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H Kühn

Publications and source records attributed to H Kühn.

At least 55 records · Page 3Linked to original sources
PubMed1998

Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits.

In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis.

Amino Acid Sequence↗
PubMed1998

The iron ligand sphere geometry of mammalian 15-lipoxygenases.

We investigated the geometry of the iron ligand sphere of the native rabbit 15-lipoxygenase (15-LOX) by X-ray absorption spectroscopy using synchrotron radiation. The soybean LOX-1 was used as a reference compound because its iron ligand sphere is well characterized. For structural information the X-ray absorption spectra were evaluated using the Excurve Program (CCLRC Daresbury Laboratory, Warrington, U.K.). From the positions of the absorption edges and from the intensities of the 1s-3d pre-edge transition peaks a six-coordinate ferrous iron was concluded for the rabbit 15-LOX. Evaluation of the extended region of the absorption spectra suggested six nitrogen and/or oxygen atoms as direct iron ligands, and the following binding distances were determined (means+/-S.D.; estimated accuracy is +/-0.001nm for bond distances, on the basis of more than 22 X-ray absorption spectra): 0.213+/-0.001nm, 0.213+/-0. 001 nm, 0.236+/-0.001 nm, 0.293+/-0.001 nm, 0.189+/-0.001 nm and 0. 242+/-0.001. Lyophilization of the LOX altered the binding distances but did not destroy the octahedral iron ligand sphere. For construction of a structural model of the iron ligand sphere the binding distances extracted from the X-ray spectra were assigned to specific amino acids (His-360, -365, -540, -544 and the C-terminal Ile-662) by molecular modelling using the crystal coordinates of the soybean LOX-1 and of a rabbit 15-LOX-inhibitor complex.

Amino Acid Sequence↗
PubMed1998

Characterization of a methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves.

We found three methyl jasmonate-induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX-92, -98 and -100) [Feussner, I., Hause, B., Vörös, K., Parthier, B. & Wasternack, C. (1995) Plant J. 7, 949-957]. At least two of them (LOX-92 and LOX-100), were shown to be localized within chloroplasts of barley leaves. Here, we describe the isolation of a cDNA (3073 bp) coding for LOX-100, a protein of 936 amino acid residues and a molecular mass of 106 kDa. By sequence comparison this lipoxygenase could be identified as LOX2-type lipoxygenase and was therefore designated LOX2:Hv:1. The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13-LOX and arachidonate 15-LOX, respectively. The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid. The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h. Its expression was not affected by exogenous application of abscisic acid. Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2:Hv:1, suggesting a separate signalling pathway compared with other jasmonate-induced proteins of barley. The properties of LOX2:Hv:1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far.

Acetates↗
PubMed1998

Membrane translocation of 15-lipoxygenase in hematopoietic cells is calcium-dependent and activates the oxygenase activity of the enzyme.

Mammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 -treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.

Animals↗
PubMed1998

Intra- and extracellular expression of rabbit reticulocyte 15-lipoxygenase in the Baculovirus/insect cell system.

Rabbit reticulocyte 15-lipoxygenase was expressed as intracellular enzyme and as export protein in High Five cells. While intracellular expression in the baculovirus/insect cell system was already reported for various mammalian lipoxygenases, we have developed a strategy for secretion of this cytosolic enzyme using the signal sequence of human interleukin-2. Expression levels of 10 mg/liter (intracellular strategy) and 18 mg/liter (extracellular strategy) were obtained. The recombinant enzyme expressed as intracellular protein was purified to apparent homogeneity by anion-exchange chromatography on a Mono-Q column with an overall recovery of 80% enzyme activity. For the final enzyme preparation, a specific linoleic acid oxygenase activity of 16.7 micromol 13-hydroperoxyoctadeca-9,11-dieic acid formation mg-1 min-1 was determined, which corresponds to a molecular turnover number of 21 s-1. Similar turnover rates have been reported for the native rabbit 15-lipoxygenase. Extracellularly expressed recombinant 15-lipoxygenase exhibited a heterogeneity in anion-exchange chromatography. Three major peaks of 15-lipoxygenase activity were eluted from a Mono-Q column and the relative amounts of these isoforms varied from batch to batch of enzyme expression. One of the major isoenzymes which cochromatographed with the native 15-lipoxygenase was purified to homogeneity from the cell-free culture supernatant and exhibited a specific activity of 5.1 micromol 13-hydroperoxyoctadeca-9,11-dienoic acid formation mg-1 min-1 (turnover rate of 6.1 s-1). The recombinant enzyme species were characterized with respect to their protein-chemical and enzymatic properties and no differences to the native rabbit 15-lipoxygenase were detected.

Animals↗
PubMed1997

Co-oxidation of NADH and NADPH by a mammalian 15-lipoxygenase: inhibition of lipoxygenase activity at near-physiological NADH concentrations.

The purified 15-lipoxygenase from rabbit reticulocytes is capable of oxidizing NADH in the presence of linoleic acid and oxygen. This co-oxidation proceeds at a rate that amounts to approx. 7% of linoleic acid oxygenation rates. Although NADH inhibits the lipoxygenase reaction with linoleic acid as substrate (46% inhibition at 0.2 mM NADH), the reaction specificity of the enzyme was not altered since (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid was identified as the major reaction product. NADH oxidation was inhibited by NAD+ (uncompetitive with respect to linoleate and mixed/competitive with respect to NADH), and NADPH or NMNH could substitute for NADH with slightly different apparent Km values. NADH oxidation was enhanced at lower oxygen tension, but was completely prevented under anaerobic conditions. Computer-assisted modelling of 15-lipoxygenase/NADH interaction and sequence alignments of mammalian lipoxygenases with NADH-dependent enzymes suggested that there is no specific binding of the coenzyme at the putative fatty acid-binding site of lipoxygenases. These results suggest that NAD(P)H might be oxidized by a radical intermediate formed during the dioxygenase cycle of the lipoxygenase reaction but that NADH oxidation might not proceed at the active site of the enzyme. The mechanism and possible biological consequences of 15-lipoxygenase-catalysed NAD(P)H oxidation are discussed.

Animals↗
PubMed1997

Structural elucidation of oxygenated storage lipids in cucumber cotyledons. Implication of lipid body lipoxygenase in lipid mobilization during germination.

At early stages of germination, a special lipoxygenase is expressed in cotyledons of cucumber and several other plants. This enzyme is localized at the lipid storage organelles and oxygenates their storage triacylglycerols. We have isolated this lipid body lipoxygenase from cucumber seedlings and found that it is capable of oxygenating in vitro di- and trilinolein to the corresponding mono-, di-, and trihydroperoxy derivatives. To investigate the in vivo activity of this enzyme during germination, lipid bodies were isolated from cucumber seedlings at different stages of germination, and the triacylglycerols were analyzed for oxygenated derivatives by a combination of high pressure liquid chromatography, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy. We identified as major oxygenation products triacylglycerols that contained one, two, or three 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid residues. During germination, the amount of oxygenated lipids increased strongly, reaching a maximum after 72 h and declining afterward. The highly specific pattern of hydroperoxy lipids formed suggested the involvement of the lipid body lipoxygenase in their biosynthesis. These data suggest that this lipoxygenase may play an important role during the germination process of cucumber and other plants and support our previous hypothesis that the specific oxygenation of the storage lipids may initiate their mobilization as a carbon and energy source for the growing seedling.

Chromatography, High Pressure Liquid↗
PubMed1997

A kinetic model for the interaction of nitric oxide with a mammalian lipoxygenase.

Nitric oxide (NO) has been known for a long while to act as an inactivator of the soybean lipoxygenase-1 and cyclooxygenase. More recently, NO was shown to interact also with a mammalian 15-lipoxygenase [Wiesner, R., Rathmann, J., Holzhütter, H. G., Stosser, R., Mader, K., Nolting, H. & Kühn, H. (1996) Nitric oxide oxidises ferrous mammalian lipoxygenases to a pre-activated ferric species, FEBS Lett. 389, 229-232]. In this paper we present a detailed kinetic analysis of the interaction of NO with the 15-lipoxygenase from reticulocytes. Time courses of hydroperoxide formation were monitored after an anaerobic incubation of the enzyme with varying concentrations of NO and for varying length of the incubation period. Owing to the presence of O2 during the enzymatic reaction, the inhibitory effect of NO declines in a time-dependent manner since NO is converted into non-reactive NO2. This is manifested as a lag phase of the time course. The lag phase becomes more pronounced if the enzyme is incubated over a fixed period (15 s) with increasing concentrations of NO. In contrast, increasing the length of the incubation period at fixed concentrations of NO diminishes the duration of the lag phase. These experimental findings can be accounted for by a kinetic model which assumes (a) fast reversible binding of NO to the inactive ferrous Fe(II) form of the enzyme and (b) slow irreversible conversion of the Fe(II)-NO complex into an activated ferric form of the enzyme which is susceptible to peroxide activation. The rate constants for these two different kinetic modes of NO action were estimated by fitting the solutions of the model equations to the experimental time courses. The dissociation constant of the Fe(II)-NO complex amounts to 2.5 microM. Computer simulations performed on the basis of the model indicate that the response of the lipoxygenase to increasing intracellular NO concentrations depend upon the available peroxide tone: at low peroxide concentration the enzyme is initially trapped in the inactive ferrous form and thus may be activated by NO via the activated ferric species. On the other hand, at high peroxide concentrations, a partial inhibition of the initially active enzyme is expected.

Animals↗
PubMed1997

Do specific linoleate 13-lipoxygenases initiate beta-oxidation?

The germination process of oilseed plants is characterized by a mobilization of the storage lipids which constitute the major carbon source for the growing seedling. Despite the physiological importance of the lipid mobilization, the mechanism of this process is not well understood. Recently, it was found that a specific linoleate 13-lipoxygenase is induced during the stage of lipid mobilization in various oilseed plants and that this enzyme is translocated to the membranes of the lipid storage organelles, the so called lipid bodies. Lipoxygenase expression was paralleled by the occurrence of enantiospecific hydro(pero)xy polyenoic fatty acid derivatives in the storage lipids suggesting the in vivo action of the enzyme. Furthermore, it was reported that oxygenated polyenoic fatty acids, in particular as 13(S)-hydro(pero)xy-9(Z),11(E)-octadecanoic acid [13(S)-H(P)ODE], are cleaved preferentially from the storage lipids when compared with their non-oxygenated linoleate residues. These findings may suggest that 13(S)-H(P)ODE may constitute the endogenous substrate for beta-oxidation during lipid mobilization of oilseed plants.

Cucumis sativus↗
PubMed1997

In vivo action of 15-lipoxygenase in early stages of human atherogenesis.

Oxidative modification of low density lipoprotein has been suggested as patho-physiologically relevant process in atherogenesis and the lipid peroxidizing enzyme 15-lipoxygenase may be involved. For experimental evidence on the in vivo action of this enzyme in the time course of plaque formation we analyzed the lipid extracts of lesional areas representing various stages of human atherogenesis for the occurrence of specific 15-lipoxygenase products. In advanced human lesions the degree of oxygenation of the lesion lipids measured as hydroxy linoleic acid/linoleic acid ratio varied between 0.2 and 3.2%. Here an unspecific pattern of oxygenated lipids that did not differ from the pattern formed during copper-catalyzed LDL oxidation was detected. In both cases an enantiomer ratio (S/R-ratio) of 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) of approximately 1:1 was found. In young human lesions which were obtained from the collection of the pathological determinants of atherosclerosis in youth (PDAY) program the hydroxy linoleic acid/linoleic acid ratio was much smaller (variation between 0.05 and 0.6%), and a significant share of specific 15-lipoxygenase products was detected (S/R-ratio of 13-hydroxy linoleic acid of 54 +/- 3.1/46 +/- 3.1 [mean +/- SD]). These data suggest that the 15-lipoxygenase is enzymatically active on endogenous substrates in young human lesions and thus, may be of patho-physiological importance for early atherogenesis. In advanced human plaques the 15-lipoxygenase may be functionally silent and specific lipoxygenase products formed in earlier stages may be decomposed or superimposed by large amounts of nonenzymatic lipid peroxidation products.

Adolescent↗
PubMed1997

A comparative histopathological study of the influence of chemo- and radiotherapy on human prostatic carcinoma grown in nude mice.

Histological investigations were carried out to determine the effects of various kinds of treatment on the heterotransplanted prostatic carcinoma PC EW in athymic nude mice. Morphological changes after chemotherapy, radiotherapy, and combined radiochemotherapy were analyzed qualitatively and quantitatively. Effects of chemo- and radiotherapy like reduction of mitotic activity and increase of necrotic resp. fibrotic areas cumulated under the combined therapy. Results after radiotherapy or combined therapy corresponded to those reported for human prostatic carcinoma. Our observations indicate that the heterotransplanted tumor line PC EW represents a well suited preclinical test system for treatment of human prostatic carcinoma.

Adenocarcinoma↗
PubMed1997

The role of 15-lipoxygenase in atherogenesis: pro- and antiatherogenic actions.

Oxidative modification converts LDL to an atherogenic form and 15-lipoxygenase has been implicated in this process. Several lines of experimental evidence support such a proatherogenic action of the enzyme. However, recent data obtained with transgenic rabbits overexpressing 15-lipoxygenase suggest also an antiatherogenic effect. In this paper the pathophysiological relevance of 15-lipoxygenase in atherogenesis is critically reviewed.

Animals↗
PubMed1997

Prostaglandin F2-like compounds, F2-isoprostanes, are present in increased amounts in human atherosclerotic lesions.

Oxidative modification of LDL is believed to play a major role in atherogenesis. As major lipid peroxidation products oxygenated linoleic acid derivatives and oxysterols have been described in human atherosclerotic lesions. Here we report that human lesions contain isoprostanes as peroxidation products of arachidonic acid at a level of 27.1 +/- 21.2 pg/mg wet weight (n = 10), which corresponds to 75.9 +/- 59.3 pg/mg dry weight, n contrast, human umbilical veins (n = 10), which were used as nonatherosclerotic control vessels, contain much smaller amounts of isoprostanes (1.4 +/- 0.7 pg/mg wet weight, which corresponds to 11.7 +/- 6.2 pg/mg dry weight), and there are significant differences between the two types of vessels. As major products of linoleic acid oxidation, racemic hydroxy linoleate isomers were detected in the lesional ester lipids. In human lesions, the hydroxy linoleic acid/linoleic acid ratio was about 0.5%, a result indicating that 5 out of 1000 linoleate residues are present as hydroxylated derivatives. In umbilical veins, no hydroxy linoleic acid could be detected. These data show that human atherosclerotic lesions contain increased amounts of hydroxy linoleic acid isomers and isoprostanes when compared with nonatherosclerotic vessel wall and suggest a link between local lipid peroxidation and progression of atherosclerosis. For evaluation of the degree of lipid peroxidation, the determination of the hydroxy linoleic acid/linoleic acid ratio appears to be more suitable than the isoprostane content.

Aged↗