMed1991

Synthesis of some 3',5'-dideoxy-5'-C-phosphonomethyl nucleosides.

Ammonium [1-(3',5',6'-trideoxy-beta-D-erythro-hexofuranosyl)thymine]-6'- phosphonate (1), ammonium 3',5'-dideoxycytidine-5'-C-methylphosphonate (2) and 3',5'-dideoxyadenosine-5'-C-methyl phosphonic acid (3) have been synthesized and tested for anti-HIV activity. The key steps involved an Arbuzov reaction between triethyl phosphite and 3,5,6-trideoxy-6-iodo-1,2-O-isopropylidene-alpha-D-erythro- hexofuranose (7), followed by condensation with the appropriate nucleoside bases. The substances 1, 2 and 3 have been tested in vitro against HIV.

Antiviral Agents↗
PubMed1991

Arachidonic acid metabolism: role in inflammation.

Arachidonic acid is oxygenated and further transformed into a variety of products which mediate or modulate inflammatory reactions. The cyclo-oxygenase pathway, which is inhibited by nonsteroidal anti-inflammatory drugs, produces vasodilator prostaglandins such as PGE2 and prostacyclin (PGI2) and also thromboxane A4, a potent platelet aggregating agent. Recent work has demonstrated the importance of products formed via various lipoxygenase-catalyzed reactions. Leukotrienes are formed by initial oxygenation at C-5 followed by further transformation to an unstable epoxide intermediate, leukotriene A4 (LTA4). The intermediate can be converted into LTB4 by hydrolysis and into LTC4 by conjugation with glutathione. The formation of the leukotriene, LTA4, is catalyzed by a 5-lipoxygenase possessing both lipoxygenase and LTA4-synthase activities. Unlike other lipoxygenases, this enzyme requires multiple stimulatory factors for maximal activity. These include Ca2+, ATP, and three non-dialyzable cellular components. The cDNAs for human 5-lipoxygenase and LTA4-hydrolase have recently been cloned and the amino acid sequences for the enzymes have been deduced. LTC4 and its metabolites, LTD4 and LTE4, increase vascular permeability and contract airway smooth muscle. Leukotriene B4 causes adherence of neutrophils to endothelial cells and is a potent chemotactic agent. It also stimulates release of lysosomal enzymes and generation of superoxide anion in neutrophils. Oxygenation of arachidonic acid at C-12 and C-15 generates products (5S, 12S-DHETE and 14,15-DHETE) which can modulate various neutrophil functions. A new group of arachidonic acid-derived products was recently discovered. These compounds (lipoxins), formed by oxygenation at C-5 and C-15 and additional reactions, contain a conjugated tetraene structure and three alcohol groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic Acids↗