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Biomedical subjects

T Schewe

Publications and source records attributed to T Schewe.

At least 91 records · Page 5Linked to original sources
PubMed1985

Possible involvement of endogenous free fatty acids in the selective inactivation of the mitochondrial electron transfer system during hyperthermic incubation of rabbit reticulocytes.

Preincubation of rabbit reticulocyte-rich red cell suspensions at 45 degrees C for 1 h led to a lowering of the total endogenous oxygen uptake by 40%, a decrease of the CCCP-stimulated antimycin-A-sensitive part of the oxygen uptake by 72%, an inhibition of the antimycin A-resistant oxygen uptake by only one-third, the loss of the response of cell respiration to the uncoupler CCCP and a slight enhancement of the degree of uncoupling which is however of minor importance for the changes in the cell respiration during hyperthermic incubation. The degree of inactivation of the respiratory chain increased continuously with the temperature of preincubation beginning at 39 degrees C. Addition of defatted human serum albumin to the cells caused protection from thermal inactivation. The protective action of serum albumin was lowered by loading it with myristic acid. It is concluded that the thermal inactivation of the respiratory chain in reticulocytes may be mediated by endogenous free fatty acids.

Animals↗
PubMed1984

Pentane formation during the anaerobic reactions of reticulocyte lipoxygenase. Comparison with lipoxygenases from soybeans and green pea seeds.

The lipoxygenases from reticulocytes, soybeans and green pea seeds produce pentane in an anaerobic assay containing 13Ls-hydroperoxy-9-cis, 11-trans-octadecadienoic acid and 9,12-all-cis-octadecadienoic acid. The presence of oxygen strongly inhibits pentane formation by the three enzymes. Relative to the lipoxygenase activity with linoleic acid as substrate, soybean lipoxygenase is 4-times as effective in pentane formation as the lipoxygenases from reticulocytes or green pea seeds. Pentane formation by the reticulocyte lipoxygenase is completely inhibited by lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid, 3-t-butyl-4-hydroxyanisol) but only partially by radical scavengers which do not influence the oxygenase activity (2,6-di-t-butyl-4-hydroxytoluene). From the temperature dependence below 20 degrees C an activation energy of the pentane production by the reticulocyte lipoxygenase of about 28 kJ/mol was calculated, which is somewhat higher than that for the oxygenase activity. During the anaerobic reaction of both reticulocyte and soybean lipoxygenase C18-oxodienes, C13-oxodienes, linoleic acid dimers and a polar compound proposed to be epoxy-hydroxyoctadecenoic acid are produced in a similar pattern. Reticulocyte lipoxygenase produces pentane with submitochondrial particles only under anaerobic conditions after an aerobic preincubation. During the incubation of intact reticulocytes with the calcium ionophore A23187 or arachidonic acid, pentane is released. Preincubation of the cells with lipoxygenase inhibitors completely abolishes the pentane formation. Erythrocytes do not form any pentane under the same experimental conditions.

Anaerobiosis↗
PubMed1984

The mechanism of inactivation of lipoxygenases by acetylenic fatty acids.

The inactivation of soybean lipoxygenase by 5,8,11,14-eicosatetraynoic acid was studied in detail. The inactivation was found to be time-dependent and irreversible. A kinetic scheme, based on the assumption of a rapid inactivation of the enzyme-product complex, yielded a Km value for 5,8,11,14-eicosatetraynoic acid of 1.3 microM, which is about a tenth of that described for arachidonic acid, and a reaction constant k+2 of 0.006s-1, which is four orders of magnitude lower. The reasons for these differences are discussed. Several types of experimental evidence indicate that the first step of the enzyme inactivation is the conversion of 5,8,11,14-eicosatetraynoic acid via a lipoxygenase reaction: (a) the conversion of radioactively labelled methyl ester of 5,8,11,14-eicosatetraynoic acid to other products; (b) the oxygen requirement of the inactivation; (c) the competitive protective effect of linoleic acid; (d) the similarity of the activation energy for both the dioxygenation of linoleic acid and the enzyme inactivation by 5,8,11,14-eicosatetraynoic acid; (e) the formation of one mole methionine sulfoxide/mole enzyme during the reaction with 5,8,11,14-eicosatetraynoic acid, similar to the suicidal reaction of reticulocyte lipoxygenase with 13LS-hydroperoxy-linoleic acid. These results, as well as the lack of covalent binding of 14C-labelled 5,8,11,14-eicosatetraynoic acid methyl ester, contradict the allene mechanism postulated by others [D.T. Downing, D.G. Ahern, and M. Bachta (1970) Biochem. Biophys. Res. Commun. 40, 218-223; K.H. Gibson (1977) Chem. Soc. Rev. 6, 489-510]. It is assumed that the susceptible methionine is located at the active centre of the enzyme.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1984

The possible biological importance of lipoxygenase pathway in aorta endothelial cells.

Cultured calf aortic endothelial cells metabolize exogenous 1-[14C] arachidonic acid via the lipoxygenase pathway to 12-hydroxyeicosatetraenoic acid and dihydroxyeicosatetraenoic acid(s). Disruption of the cells by freezing and thawing strongly increases the formation of lipoxygenase metabolites. Under these conditions mainly 15-hydroxy-eicosatetraenoic acid was formed in addition to 12-hydroxyeicosatetraenoic acid. From the calculation of the specific radioactivity of the lipoxygenase products formed it was concluded that not only the exogenous but also endogenous arachidonic acid released from intracellular sources such as phospholipids, triglycerides or cholesterol esters is oxygenated. The possible biological significance of the lipoxygenase metabolites is discussed.

Animals↗
PubMed1984

The inactivation of lipoxygenases by acetylenic fatty acids.

5,8,11,14-eicosatetraynoic acid (ETYA) inactivates irreversibly the lipoxygenases from soybeans and reticulocytes in a time-dependent manner. 5,8,11-eicosatriynoic acid (ETrYA) is a powerful inactivator only for reticulocyte lipoxygenase. Several types of experimental evidence indicate that the acetylenic fatty acids act as suicidal substrates which are converted during the lipoxygenase reaction probably to allene hydroperoxides. These highly reactive intermediates or radical precursors react immediately with a methionine residue at the active centre of the enzyme forming one methionine sulfoxide per molecule. Experiments with 14C-labelled ETYA-methylester show that despite complete enzyme inactivation no covalent binding of the suicidal substrate to the lipoxygenases occurred.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1983

Reticulocyte lipoxygenase changes the passive electrical properties of bovine heart submitochondrial particles.

Purified reticulocyte lipoxygenase oxygenates the polyunsaturated phospholipids of sonified submitochondrial particles from bovine heart as measured by a burst of oxygen uptake. Over the frequency range of 0.5 to 100 MHz, the complex impedance of the submitochondrial particles as a function of the frequency before and after lipoxygenase attack was measured. From these data, the membrane capacity, the conductivity of the membrane and the conductivity inside the particles were calculated. Lipoxygenase action causes a 4-fold increase in the membrane capacity and a 2-fold increase in the membrane conductivity. Using the method of deformation of electric pulses, kinetic measurements were performed. In parallel to the changes of the passive electric properties, a partial inhibition of NADH oxidase and succinate oxidase was caused by the lipoxygenase attack. Oxygen uptake, changes of the passive electric properties and the inhibition of respiratory enzymes were prevented by lipoxygenase inhibitors. Owing to the high oxygen consumption produced by the lipoxygenase reaction, anaerobiosis was reached within the first 30 s in the closed chamber. Therefore, it must be concluded that the changes in passive electric properties and the inhibition of the respiratory enzymes are due to secondary anaerobic processes such as the hydroperoxidase reaction catalyzed by the lipoxygenase or a slow redistribution of peroxidized membrane lipids. The results are discussed in relation to the breakdown of mitochondria during the maturation process of red cells.

Animals↗
PubMed1983

Reticulocyte lipoxygenase exhibits both n-6 and n-9 activities.

Purified reticulocyte lipoxygenase converts arachidonic acid to both 15- and 12-hydroxyperoxyeico-satetraenoic acids. The proportion of the two reaction products does not change during the purification procedure as shown by HPLC analysis. By means of isoelectric focusing it was not possible to separate the n-6 and n-9 activities. Reticulocyte lipoxygenase was completely inactivated by both 5,8,11-eicosatriynoic and 5,8,11,14-eicosatetraynoic acids in contrast to soybean lipoxygenase-1 which was inactivated only by 5,8,11,14-eicosatetraynoic acid. These results indicate that reticulocyte lipoxygenase exhibits both n-6 and n-9 activities. A contamination of the enzyme preparation with other lipoxygenases, e.g., the n-9 lipoxygenase from thrombocytes appears to be excluded.

Animals↗
PubMed1983

Haemoglobin potentiates the respiration-inhibitory action of lipoxygenases via its pseudolipohydroperoxidase activity.

The inhibition of the NADH oxidase and of the succinate-cytochrome c oxidoreductase activities of beef heart submitochondrial particles (ETP) induced by reticulocyte lipoxygenase is strongly potentiated by haemoglobin added simultaneously or subsequently to lipoxygenase. Half-maximal potentiation was observed with 0.9 microM haemoglobin. Haemoglobin augments also the lipoxygenase-induced loss of acid-labile sulphur in ETP not related to respiratory inhibition. Soybean lipoxygenase produces only weak respiratory inhibition even at high concentrations in the absence of haemoglobin; strong inhibition is obtained, however, in the presence of haemoglobin. Among other haem compounds tested only free haemin but not cytochrome c and catalase exhibit identical effects. The action of haem compounds on the system ETP plus lipoxygenase is accompanied by their destruction. The experimental evidence indicates that the effects of haemoglobin described here are due to its ability to decompose catalytically hydroperoxy lipids generated by lipoxygenase. This catalytic property (pseudolipohydroperoxidase activity) was demonstrated with 13L-hydroperoxylinoleic acid as substrate. The relations between the various catalytic actions of haemoglobin related to lipid peroxidation are discussed.

Animals↗
PubMed1983

In vitro maturation of rabbit reticulocytes. III. Response of lipoxygenase.

Lipoxygenase activity with linoleic acid as substrate and the immunologically detectable amount of lipoxygenase protein were estimated in the course of in vitro maturation of rabbit reticulocytes withdrawn at the sixth day of an experimental bleeding anaemia. With unseparated cell mixture there was a significant increase in the lipoxygenase activity of 67 +/- 15% after a maturation period of 4 h followed by a decrease up to the initial level. The maturational changes were more pronounced when the fraction of youngest reticulocytes after buoyant density separation in a serum albumin gradient was used, whereas the cells of medium density failed to show the intermittent increase. The lipoxygenase activity was largely paralleled by the amount of lipoxygenase protein. The increase of lipoxygenase was prevented by either anaerobiosis or addition of oligomycin. The protein synthesis was greatly decreased after 4 h of incubation. The decline of the amount of lipoxygenase between 4 and 24 h incubation is probably largely caused by proteolysis. The results support former interpretations as to the synthesis and biological dynamics of lipoxygenase in reticulocytes.

Animals↗
PubMed1983

Lung strips from guinea pigs as test system for lipoxygenase inhibitors. Inhibition of arachidonic acid-induced contractions by 3-t-butyl-4-hydroxyanisole and nordihydroguaiaretic acid.

Lung strips and tracheal spirals from guinea pigs and rats were compared as test systems for lipoxygenase inhibitors. The contraction of the smooth muscle was induced by addition of arachidonic acid. Lung strips of guinea pigs proved to be the most suitable in vitro model. Under basal conditions half-maximal contraction was produced by 13 microM arachidonic acid (AA) which was strongly inhibited by 50 microM nordihydroguaiaretic acid (NDGA) or 100 microM 3-t-butyl-4-hydroxyanisole (BHA). NDGA and BHA caused a shift of response towards higher concentrations of AA. BHA also produced pronounced dilatations of both basal tonus and carbachol-induced contractions of the preparations from the respiratory tract. It is concluded that the actions of NDGA and BHA on the airway preparations are due to inhibition of the lipoxygenase reaction and, therefore, of leukotriene synthesis. The assumption of the lipoxygenase inhibition is supported by their action on purified lipoxygenase from rabbit reticulocytes which was inhibited by 50% by 0.5 microM NDGA and 160 microM BHA. In contrast, the antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT) did not inhibit at all at 1 mM. To the knowledge of the authors this is the first evidence so far reported for the bronchodilating action of BHA.

Animals↗
PubMed1983

Catalytic activities of haemoglobin related to lipid peroxidation.

The catalytic activities of haemoglobin and other haemoproteins related to lipid peroxidation, especially the haemin-catalyzed lipid peroxidation, the quasi-lipoxygenase activity and the hydroperoxidase activity are briefly reviewed. Important characteristics of these actions are compared. The reaction products of the quasi-lipoxygenase activity are reported.

Animals↗
PubMed1983

A structural model for the interaction of haem with unsaturated fatty acids explaining its quasi-lipoxygenase activity. Quantum chemical calculations.

The quasi-lipoxygenase activity of haemoglobin differs in many respects from the well-known haemin-catalyzed lipid peroxidation (1-4), especially in its high substrate specificity for unsaturated fatty acids containing one 1,4-pentadiene system (dienoic fatty acids). In this report a structural model for the fatty acid haem interaction based on quantum-chemical calculations is presented which show that only dienoic fatty acids are bound to the haem in such a way that the initial hydrogen abstraction that is necessary for the over-all reaction, is favoured sterically and energetically.

Animals↗
PubMed1982

Self-inactivation by 13-hydroperoxylinoleic acid and lipohydroperoxidase activity of the reticulocyte lipoxygenase.

1. The self-inactivation of lipoxygenase from rabbit reticulocytes with linoleic acid at 37 degrees C is caused by the product 13-hydroperoxylinoleic acid. This inactivation is promoted by either oxygen or linoleic acid. 2. Lipohydroperoxidase activity was demonstrated with 13-hydroperoxylinoleic acid plus linoleic acid as hydrogen donor under anaerobic conditions at 2 degrees C. The products were 13-hydroxylinoleic acid, oxodienes and compounds of non-diene structure similar to those produced by soybean lipoxygenase-1. 3. 13-Hydroperoxylinoleic acid also changed the absorbance and fluorescence properties of reticulocyte lipoxygenase. The results indicate that one equivalent of 13-hydroperoxylinoleic acid converts the enzyme from the ferrous state into the ferric state as described for soybean lipoxygenase-1. The spectral changes were reversed by sodium borohydride at 2 degrees C, but not at 37 degrees C; it is assumed that the ferric form of reticulocyte lipoxygenase suffers inactivation.

Animals↗
PubMed1981

On the site of action of the inhibition of the mitochondrial respiratory chain by lipoxygenase.

Treatment of beef-heart submitochondrial particles with reticulocyte lipoxygenases gives rise to a strong irreversible inhibition of the NADH and succinate oxidase activities. This is not accompanied by any loss of the Fe-S clusters of the respiratory chain as determined by EPR spectroscopy. The inhibitory blockage is located between both the NADH and succinate dehydrogenases and Q-10. The inhibitory action of treatment with lipoxygenase also takes place in the absence of Q-10. The Fe-S clusters of the mitochondrial outer membrane are destroyed by lipoxygenase treatment, without any effect on the rotenone-insensitive NADH: cytochrome c oxidoreductase activity. It is concluded that these clusters are not involved in this enzyme.

Animals↗