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O Rådmark

Publications and source records attributed to O Rådmark.

At least 73 records · Page 4Linked to original sources
PubMed1989

Formation and effects of leukotriene B4 in human lymphocytes.

Incubation of human tonsillar B lymphocytes or peripheral blood T lymphocytes with leukotriene (LT) A4 led to the formation of LTB4. Stimulation of these cells with ionophore A23187 did not lead to the synthesis of detectable amounts of leukotrienes. Formation of LTB4 was observed in several monoclonal B- and T-cell lines after incubation with LTA4, but not after stimulation with ionophore A23187. The Burkitt lymphoma cell line Raji was found to possess higher LTA4-hydrolase activity than normal lymphocytes. The expression of the LTA4-hydrolase gene but not the 5-lipoxygenase gene was demonstrated on the transcriptional level in Northern blots and on the translational level by Western blots. Stimulation of human monocytes with ionophore A23187 resulted in the release of LTA4. Coincubations of transformed lymphocytes and monocytes stimulated with ionophore A23187 produced increased amounts of LTB4 as compared with monocytes alone. LTB4 influence on lymphocyte activation was studied and CD23 expression was used as a marker. The expression of this antigen was enhanced on resting B lymphocytes in synergy with B-cell growth-promoting factors. LTB4 also augmented DNA synthesis, cell replication and IgG secretion. These results indicate that extracellular LTA4, released from activated monocytes, is converted by lymphocytes into LTB4 which might cause activation and differentiation of B lymphocytes.

Antigens, CD↗
PubMed1988

Leukotriene A4: metabolism in different rat tissues.

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

Animals↗
PubMed1988

Guinea-pig liver leukotriene A4 hydrolase. Purification, characterization and structural properties.

Leukotriene A4 hydrolase from perfused guinea-pig liver was purified 1200-fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37 degrees C (27 microM and 68 mumol x mg-1 x min-1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at -10 degrees C, 0 degree C and +10 degrees C (Arrhenius plots). Physical properties including Mr (67,000-71,000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N-terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S,6R)-5,6-dihydroxy-7,9-trans-11,14-cis-icosatetraenoic acid.

Amino Acid Sequence↗
PubMed1988

14,15-Dihydroxy-5,8,10,12-eicosatetraenoic acid. Enzymatic formation from 14,15-leukotriene A4.

When 14C-labeled (14S, 15S)-14,15-trans-oxido-5,8-cis-10,12-trans-eicosatetraenoic acid (14,15-leukotriene A4) was incubated with cytosolic epoxide hydrolase purified from mouse liver, one major radiolabeled product appeared. The structure was assigned as (14R, 15S)-14,15-dihydroxy-5,8-cis-10,12-trans-eicosatetraenoic acid (14,15-DHETE), based on analytical data as well as enzyme mechanistic considerations. The formation of this compound was dependent on time and enzyme concentration and was abolished after heat treatment of the enzyme. The apparent Km and Vmax values at 37 degrees C were 11 microM and 900 nmol X mg-1 X min-1 respectively. This enzymatic hydrolysis of 14,15-leukotriene A4 represents an additional mode of formation for 14,15-DHETE, a compound previously found to modulate functions of human leukocytes.

Animals↗
PubMed1988

Enzymatic formation of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid: kinetics of the reaction and stereochemistry of the product.

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

Animals↗
PubMed1988

Leukotriene A4, conversion to leukotriene B4 in human T-cell lines.

Human T-cell lines (HSB, MOLT-4 and CCRF-CEM) produced leukotriene B4 when incubated with leukotriene A4. The product was characterized by chromatographic properties, UV-spectroscopy and gas chromatography mass spectrometry. About 10 pmol of leukotriene B4 was obtained per 10(6) cells. When incubated with arachidonic acid plus the calcium ionophore A23187 however, no leukotriene B4 was found, indicating that the T-cell lines lack 5-lipoxygenase yet contain LTA4 hydrolase.

Calcimycin↗
PubMed1988

Molecular cloning and amino acid sequence of human 5-lipoxygenase.

5-Lipoxygenase (EC 1.13.11.34), a Ca2+-and ATP-requiring enzyme, catalyzes the first two steps in the biosynthesis of the peptidoleukotrienes and the chemotactic factor leukotriene B4. A cDNA clone corresponding to 5-lipoxygenase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antibody. Additional clones from a human placenta lambda gt11 cDNA library were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. Sequence data obtained from several overlapping clones indicate that the composite cDNAs contain the complete coding region for the enzyme. From the deduced primary structure, 5-lipoxygenase encodes a 673 amino acid protein with a calculated molecular weight of 77,839. Direct analysis of the native protein and its proteolytic fragments confirmed the deduced composition, the amino-terminal amino acid sequence, and the structure of many internal segments. 5-Lipoxygenase has no apparent sequence homology with leukotriene A4 hydrolase or Ca2+ -binding proteins. RNA blot analysis indicated substantial amounts of an mRNA species of approximately equal to 2700 nucleotides in leukocytes, lung, and placenta.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of a cDNA coding for human leukotriene A4 hydrolase. Complete primary structure of an enzyme involved in eicosanoid synthesis.

We have isolated a near full-length cDNA encoding human leukotriene A4 hydrolase, which synthesizes a potent chemotactic and spasmogenic compound, leukotriene B4. A human spleen cDNA library was screened with a 48-mer oligonucleotide probe, synthesized according to the partial amino acid sequence of the human leukocyte enzyme. The nucleotide sequence of the cDNA had an open reading frame of 1,833 base pairs, which contained regions coding for the N-terminal amino acid sequence, the amino acid sequence for the probe design, and several other peptide sequences of the enzyme. The complete primary structure of the enzyme composed of 610 amino acid residues (molecular weight, 69,153) was deduced from the cDNA.

Amino Acid Sequence↗
PubMed1987

Enzymatic hydrolysis of leukotriene A4 into 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid and LTB4 by mammalian kidney.

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

Animals↗
PubMed1987

Molecular cloning and amino acid sequence of leukotriene A4 hydrolase.

A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region. From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140. No apparent homologies with microsomal epoxide hydrolases were found. RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes.

Amino Acid Sequence↗
PubMed1986

Leukotriene A4. Enzymatic conversion into 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid by mouse liver cytosolic epoxide hydrolase.

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

Animals↗
PubMed1986

A simultaneous quantitation of leukotriene B4 and its omega-oxidized products by gas chromatography-mass spectrometry.

We developed a highly sensitive and specific method for the simultaneous quantitation of leukotriene B4 (LTB4) and its omega-oxidized metabolites (20-hydroxy-LTB4 and 20-carboxy-LTB4) by mass fragmentography using deuterated compounds as internal standards. The ions produced by the cleavage of the C12-13 bond of the methyl ester dimethylisopropylsilyl ether derivatives of LTB4 and its metabolites were measured by selective ion monitorings. The detection limit of LTB4 was less than 10 pg and about 100-fold lower than that by high performance liquid chromatography. By using this method, the synthesis and further metabolism of LTB4 in human polymorphonuclear leukocytes were investigated.

Gas Chromatography-Mass Spectrometry↗
PubMed1986

Characterization of leukotriene A4 synthase from murine mast cells: evidence for its identity to arachidonate 5-lipoxygenase.

Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was identified by demonstration of two epimers of 6-transleukotriene B4, methanol trapping, as well as further transformation to leukotriene B4 by leukotriene A4 hydrolase. Leukotriene A4 synthase stereospecifically eliminated the D-hydrogen at C-10 (pro-R) in the synthesis of leukotriene A4 when incubated with [10D-3H;3-(14)C]5-HPETE. The purified enzyme also exhibited 5-lipoxygenase activity toward arachidonic acid and 8-lipoxygenase activity towards 8,11,14-cisicosatrienoic acid. All of these activities required Ca2+ and ATP for their maximal velocities. The effects of heat treatment and of several lipoxygenase inhibitors on these enzyme activities as well as coelution in various chromatographic systems strongly suggest that lipoxygenase and leukotriene A4 synthase activities reside in the same enzyme molecule.

Animals↗
PubMed1985

Leukotriene A4-hydrolase activity in guinea pig and human liver.

Guinea pig and human liver homogenates transformed leukotriene A4 into leukotriene B4. In both species, the enzymatic activity was recovered in the 105000 X g supernatant, and it was found to be susceptible to heat treatment (56 degrees C, 1 h). Digestion with a proteolytic enzyme also resulted in loss of enzymatic activity. The formation of leukotriene B4 was pH-dependent, with an optimum between pH 7 and pH 8.5. In addition, two other organs from the guinea-pig, lungs and kidneys, contained leukotriene A4-hydrolase activity. The identity of leukotriene B4 was ascertained by high-performance liquid chromatography, ultraviolet spectrometry, gas chromatography-mass spectrometry and bioassay. We have recently demonstrated the presence of leukotriene A4-hydrolase activity in mammalian plasma (Fitzpatrick et al. (1983) Proc. Natl. Acad. Sci. USA 80, 5425-5429). The results of the present study suggest several possible origins of this plasma leukotriene A4 hydrolase.

Animals↗