Maturational breakdown cascade of mitochondria in reticulocytes.
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Animals↗
Biomedical subjects
T Schewe
Publications and source records attributed to T Schewe.
The maturational breakdown of mitochondria in reticulocytes.
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Adenosine Triphosphate↗
Formation of lipoxin B by the pure reticulocyte lipoxygenase.
The pure reticulocyte lipoxygenase converts 5,15-DiHETE via a lipoxygenase reaction to 5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15-HETE and 15-HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.
Gas Chromatography-Mass Spectrometry↗
Requirement of monohydroperoxy fatty acids for the oxygenation of 15LS-HETE by reticulocyte lipoxygenase.
The lipoxygenase from reticulocytes oxygenates 15LS-HETE to 8-hydroperoxy-15-hydroxy-5,9,11,13-eicosatetraenoic acid and 5-hydroperoxy-15-hydroxy-6,8,11,13-eicosatetraenoic acid only in the presence of catalytic concentrations of monohydroperoxy fatty acids. During this reaction the hydroperoxy fatty acids are converted to more polar products including hydroxy fatty acids. From kinetic measurements of 15LS-HETE oxygenation it was calculated that 1 mol monohydroperoxy fatty acid is consumed during the oxygenation of about 9 mol 15LS-HETE.
Fatty Acids↗
Hydroperoxyfatty acids inactivate the reticulocyte lipoxygenase independently of a hydroperoxidase reaction.
From a comparison of 9Ds-HPODE and 13Ls-HPODE and their methyl esters as substrates and inactivating agents of reticulocyte lipoxygenase it is concluded that the compounds inactivate the enzyme independently of any hydroperoxidase reaction. The protective effect of 4-nitrocatechol indicates the formation of Fe(III) complexes of the enzyme with the hydroperoxyfatty acid compounds prior to inactivation.
Anaerobiosis↗
The stoichiometry of oxygen uptake and conjugated diene formation during the dioxygenation of linoleic acid by the pure reticulocyte lipoxygenase. Evidence for aerobic hydroperoxidase activity.
Simultaneous measurements of oxygen uptake and conjugated diene formation (increase in the absorbance at 234 nm) during the dioxygenation of linoleic acid by the pure reticulocyte lipoxygenase gave a nearly theoretical stoichiometry of 1.1 in a temperature range from 5 to 30 degrees C and a wide range of concentrations of both oxygen and linoleic acid. At low concentrations of either oxygen or linoleic acid or both, secondary processes occurred such as linoleic acid-supported lipohydroperoxidase reactions leading to the disappearance of conjugated dienes and to the formation of oxodienes, linoleic acid dimers and epoxyhydroxy derivatives. Under these conditions marked deviations of the stoichiometry between oxygen uptake and conjugated diene formation appeared. The formation of conjugated oxodienoic fatty acids absorbing at 285 nm occurred only under conditions of high concentrations of linoleic acid and limiting oxygen supply. The results indicate that lipohydroperoxidase reactions catalyzed by the pure reticulocyte lipoxygenase do not only take place under strictly anaerobic conditions but also under conditions of limiting concentrations of either linoleic acid or oxygen or both.
Aerobiosis↗
Enzymology and physiology of reticulocyte lipoxygenase: comparison with other lipoxygenases.
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Animals↗
The stereochemistry of the reactions of lipoxygenases and their metabolites. Proposed nomenclature of lipoxygenases and related enzymes.
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Animals↗
Positional specificity of lipoxygenases and their suitability for testing potential drugs.
Comparative studies on isolated lipoxygenases revealed the following features: variability of their positional specificity, diversity of enzymatic properties including sensitivity to inhibitors, a common general mechanism of action. The molecular basis of the positional specificity of lipoxygenases is discussed. The lipoxygenase of reticulocytes proved to be an excellent model in the search for inhibitors of the arachidonate 5-lipoxygenase. Reticulocyte lipoxygenase may form a wide spectrum of products belonging to all important classes of primary lipoxygenase metabolites hitherto known (HPETE's, diHPETE's, lipoxins, epoxyleukotrienes and others). This enzyme was used as basal test in a screening hierarchy for lipoxygenase-inhibitory antiasthmatic drugs and led to the discovery of new orally acting potential antiasthmatics. There was a high correlation between inhibition of reticulocyte lipoxygenase and antibronchoconstrictory activity with airway preparations in vitro and in vivo.
Animals↗
The occurrence of a lipoxygenase pathway in reticulocytes of various species.
Reticulocytes from various species (rat, mouse, rhesus monkey) obtained by phenylhydrazine treatment of the animals metabolized polyenoic fatty acids via a lipoxygenase pathway. Linoleic acid was converted to 13-hydro(pero)xy-9,11(Z,E)octadecadienoic acid [13-H(P)ODE] and 9-hydro(pero)xy-10,12(E,Z)octadecadienoic acid [9-H(P)ODE], whereas arachidonic acid was oxygenated to 15-hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid (15-HETE) as shown by straight-phase high-pressure liquid chromatography (SP-HPLC). Addition of calcium and ionophore A 23,187 strongly enhanced the formation of lipoxygenase products, whereas 5,8,11,14eicosatetraenoic acid (ETYA) completely inhibited their formation. Estimates of the specific radioactivities of the lipoxygenase products indicate differences in the metabolization of externally added and endogenously released polyenoic fatty acids. These results strongly suggest that lipoxygenases generally occur in immature red blood cells.
5,8,11,14-Eicosatetraynoic Acid↗
Pure enzymes in a test hierarchy for antibronchoconstrictory lipoxygenase inhibitors.
A test hierarchy for potential antibronchoconstrictive drugs acting as lipoxygenase inhibitors was developed which includes a purified mammalian lipoxygenase/leukotriene A4 synthase, purified cyclooxygenase, arachidonic acid metabolism of polymorphonuclear leukocytes, arachidonic acid-induced contraction of lung strips, isolated human bronchi, and ovalbumin-induced bronchoconstriction with sensitized guinea pigs. The lipoxygenase from rabbit reticulocytes turned out to be superior to a variety of other animal and plant lipoxygenases. Among the various test systems with respiratory tract preparations, the arachidonic acid-induced contraction of guinea pig lung strips gave the most reliable results in the search for new antiasthmatic drugs. The validity of the test hierarchy was ascertained in screening investigations. Novel classes of antibronchoconstrictory lipoxygenase inhibitors were found.
Animals↗
Two modes of irreversible inactivation of the mitochondrial electron-transfer system by tetradecanoic acid.
Bovine heart submitochondrial particles were incubated for 2-6 h at 37 degrees C with various concentrations of tetradecanoic acid, and the effects on the activities, the total acid-labile sulphide content and EPR spectra of the electron transfer system were studied. Two distinct time-dependent processes of the slow irreversible inactivation of the electron-transfer system were found. They differ in the concentration of tetradecanoic acid required. The more specific effect, induced by 100-400 nmol tetradecanoic acid per mg protein, consists of a selective blockage of electron transfer between the Fe-S clusters of the NADH dehydrogenase and ubiquinone, without damage to any of the Fe-S clusters. Higher concentrations of tetradecanoic acid caused gradual destruction of all Fe-S clusters of NADH dehydrogenase and of the 3-Fe cluster of succinate dehydrogenase, leading to complete inactivation of both NADH and succinate oxidation.
Animals↗
Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14, 15-leukotriene A4.
The purified lipoxygenase of rabbit reticulocytes converts arachidonic acid at 0 degrees C to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) via reactions which involve hydrogen abstraction at C-13 and C-10, respectively. At 37 degrees C the enzyme converts arachidonic acid to additional products which were identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, 8,15-dihydroperoxy-5,9,11,13- and 5,15-dihydroperoxy-6, 6,8,11,13-eicosatetraenoic acids (8,15-diHPETE and 5,15-HPETE, respectively) and diastereoisomers of 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-diHPETEs). The 8,15- and 5,15-diHPETEs were formed by double lipoxygenation since each incorporated 2 molecules of 18O2 and since their synthesis from 15-HPETE was blocked under anaerobic conditions. The 8,15-diHETEs each incorporated 18O from 18O2 at C-15 and were found to arise from nonenzymatic hydrolysis of an epoxytriene which was identified as 14,15-leukotriene A4 by trapping in acidic methanol. This compound was a major product of 15-HPETE in anaerobic incubations. The conversion of 15-HPETE to 14,15-leukotriene A4 was inhibited by the lipoxygenase inhibitors nordihydroguairetic acid and 5,8,11,14-eicosatetraynoic acid. The 14,15-leukotriene A4 synthase and 15-lipoxygenase activities were inhibited by 5,8,11,14-eicosatetraynoic acid in a similar time-dependent manner. The results support a mechanism whereby 14,15-leukotriene A4 is synthesized from 15-HPETE by a further enzymatic step carried out by the reticulocyte 15-lipoxygenase via hydrogen abstraction at C-10 and a redox cycle of the non-heme iron atom of the enzyme.
Animals↗
Metabolism of [1-14C]-arachidonic acid by cultured calf aortic endothelial cells: evidence for the presence of a lipoxygenase pathway.
Intact cultured calf aortic endothelial cells from a 10th-14th subculture rapidly metabolize exogenous [1-14C]-arachidonic acid by three different routes: i) incorporation into triglycerides and phosphilipids in a ratio of about 2:1, ii) formation of lipoxygenase metabolites (12-hydroxy-5,8,10,14-eicosatetraenoic acid) and iii) formation of cyclooxygenase metabolites (6-keto-PG F1 alpha and PG F2 alpha). From analyses by thin-layer chromatography and high-pressure liquid chromatography it was established that the main lipoxygenase metabolites in intact cells are 12-hydroxy-5,8,10,14-eicosatetraenoic acid and a compound proposed to be (a) dihydroxyeicosatetraenoic acid(s). In frozen and thawed cells the incorporation of arachidonic acid into cellular lipids is abolished, whereas the lipoxygenase pathway is strongly enhanced. Under these conditions the cells produce predominantly 15-hydroxy-5,8,11,13-eicosatetraenoic acid in addition to 12-hydroxy-5,8,10,14-eicosatetraenoic acid. The formation of lipoxygenase products was inhibited by heating the cells or by preincubation with nordihydroguaiaretic acid or BW 755 degrees C, whereas indomethacin was without effect. The formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid by intact cells was inhibited by 5,8,11-eicosatriynoic acid. Indomethacin and acetylsalicylic acid inhibited the formation of cyclooxygenase metabolites. 15Ls-hydroxy-5,8,11,13-eicosatetraenoic acid was incorporated into cellular lipids, but not dihydroxyeicosatetraenoic acids. Exogenous [3H]-labelled prostacyclin and TxB2 were not incorporated but were metabolized to less polar products.
Animals↗
Improved procedure for the detection of activity of lipoxygenases on electropherograms.
Several procedures for activity staining of lipoxygenases were compared. The best results were obtained with the o-dianisidine method of De Lumen and Kazeniac which had to be modified, however, with respect to the conditions of electrophoretic separation, treatment of the gel after separation, incubation conditions and staining solution, to achieve satisfactory detection of the lipoxygenases from wheat grains, pea seeds, soybeans and rabbit reticulocytes.
Animals↗
The share of lipoxygenase in the antimycin-resistant oxygen uptake of intact rabbit reticulocytes.
The endogenous oxygen uptake of rabbit reticulocyte-rich red cell suspensions obtained by bleeding anaemia of rabbits amounted to 7.85 +/- 0.87 mumoles/h . ml of packed cells and was inhibited by antimycin A to 77.2 +/- 1.1%. The antimycin A-resistant oxygen uptake was further inhibited by the lipoxygenase inhibitors salicylhydroxamic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, 4-nitrocatechol or propylgallate by about 20-30% corresponding to 5-7% of the total oxygen uptake. The effects of the lipoxygenase inhibitors were most pronounced during the first period of bleeding anaemia (5th-9th day). 3-amino-1,2,4-triazole inhibited another part of the non-respiratory oxygen uptake and acted additively to 5,8,11,14-eicosatetraynoic acid; the two inhibitors together caused inhibition by one-half. Influx of calcium ions mediated by the ionophore A 23187 led to a two-fold increase in the non-respiratory oxygen uptake which was mainly due to stimulation of the lipoxygenase reaction. The rate of the lipoxygenase-mediated oxygen uptake was sufficient for a complete dioxygenation of the polyenoic fatty acids present in mitochondrial phospholipids during the maturational degradation of mitochondria in reticulocytes.
Animals↗
On the nature of the stimulation of the lipoxygenase from rabbit reticulocytes by biological membranes.
The oxygenation of concentrated emulsions (about 260 microM) of arachidonic acid or linoleic acid catalyzed by the purified lipoxygenase from rabbit reticulocytes is strongly stimulated in the presence of low concentrations of beef heart submitochondrial particles or other membrane preparations. Maximal stimulation was observed at a proportion of about 5 mumoles polyenoic acid per mg of membrane protein. Whereas at a constant ratio of arachidonic acid and membranes the reaction rate obeyed the Michaelis-Menten kinetics with an apparent Km value of 75 microM for arachidonic acid, sigmoid kinetics was observed under conditions of constant concentration of membranes and, consequently, varying proportions of arachidonate and membranes. Under conditions of maximal stimulation binding of reticulocyte lipoxygenase to the membranes was insignificant. Moreover, one-third of the arachidonic acid sedimented during ultracentrifugation of the membranes as judged from experiments with [14C] arachidonic acid. From other sedimentation experiments and from the comparison with soybean lipoxygenase which is not stimulated by membranes it is concluded that the emulsion droplets interacting with the membranes are the substrate of the stimulated reaction. The stimulation may be brought about by facilitating the susceptibility of the fatty acids to reticulocyte lipoxygenase. The membrane-stimulated reaction of reticulocyte lipoxygenase was not inhibited by the antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT) excluding the participation of free radicals. The stimulatory effect of membranes seems not to be related to their susceptibility towards lipoxygenase attack which varied strongly in order mitochondrialmembranes greater than endoplasmic membranes greater than erythrocyte ghosts. The stimulation by membranes resembles that produced by the detergent sodium cholate at concentrations near to the critical micellar concentration. Moreover, the stimulations by membranes and by cholate do not behave synergistically or abolish each other. The stimulatory effect of membranes did not occur at a proportion of 200 nmoles per mg of membrane protein.
Animals↗
Enzymatic properties of the lipoxygenase from pea seeds.
A lipoxygenase preparation was obtained from dried green pea seeds. Disc electrophoresis with enzyme staining indicated the presence of only one main isoenzyme corresponding to the isoenzyme PL I according to Yoon and Klein (J. Agric. Food Chem. 24, 955 (1979)), whereas PL II was absent. The assay for pea lipoxygenase has been optimized by using a final concentration of 0.53 mM potassium linoleate in the presence or absence of 0.2% sodium cholate. Without detergent the rho H optimum was 5.9, in its presence 6.8. The formation of conjugated dienes absorbing at 234 nm accounted for 75% of the oxygen uptake. The difference is mainly due to the aerobic formation of oxodienoic acids absorbing at 285 nm via a lipohydroperoxidase activity concomitant with the dioxygenase reaction. Other lipohydroperoxidase products were formed only to a minor extent under aerobic conditions, whereas in the anaerobic lipohydroperoxidase reaction of pea lipoxygenase in the system 13L8-hydroperoxylinoleic acid/linoleic acid fatty acid dimers containing conjugated double bonds were formed additionally. The pea enzyme showed self-inactivation at 37 degrees C, but in contrast to the lipoxygenase from rabbit reticulocytes the self-inactivation appeared only syncatalytically during the aerobic reaction. The antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT, 1 mM) did not protect from self-inactivation. In contrast to the lipoxygenase from soybeans, wheat and rabbit reticulocytes the pea lipoxygenase caused a co-oxidation of Cu-chlorophyllin in the presence of linoleate at 5 degrees C. The co-oxidation was completely inhibited by 1 mM BHT which did not inhibit the dioxygenation of linoleate at this temperature. Unlike the reticulocyte enzyme the pea lipoxygenase failed to attack mitochondrial membranes or to produce inhibition of the respiratory chain. The results lead to the conclusion that a simple classification of lipoxygenases in type I and type II enzymes is not justified. A reaction scheme is proposed to explain both the co-oxidative activity and the aerobic formation of oxodienoic acids by pea lipoxygenase, presuming the dissociation of a linoleic acid radical from the ferrous lipoxygenase as a side reaction of the catalytic cycle.
Animals↗