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O Rådmark

Publications and source records attributed to O Rådmark.

At least 55 records · Page 3Linked to original sources
PubMed1991

Leukotriene A4 hydrolase in the human B-lymphocytic cell line Raji: indications of catalytically divergent forms of the enzyme.

Leukotriene A4 hydrolase was purified 1400-fold, with an approximate yield of 25%, to apparent homogeneity from the human B-lymphocytic cell line Raji. The purification included ammonium sulfate precipitations followed by anion exchange, hydrophobic interaction, and molecular exclusion fast protein liquid chromatography. Kinetic properties at 2 degrees C varied between different enzyme preparations. Two patterns were observed, one with a Km of about 12 microM and Vmax of about 1.1 mumol LTB4/mg protein/min which correlated well with the properties of the human leukocytic LTA4 hydrolase. In other enzyme preparations a higher catalytic activity was observed. These enzyme batches did not obey Michaelis-Menten kinetics but were compatible with a mixture of enzymatic species. Heat treatment (60 degrees C) led to a time-dependent decline in catalytic activity. However, certain enzyme preparations contained a subfraction of enzymatic activity which was more resistant to heat treatment, yielding a biphasic inactivation pattern. It is thus suggested, on the basis of the kinetic properties and the heat-inactivation pattern, that these enzyme preparations contained an addition form of LTA4 hydrolase.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and expression of mouse leukotriene A4 hydrolase cDNA.

A cDNA clone for mouse leukotriene A4 hydrolase encoding the full sequence of the enzyme was isolated from a mouse spleen lambda ZAP-II library. The identification was ascertained by expression of enzyme activity in Escherichia coli. The encoded protein has 610 amino acids and exhibits an extensive identity (93%) with the human leukotriene A4 hydrolase. A region spanning between residues 233 and 340, where the zinc binding site is located, was found to be perfectly conserved between the two species. We found six sites of polymorphism in the cDNA sequence of mouse LTA4 hydrolase, one of which leads to a difference in the encoded amino acid. The polymorphism of cDNA was confirmed by reverse transcription-PCR sequencing of mouse spleen total RNA, prepared as a mixture from ten different strains.

Amino Acid Sequence↗
PubMed1990

Human fibroblasts show expression of the leukotriene-A4-hydrolase gene, which is increased after simian-virus-40 transformation.

Human fibroblasts in cell culture converted the epoxide intermediate leukotriene A4 into the potent chemotaxin leukotriene B4. The identity of leukotriene B4 was ascertained by its mobility in reverse-phase high performance liquid chromatography, ultraviolet spectroscopy and gas chromatography/mass spectrometry. The presence of the enzyme responsible for the conversion (i.e. leukotriene A4 hydrolase), as well as the corresponding mRNA, were demonstrated by Western and Northern blot analyses. Leukotriene-A4-hydrolase enzyme activity, protein and mRNA were all enhanced (approximately threefold) in human fibroblasts that had been transformed by simian virus 40.

Cell Line↗
PubMed1990

Cloning of the cDNA for human 12-lipoxygenase.

A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.

Amino Acid Sequence↗
PubMed1990

Characterization of the human 5-lipoxygenase gene promoter.

Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase gene and determination of chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5'-flanking sequences up to 5.9 kilobase pairs 5' of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence (-179 to -56 from ATG), which includes five repeated GC boxes (the putative Sp1 binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment -212 to -88) revealed that the transcriptional factor Sp1 could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts.

Amino Acid Sequence↗
PubMed1989

Leukotriene A4 hydrolase: analysis of some human tissues by radioimmunoassay.

Leukotriene A4 hydrolase was quantitated by radioimmunoassay, in extracts from eight human tissues. The enzyme was detectable in all tissues, with the highest level (2.6 mg per g soluble protein) in leukocytes, followed by lung and liver. The polyclonal antiserum did not cross-react with cytosolic epoxide hydrolase purified from mouse or human liver. When incubated with leukotriene A4, formation of leukotriene B4 was evident in all tissues. Furthermore, enzymatic formation of (5S,6R)-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid from leukotriene A4, was found in extracts from liver, kidney and intestines.

Blotting, Western↗
PubMed1989

B-lymphocytic cell line Raji expresses the leukotriene A4 hydrolase gene but not the 5-lipoxygenase gene.

The expression of LTA hydrolase and 5-lipoxygenase genes was studied in Raji cells, a Burkitt lymphoma derived B-cell line. Northern and Western blot analyses clearly showed the expression, both at the transcriptional and translational level, of the LTA4 hydrolase gene in these cells. However, expression of the 5-lipoxygenase gene was undetectable. Thus, the genes coding for the two enzymes required for biosynthesis of leukotriene B4 from arachidonic acid, 5-lipoxygenase and LTA4 hydrolase, were differentially expressed in the Raji cells.

Arachidonate 5-Lipoxygenase↗