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B Samuelsson

Publications and source records attributed to B Samuelsson.

At least 73 records · Page 4Linked to original sources
PubMed1995

Mutations at the C-terminal isoleucine and other potential iron ligands of 5-lipoxygenase.

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.

Arachidonate 5-Lipoxygenase↗
PubMed1995

Chemical modification of leukotriene A4 hydrolase. Indications for essential tyrosyl and arginyl residues at the active site.

We have employed chemical modification to identify amino acids essential for the catalytic activities of the bifunctional zinc metalloenzyme leukotriene A4 hydrolase (EC 3.3.2.6). The epoxide hydrolase and the peptidase activity were both rapidly inactivated by N-acetylimidazole and tetranitromethane. Furthermore, treatment with 2,3-butanedione and phenylglyoxal also resulted in loss of both activities. Leukotriene A4 hydrolase could be protected from inactivation by these tyrosyl and arginyl reagents by the competitive inhibitors bestatin and captopril, respectively. Two tyrosyl and three arginyl residues were found by differential labeling techniques to be protected by the inhibitors, which thus suggested that these amino acids are located close to or at the active center of the enzyme. Limited modification by thiol reagents and particularly methyl methanethiosulfonate led to a > 10-fold increase in the peptidase activity and a decreased epoxide hydrolase activity, whereas prolonged treatment inhibited both activities. Kinetic analysis of modified enzyme, using the substrate alanine p-nitroanilide, revealed that the stimulatory effect on the peptidase activity was due to increased enzyme displayed a reduced apparent affinity constant for chloride ions, which strongly stimulate the peptidase activity. Neither activation nor inactivation by methyl methanethiosulfonate was influenced by the presence of competitive inhibitors, which suggested that this compound did not react with amino acids at the active center but rather with residues of importance for the overall enzyme conformation.

Arginine↗
PubMed1995

Sequential induction of 5-lipoxygenase gene expression and activity in Mono Mac 6 cells by transforming growth factor beta and 1,25-dihydroxyvitamin D3.

5-Lipoxygenase (5-LO; EC 1.13.11.34) activity in the human monocytic cell line Mono Mac 6 was upregulated by combined treatment with transforming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D3 (VD3). In undifferentiated cells, 5-LO enzyme activity was undetectable. After the addition of TGF-beta plus VD3, the activity of intact cells was 800 ng per 10(6) cells--500 times more than the assay detection limit. Also 5-LO protein and mRNA expression were induced > 128-fold and 64-fold, respectively, as compared to undifferentiated cells. Both TGF-beta and VD3 were required for these prominent responses. Either agent alone gave small amounts of 5-LO protein and mRNA but very low 5-LO activities. After the addition of TGF-beta and VD3, the induction of 5-LO protein was obvious after 1 day, but the increase in activity was delayed and did not appear until the second day. Pretreatment of cells with TGF-beta or VD3 alone for 2 days led to 5-LO protein expression but very low enzyme activity. Addition of the lacking second inducer was required for full induction of 5-LO protein expression and for upregulation of enzyme activity. Partial purification of 5-LO from Mono Mac 6 cells and recombination with soluble cellular proteins from different sources indicated the presence of cytosolic factors that affect the activity of 5-LO.

Arachidonate 5-Lipoxygenase↗
PubMed1995

Investigation of the inhibition of leukotriene A4 hydrolase.

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.

Computer Graphics↗
PubMed1995

Amino hydroxamic acids as potent inhibitors of leukotriene A4 hydrolase.

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).

Binding Sites↗
PubMed1995

Lewis histo-blood group system and associated secretory phenotypes.

This review summarises present knowledge of the chemistry, immunology, genetics and clinical significance of antibodies in the Lewis and secretor histo-blood group systems. Although red cell serology has laid the foundations for these systems, more recent advances have been made by studying Lewis and related glycoconjugates with monoclonal antibodies, determining structures by mass spectrometry and NMR spectroscopy, identifying enzymes and their specificities, and identifying the genes by molecular biology. The expression of Lewis system antigens is dependent on Lewis and secretor loci. Fucosyltransferases coded by genes at these loci compete and interact with each other and with other transferases to determine an individual's Lewis and secretor phenotype. Exocrine epithelial cells, mostly of endodermal origin, synthesise the Lewis antigens which, as plasma glycolipids, are secondarily acquired by cells of the peripheral circulation. Phenotyping red cells is often regarded as a simple way of determining the Lewis and sometimes the secretor status of an individual; however, the red cell phenotype is influenced by many factors and may not necessarily reflect someone's Lewis and secretor genotypes. Two main red cell Lewis groups are usually found, Lewis negative and Lewis positive. In Lewis-negative individuals, the secretor genotype does not affect the Lewis phenotype, but in Lewis-positive individuals, the non-secretor genotype generates the Le(a+b-) phenotype, the secretor genotype causes the Le(a-b+) phenotype, and the partial secretor genotype gives rise to the Le(a+b+) phenotype.

Carbohydrate Sequence↗
PubMed1995

Potent and selective inhibitors of leukotriene A4 hydrolase: effects on purified enzyme and human polymorphonuclear leukocytes.

Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into the proinflammatory substance LTB4 and also exhibits an amidase/peptidase activity toward synthetic substrates. Based on proposed reaction mechanisms for other zinc hydrolases, we have synthesized inhibitors of LTA4 hydrolase and evaluated their effects on the formation of LTB4 from LTA4 using both purified enzyme and intact polymorphonuclear leukocytes. The two most effective inhibitors, an alpha-keto-beta-amino ester (compound IV) and a thioamine (compound VIII), exhibited IC50 values of 1.9 +/- 0.9 and 0.19 +/- 0.12 microM (mean +/- SD, n = 4), respectively. Compounds IV and VIII were also potent inhibitors of LTB4 biosynthesis in ionophore stimulated polymorphonuclear leukocytes with IC50 < 200 nM. At higher concentrations, the biosynthesis of 5-hydroxy-eicosatetraenoic acid was also inhibited with IC50 approximately 10 microM for both substances. In contrast, leukocyte 15-lipoxygenase and platelet LTC4 synthase activity were not inhibited by these substances at the highest concentrations tested, 50 and 10 microM, respectively. Compounds IV and VIII thus exhibit selectivity among enzyme activities in the arachidonic acid cascade. In conclusion, we describe two compounds that are among the most potent and selective inhibitors of LTA4 hydrolase and LTB4 biosynthesis by intact polymorphonuclear leukocytes, described thus far.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1994

On the induction of 5-lipoxygenase expression and activity in HL-60 cells: effects of vitamin D3, retinoic acid, DMSO and TGF beta.

Induction of 5-LO activity in DMSO differentiated HL-60 cells, by the serum protein TGF beta, was partially dependent on the presence of serum lipids (Steinhilber, D., Hoshiko, S., Grunewald, J., Rådmark, O., and Samuelsson, B. (1993) Biochim. Biophys. Acta 1178, 1-8). Here we demonstrate that the serum lipid fraction can be substituted by picomolar concentrations of 1,25-dihydroxyvitamin D3 (VD3). A high concentration of VD3 (24 nM) gave a 4-fold induction of 5-LO mRNA, a 14-fold increase in 5-LO protein, and a 38-fold upregulation of the 5-LO activity of intact HL-60 cells after differentiation in the presence of DMSO and serum proteins for 4 days. Also VD3 alone gave a substantial upregulation of 5-LO protein expression and activity. On the other hand, TGF beta alone was a poor inducer of the 5-LO pathway, the presence of a differentiation inducer (retinoic acid, DMSO or VD3) was required. The most prominent induction of 5-LO protein expression and activity in cell homogenates and intact cells was observed when VD3 and TGF beta were combined.

Arachidonate 5-Lipoxygenase↗
PubMed1994

Stabilization of purified human 5-lipoxygenase with glutathione peroxidase and superoxide dismutase.

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.

Arachidonate 5-Lipoxygenase↗
PubMed1994

Novel structural and functional properties of leukotriene A4 hydrolase. Implications for the development of enzyme inhibitors.

Recent work in our laboratory, some of which is described in this report, has established that LTA4 hydrolase is a bifunctional metalloenzyme that contains one zinc atom, essential for both catalytic activities. The well-characterized epoxide hydrolase activity, i.e., the conversion of LTA4 into LTB4 is inhibited by exposure to LTA4, and this irreversible enzyme inactivation also affects the peptidase activity. In contrast, the peptide hydrolysis proceeds without any signs of enzyme inactivation, can be stimulated by physiologic concentrations of chloride ions, and is critically dependent on the presence of a Glu residue in position 296 of the protein. A model of the active center, which summarizes these novel structural and functional properties of LTA4 hydrolase, is presented in Fig. 6.

Amino Acid Sequence↗
PubMed1993

Serum factors regulate 5-lipoxygenase activity in maturating HL60 cells.

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 microM). 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.

Arachidonate 5-Lipoxygenase↗
PubMed1993

Transforming growth factor beta upregulates 5-lipoxygenase activity during myeloid cell maturation.

Transforming growth factor beta (TGF beta) increased the arachidonate 5-lipoxygenase (5-LO; EC 1.13.11.34) activity in HL-60 cells induced to granulocytic differentiation by dimethyl sulfoxide. The presence of a factor in human serum that caused a similar increase was recently demonstrated. Several observations indicate that the serum factor consists of isoforms of TGF beta. Heat-treated serum and TGF beta both resulted in approximately 10-fold increased 5-LO activity of HL-60 cells, antiserum to TGF beta neutralized the 5-LO-increasing activity in serum, and physical properties of the serum factor (lipophilic nature, alkaline pI, stability to heat and acid) coincided with those of TGF beta. The pattern of activity of native and heat-treated sera is compatible with activation of a latent form of TGF beta in serum. This activity was specific for TGF beta, since none of several other cytokines could increase 5-LO activity in differentiating HL-60 cells. However, granulocyte/macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha enhanced the effect of TGF beta. The most prominent effects of TGF beta, whether alone or together with GM-CSF, were observed for 5-LO activity in intact cells (10-fold or 30-fold induction, respectively). 5-LO protein levels were less affected (up to 2- or 5-fold, respectively, as judged from Western blots). There was no appreciable effect of TGF beta, or a combination of TGF beta and GM-CSF, on 5-LO mRNA expression.

5-Lipoxygenase-Activating Proteins↗
PubMed1993

A heat stable serum factor upregulates 5-lipoxygenase activity in HL-60 cells, modulation by TNF alpha or GM-CSF.

5-Lipoxygenase (5-LO) activity in differentiating HL-60 cells was upregulated by a heat stable protein in human serum. Hematopoietic cytokines were evaluated for this effect on 5-lipoxygenase activity, none of the compounds tested could replace serum. However, TNF alpha or GM-CSF were able to augment the effect of heat treated serum, giving 5-LO activities that (per cell) were higher than for granulocytes isolated from peripheral blood. The serum factor primarily upregulated 5-LO activity. The amounts of 5-LO protein, or mRNA for 5-LO or 5-lipoxygenase activating protein (FLAP), were less affected.

5-Lipoxygenase-Activating Proteins↗