Occurrence of lipoxygenase products in membranes of rabbit reticulocytes. Evidence for a role of the reticulocyte lipoxygenase in the maturation of red cells

A lipoxygenase has been found in the reticulocytes of all mammalian species tested so far (rabbit, rat, mouse, monkey, and humans); evidence from in vitro studies suggests that the lipid-peroxidizing effects of this enzyme could render the mitochondrion and other intracellular organelles prone to the proteolytic degradation which is a natural step in development of the reticulocyte to the mature red cell. In this study we sought evidence of an active lipoxygenase in vivo. A bleeding anemia was induced in rabbits, and in the course of the subsequent reticulocytosis the red cell membranes were examined for the presence of the characteristic lipoxygenase products of linoleic and arachidonic acids. Erythrocyte membranes from control collections contained only small amounts of hydroxy fatty acids (0.03-0.08% of the polyenoic fatty acids). In contrast, reticulocyte-enriched red cells contained up to 3.3% of the polyenoic acids as hydroxylated derivatives. The main hydroxy fatty acid in reticulocyte membranes was identified as 13-L(S)-hydroxy-9Z,11E-octadecadienoic acid. Small amounts of other hydroxy derivatives including 15-hydroxy-5,8,11,13-(Z,Z,Z,E)eicosatetraenoic acid were also detected. These products appeared about 3 days after development of reticulocytosis. The precise structures of the hydroxylated polyenoic fatty acids and the time course of their appearance strongly suggest that their formation is due to the intracellular action of the cell-specific reticulocyte lipoxygenase. These findings are the first evidence for an activity of this enzyme in vivo, and the results support the hypothesis that enzymic peroxidation of reticulocyte intracellular membranes is a step in preparation of the intracellular organelles for proteolytic degradation.     

Oxygenation of mitochondrial membranes by the reticulocyte lipoxygenase. Action on monoamine oxidase activities A and B

Incubation of isolated rat liver mitochondria with the pure rabbit reticulocyte lipoxygenase caused a time-dependent inactivation of the monoamine oxidase activities A and B. Furthermore, a conversion of the monoamine oxidase into a diamine oxidase was observed. The inactivation kinetics for both monoamine oxidase activities A and B showed a biphasic behaviour; a reversible short-term inhibition during the first 5 min of incubation was followed by an irreversible inactivation of the enzyme. The kinetic studies suggest that the slow irreversible inactivation of the monoamine oxidase activities is due to secondary reactions subsequent to the initial attack of the lipoxygenase on the mitochondrial outer membrane. During the interaction of the lipoxygenase with the mitochondria, only about 1.5% of the polyenoic fatty acids present in the mitochondrial membranes were oxygenated. The predominant products formed during the interaction of the lipoxygenase with the mitochondrial membranes are (13S)-hydro(pero)xy-9Z,11E-octadecadienoic acid and (15S)-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid.     

Oxygenation of biological membranes by the pure reticulocyte lipoxygenase

We find that the reticulocyte lipoxygenase can oxygenate rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes (inside-out and right side-out ghosts) without prior action of a phospholipase. After alkaline hydrolysis of the ester lipids, the main products were identified as 15S-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetr aenoic acid, 17S-hydro(pero)xy-4Z,7Z,10Z,13Z,15E, 19Z,-docosahexaenoic acid, 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid, 9(S/R)-hydro(pero)xy-10E,12Z-octadecadienoic acid as well as the two all-E hydro(pero)xy octadecadienoic acid isomers. At low membrane concentrations (1 mg of protein/ml), the enzyme maintains a high stereospecificity for the S-configuration, but at higher concentrations (20 mg/ml), the products were virtually racemic. Addition of the antioxidant 2,6-ditert-butyl-p-cresol counteracted this tendency to lose stereospecificity. During these enzyme-catalyzed reactions, substantially more oxygen is consumed than can be accounted for as the hydro(pero)xy products. This discrepancy is due to secondary reactions which lead to the decomposition of the primary oxygenation products, the hydroperoxy lipids, and to oxidative modifications of membrane proteins. These data indicate that the reticulocyte lipoxygenase can oxygenate polyenoic fatty acids in various types of biological membrane and that the oxidative modifications are not restricted to the membrane lipids. The results are discussed in terms of the proposed role of the enzyme in the breakdown of mitochondria and other intracellular organelles during the maturation of red blood cells.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

The lipoxygenase of reticulocytes. Purification, characterization and biological dynamics of the lipoxygenase; its identity with the respiratory inhibitors of the reticulocyte.

A lipoxygenase has been purified from rabbit reticulocyte-rich anaemic blood cells. It possesses a molecular weight of 78 000 and an isoelectric point of 5.5 and contains 5% neutral sugars and two iron atoms per enzyme molecule. The lipoxygenase has proved to be identical with the inhibitors of respiratory proteins described formerly. The actions of the lipoxygenase on linoleic acid, phospholipids, mitochondrial and erythrocyte membranes and electron transfer particles were studied. A special feature of the reticulocyte lipoxygenase is the suicidal character of its action on lipids. With electron transfer particles the reticulocyte lipoxygenase causes a loss of acid-labile sulfur which accompanies respiratory inhibition; the strong respiratory inhibition is not exerted by soybean lipoxygenase. The reticulocyte lipoxygenase acts preferably on mitochondrial membranes as compared with cell membranes of the erythrocyte; erythrocyte cytosol moderates the action on mitochondrial membranes. Furthermore, the lipoxygenase reaction can concomitantly and irreversibly inactivate sulfhydryl enzymes as demonstrated with muscle glyceraldehyde-3-phosphate dehydrogenase. The occurrence of the lipoxygenase here described is restricted to reticulocytes; very low amounts were observed in bone marrow and no lipoxygenase was detectable in normal blood. During the course of an experimental anaemia the lipoxygenase is produced owing to superinduction in large amounts, which may persist for a long time since they escape inactivation. Preliminary evidence was obtained for the occurrence of other lipoxygenases in tissues of lung, spleen, kidney and also epithelial tumours.     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

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.     

Some properties of the lipoxygenase from rabbit reticulocytes.

A lipoxygenase was enriched from the stoma-free supernatant of rabbit reticulocytes. The enzyme causes drastic deterioration of mitochondrial membranes. The release of matrix enzymes is paralleled by formation of products of lipid peroxidation. The enzyme reacts with isolated phospholipds and free cis-unsaturated fatty acids. Some properties were determined: molecular weight, isoelectric point, temperature and pH-dependence and Km value for linoleic acid. The enzyme is inhibited by reaction products and a variety of inhibitors, especially antioxidants and chelating agents.     

Selectivity of action of the lipoxygenase from rabbit reticulocytes on mitochondria and erythrocyte membranes]

Whereas the lipoxygenase from rabbit reticulocytes caused a large formation of malonyl dialdehyde (MDA) with rat liver mitochondria, erythrocyte ghosts were attacked only slightly independently of their type of preparation. The formation of MDA was not enhanced by release of spectrin-actin from the ghosts. The lipoxygenase did not give rise to hemolysis of intact erythrocytes. The formation of MDA was increased by heat treatment of the ghosts. Addition of cholesterol to a phospholipid emulsion inhibited the formation of MDA by the reticulocyte lipoxygenase. These results indicate that both lipid-protein interactions and the cholesterol content of the membranes may be involved in the preferential attack of the lipoxygenase on mitochondrial membranes.     

Relationship between the conformation of isolated rat liver mitochondria and their susceptibility to rabbit reticulocyte lipoxygenase]

Lipoxygenase from rabbit reticulocytes cause disruption of mitochondrial membranes and peroxidation of their lipids as judged by electronmicroscopy, release of matrix enzymes and formation of malonyldialdehyde. Without substrate mitochondria become orthodox and strong lysis by lipoxygenase appears. The lysis is prevented by ATP or ADP plus succinate; in this case mitochondria remain condensed or partly condensed. The protection by substrate was even observed in the presence of 2,4-DNP, although the mitochondria were transformed to the condensed state. Lysis was more pronounced in hypotonic than in hypertonic sucrose, condensed mitochondria are also attacked. No relation seems to exist between lipoxygenase attack and the conformational state of mitochondria. Lysis of mitochondria is dependent on the susceptibility of the fatty acid moiety of phospholipids, which may be influenced by both metabolic and structural events via alteration of protein-lipid interactions.     

Occurrence of free and esterified lipoxygenase products in leaves of Glechoma hederacea L. and other Labiatae

Leaves of Glechoma hederacea L. and other Labiatae contain (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid, (10E,12Z,15Z)-9-oxo-10,12,15-octadecatrienoic acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and (10E,12Z)-9-oxo-10,12-octadecadienoic acid in a ratio of 71/14/12/3 (by mass), predominantly esterified in the membrane ester lipids. The leaves contain the highest level of these products, whereas only small amounts were found in the stalk and the roots. The chemical structures of these compounds were established by ultraviolet and infrared spectroscopy, by co-chromatography with authentic standards on various types of HPLC columns including chiral-phase HPLC and gas chromatography/mass spectrometry. The stereochemical specificity indicates the enzymatic origin of the products, most probably via a lipoxygenase reaction. Freshly harvested specimens of G. hederacea L. contain only small amounts of hydroxy-polyenoic fatty acids. Air-drying causes a strong increase in the content of free and esterified (9S,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid. Up to 80% of the hydroxy fatty acids of the total lipid extracts were esterified in the cellular lipids. The data presented indicate that lipoxygenase products occur in the cellular ester lipids of G. hederacea L. and other Labiatae. The results are discussed in the light of a possible involvement of the lipoxygenase pathway in the natural senescence of leaves.     

Keto fatty acids not containing doubly allylic methylenes are lipoxygenase substrates

The soybean lipoxygenase I oxygenates the unusual substrate 12-keto-(9Z)-octadecenoic acid methyl ester as indicated by oxygen uptake and spectral changes of the incubation mixture. The main oxygenation products have been isolated by HPLC and identified as 9,12-diketo-(10E)-octadecenoic acid methyl ester and 12-keto-(10E)-dodecenoic acid methyl ester by UV and IR spectroscopy, cochromatography with an authentic standard, gas chromatography/mass spectroscopy, and 1H NMR. In the formation of both compounds the oxygenase and hydroperoxidase activities of the enzyme appear to be involved. These data and the earlier results on the oxygenation of furanoic fatty acids (Boyer et al., 1979) indicate that the lipoxygenase reaction is not restricted to substrates containing a 1,4-pentadiene structure.     

3,5-Di-t-butyl-4-hydroxytoluene (BHT) and probucol stimulate selectively the reaction of mammalian 15-lipoxygenase with biomembranes

The lipophilic antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) and the structurally-related antiatherogenic drug probucol stimulate the oxygenation of mitochondrial membranes and erythrocyte ghosts by the rabbit 15-lipoxygenase as indicated by an increase in oxygen consumption as well as by an enhanced loss of polyenoic fatty acids and by the formation of specific lipoxygenase products in the membrane phospholipids. The oxygenation of linoleic acid, phospholipids and human low-density lipoproteins was not stimulated. With mitochondrial membranes, BHT causes a quenching of the 1-anilino-8-naphthalene sulfonate fluorescence. Thus, it is suggested that the stimulation of membrane oxygenation may be due to structural changes in the membranes leading to a better susceptibility of the polyenoic fatty acid residues towards lipoxygenase attack. Owing to this unexpected effect of the antioxidants, which is not related to their radical-scavenger capacity, care should be taken in interpreting experimental data on effects of BHT and probucol.     

Long-chain fatty acids and ethanol affect the properties of membranes inDrosophila melanogaster larvae

The larval fatty acid composition of neutral lipids and membrane lipids was determined in three ethanol-tolerant strains ofDrosophila melanogaster. Dietary ethanol promoted a decrease in long-chain fatty acids in neutral lipids along with enhanced alcohol dehydrogenase (EC 1.1.1.1) activity in all of the strains. Dietary ethanol also increased the incorporation of¹⁴C-ethanol into fatty acid ethyl esters (FAEE) by two- to threefold and decreased the incorporation of¹⁴C-ethanol into free fatty acids (FFA). When cultured on sterile, defined media with stearic acid at 0 to 5 mM, stearic acid decreased ADH activity up to 33%. In strains not selected for superior tolerance to ethanol, dietary ethanol promoted a loss of long-chain fatty acids in membrane lipids. The loss of long-chain fatty acids in membranes was strongly correlated with increased fluidity in hydrophobic domains of mitochondrial membranes as determined by electron spin resonance and correlated with a loss of ethanol tolerance. In the ethanol-tolerant E2 strain, which had been exposed to ethanol for many generations, dietary ethanol failed to promote a loss of long-chain fatty acids in membrane lipids. We are grateful for the support of National Institutes of Health Grant AA06702 (B.W.G.) and National Science Foundation Grant CHE-891987 (R.G.K.).     

Characteristics of fatty acid composition of lipids in higher plant vacuolar membranes

The fatty acid composition of vacuolar membrane lipids from plant storage tissues and their genesis have been studied. A high content of unsaturated fatty acids (up to 77%) was observed in lipids of these membranes. Linoleic acid prevailed in vacuolar lipids of carrot and red beet (54.2 and 44.2%, respectively). Linolenic acid prevailed in vacuolar lipids of garden radish and turnip (39.7 and 33.9%, respectively). Regarding saturated fatty acids, vacuolar lipids of garden radish, carrot, and red beet contained predominantly palmitic acid (up to 20-24%). Unsaturated fatty acids, petroselinic (C18: 1omega12), cis-vaccenic (C18: 1omega7), hexatrien-7,-10,-13-oic (C16:3omega3) and others, were observed in vacuolar lipids of roots. These acids are usually synthesized in chloroplasts, and their presence in vacuolar lipids can be associated either with the transport of metabolites to the vacuole, or with endocytosis during vacuolar formation in the plant cell. The specific features of fatty acid composition of tonoplast lipids apparently are closely related to the tonoplast unique fluidity and mobility required for running osmotic processes in the cell and for forming transport protein assemblies.     

Incorporation of myristate and palmitate into the sheep reticulocyte transferrin receptor: evidence for identical sites of labeling

The ability of sheep reticulocytes and plasma membranes isolated from them to incorporate fatty acids into the transferrin receptor has been examined using both [3H]palmitate and [3H]myristate. Both fatty acids, when incorporated into the transferrin receptor, can be released by treating the protein with 1 M hydroxylamine at pH 7.0. After treatment of the 3H-acylated receptor with borohydride, an 3H-labeled alcohol is released, suggesting that the receptor-bound fatty acid is in thioester linkage. With both [3H]myristate and [3H]palmitate, Cleveland maps from immunoprecipitates of the transferrin receptor labeled in intact cells and isolated membranes show that identical peptides are labeled. No evidence was obtained for qualitatively different labeling with the two fatty acids. In intact reticulocytes, incorporation of [3H]palmitate into the transferrin receptor is approximately 3.5 times greater than the incorporation of [3H]myristate from equivalent concentrations of the labeled fatty acids. However, in isolated reticulocyte plasma membranes, there is much less difference between palmitate and myristate incorporation (with ATP) or between their acyl-CoA derivatives. The reason for the discrepancy between cells and membranes is unknown but may be due to the presence in intact cells of more than one enzyme for activating the fatty acids. Acylation of the receptor in isolated plasma membranes is fourfold greater with the CoA derivatives than with the free fatty acids. The fatty acid activating enzyme(s) as well as the acyltransferase(s) appear to be membrane bound in reticulocytes.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

Occurrence of the erythroid cell specific arachidonate 15-lipoxygenase in human reticulocytes

Human reticulocytes obtained from patients suffering from various haemolytic disorders convert exogenous [1-14C]-arachidonic acid to 15-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE). Immunological studies (dot blot, Western blot) indicated that human reticulocytes contain a lipoxygenase which cross-reacts with a polyclonal antiserum against the rabbit reticulocyte lipoxygenase. Northern blotting with a cloned lipoxygenase cDNA probe shows that the specific mRNA is also present. Reaction of the lipoxygenase with submitochondrial particles caused inactivation of respiratory enzymes. The occurrence of an erythroid cell specific lipoxygenase of similar type in reticulocytes of various mammals and man suggests the general role of this enzyme in the maturational degradation of mitochondria.     

Study of the action of nystatin on the plasmatic membranes of the liver of rabbits]

The effect of mystatin on the plasmic membranes of the rabbit liver after intravenous administration of the antibiotic to the animals in a dose of 5 mg/kg was studied. It was found that intravenous administration of nystatin had no effect on the quantitative content of protein, lipids and nucleic acids in the plasmic membranes of the liver. The method of electrophoresis in polyacrylamide gel revealed significant changes in the composition of the liver membrane protein due to the treatment with nystatin. The effect of nystatin on the composition of lipids and fatty acids contained in the membrane lipids was also investigated. The data of the thin layer chromatography showed that nystatin did not affect the qualitative composition and the content of separate lipid fractions in the lipids of the liver plasmic membranes. However, the fatty acid analysis of the membrane lipids after intravenous administration of nystatin revealed a number of qualitative and quantitative differences in the composition of the lipid fatty acids of the membranes tested. The results showed that nystatin affected the membrane structures of the rabbit liver cells.     

Lipid composition of organelles from germinating castor bean endosperm

Glyoxysome, endoplasmic reticulum, mitochondria, and proplastid fractions were isolated from endosperm of castor beans (Ricinus communis) germinated for 5 days at 30 C. Samples from sucrose density gradients were diluted with 0.15 m KCI and the membranes pelleted. Lipid extracts of these membranes were analyzed for phosphoglyceride, acyl lipid, and sterol content. The endoplasmic reticulum contains 1.24 mumol of phosphoglyceride per mg of protein; the mitochondria, 0.65 mumol/mg; and the glyoxysome membranes, 0.55 mumol/mg. Phosphatidyl choline and phosphatidyl ethanolamine are the most abundant lipids in all membranes studied, accounting for 70% or more of the lipid phosphorus and 50% or more of the fatty acid. Glyoxysome membranes and endoplasmic reticulum also contain phosphatidyl inositol (respectively, 9 and 17% of the lipid phosphorus) and free fatty acids (13% of the total fatty acid in each). Compared with other organelles, mitochondrial membranes have more phosphatidyl ethanolamine relative to phosphatidyl choline and are characterized by the presence of cardiolipin, in which 80% of the fatty acid is linoleate. The relative amounts of linoleate, palmitate, oleate, stearate, and linolenate in each of the phosphotoglycerides are constant regardless of the membrane source. Stimasgasterol and beta-sitosterol are present in the membranes (1-9 nmol each/mg protein).The data provide further evidence that glyoxysome membranes are derived from the endoplasmic reticulum but at the same time indicate some differentiation.