The Lipoxygenase of Reticulocytes

Under conditions leading to inactivation of reticulocyte lipoxygenase by 13~~-hydroperoxylinoleic acid a single methionine, presumably in the active center of the enzyme, is oxidized to methionine sulfoxide.Reticulocyte lipoxygenase catalyzes two successive reactions: firstly the n-6-oxygenation of fatty acids with more than one double bond [I, 21 and secondly the splitting of the hydroperoxy compounds formed by the first reaction [3]. The lipohydroperoxidation reaction is accompanied by a strongly temperature-dependent irreversible self-inactivation of the enzyme. It was also shown that nearly stoichiometric amounts of hydroperoxylinoleate were sufficient to inactivate the enzyme [3]. It appeared likely that the hydroperoxide oxidized selectively one amino acid residue. In contrast the lipoxygenase type I from soybeans does not exhibit selfinactivation.In the present communication it is shown that under conditions leading to inactivation of reticulocyte lipoxygenase, a single methionine presumably in the active center of the enzyme is oxidized to methionine sulfoxide. MATERIALS AND METHODSLipoxygenase from rabbit reticulocyte was purified according to [2]. Soybean lipoxygenase (grade I) was obtained from Sigma (St. Louis). 13r.,-Hydroperoxylinoleic acid was prepared by aerobic incubation of linoleic acid (99 % pure, Fluka) with soybean lipoxygenase in 0.1 M sodium borate buffer, pH 9.0 [4] and purified by preparative thin-layer chromatography on 0.25-mm silica gel pre-coated plates, as described earlier [3]. The concentration of 13~~-hydroperoxylinoleic acid was determined spectrophotometrically by measuring the absorption at[35S]Methionine with a specific radioactivity of about 37 MBq/nmol was obtained from the Radiochemical Centre (Amersham, UK).Lipoxygenase activity was monitored at 2 "C by the modified method of Holman [6] by measuring the increase in absorbance of conjugated dienes at 234 nm. Protein content was determined spectrophotometrically using the absorption coefficient at 280nm of 1.68Reaction with 13~~-hydroperoxylinoleic acid All incubations were performed under strictly anaerobic conditions. The reticulocyte lipoxygenase was reacted with Enzyme. Lipoxygenase (EC 1.13.11.12).13~,-hydroperoxylinoleic acid in 0.2 M potassium phosphate buffer, pH 7.8. After incubation at 37 "C or 2 "C, the excess of 131,~-hydroperoxylinoleic acid was removed by addition of excess of potassium iodide and sodium thiosulfate in 1 M sodium acetate buffer, pH 5.0, and incubated at 2 "C for 2.5 h according to the modified method of Hicks [7]. The enzyme was precipitated by addition of 10 % trichloroacetic acid, centrifuged, washed with absolute ethanol, absolute ethanolldiethyl ether (2: 1) and diethyl ether. Control incubations were carried out with soybean lipoxygenase. Methionine sulfoxide determinationProtein samples (0.5 -1 mg) were hydrolyzed under nitrogen atmosphere in sealed borosilicate glass vials with 1 ml 4 M NaOH at 110°C for 16h. Analysis for methionine sulfoxide was performed on a Biocal BC200 amino acid ...

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“…Lipoxygenase from rabbit reticulocyte was purified according to [2]. Soybean lipoxygenase (grade I) was obtained from Sigma (St. Louis).…”

Section: Methodsmentioning

confidence: 99%

Methionine sulfoxide formation: the cause of self‐inactivation of reticulocyte lipoxygenase

Rapoport

Härtel

Hausdorf

1984

European Journal of Biochemistry

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“…The actions of reticulocyte lipoxygenase on both free polyene acids and phospholipids are accompanied by its irreversible inactivation, particularly at temperatures higher than 20 "C [2]. This self-inactivation is probably involved in the loss of activity during the maturation of reticulocytes [2]. The aim of this study was to elucidate the mechanism of the selfinactivation of reticulocyte lipoxygenase.…”

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confidence: 99%

“…Like soybean-lipoxygenase-1 it converts linoleic acid predominately to 13-hydroperoxylinoleic acid (R. Wiesner and H. Kiihn, unpublished work) and arachidonic acid to (1 5s)-hydroperoxyeicosatetraenoic acid [4]. The actions of reticulocyte lipoxygenase on both free polyene acids and phospholipids are accompanied by its irreversible inactivation, particularly at temperatures higher than 20 "C [2]. This self-inactivation is probably involved in the loss of activity during the maturation of reticulocytes [2].…”

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confidence: 99%

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Self‐Inactivation by 13‐Hydroperoxylinoleic Acid and Lipohydroperoxidase Activity of the Reticulocyte Lipoxygenase

Härtel

Ludwig

Schewe

et al. 1982

European Journal of Biochemistry

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1. The self-inactivation of lipoxygenase from rabbit reticulocytes with linoleic acid at 37 "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 "C. The products were 13-hydroxylinoleic acid, oxodienes and compounds of non-diene structure similar to those produced by soybean lipoxygenase-I .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 "C, but not at 37°C; it is assumed that the ferric form of reticulocyte lipoxygenase suffers inactivation.

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“…Rabbit reticulocytes exhibit an antimycin-A-resistant oxygen uptake which was partly inhibited by various lipoxygenase inhibitors. This partial inhibition amounts to about 5% of the total oxygen uptake [9]. Recently the occurrence of oxygenated polyenoic fatty acids in the membranes of rabbit reticulocytes has been reported [lo], which indicates the intracelluIar action of the lipoxygenase on membrane lipids.…”

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Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes*

Kühn

Belkner

Wiesner

1990

European Journal of Biochemistry

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Mitochondria1 membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,1 1E)-(l3S)-13-hydroxy-9,1l-octadecadienoic acid, 9 s and 9R isomers of (IOE,12Z)-9-hydroxy-10,12-octadecadienoic acid and their all-E isomers. Furthermore (5Z,8Z,112,13E)-(1 5S)-15-hydroxy-5,8,11,13-icosatetraenoic acid, 9-and 13-oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co-chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15-lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (92,llE)-(13S)-13-hydroxy-9,1 I-octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific; a complex mixture of all positional and optical isomers was detected.The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.Reticulocytes of different species [l, 21 contain a lipoxygenase which is able to oxygenate polyenoic fatty acids esterified to membrane lipids [3]. This enzyme was proposed to play an important role in the breakdown of mitochondria during the maturation of red blood cells [4]. Free arachidonic acid was converted by the pure enzyme to a mixture of (52,8Z,lIZ,-13E)- (13S) In contrast to our knowledge of the properties of the isolated enzyme, little is known about the intracellular action of the lipoxygenase. Rabbit reticulocytes exhibit an antimycin-A-resistant oxygen uptake which was partly inhibited by various lipoxygenase inhibitors. This partial inhibition amounts to about 5% of the total oxygen uptake [9]. Recently the occurrence of oxygenated polyenoic fatty acids in the membranes of rabbit reticulocytes has been reported [lo], which indicates the intracelluIar action of the lipoxygenase on membrane lipids.In this study, we report the subcellular distribution of the oxygenated polyenoic fatty acids in various membrane fractions of rabbit reticulocytes and in reticulocytes at different stages of maturation.

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The Lipoxygenase of Reticulocytes

Cited by 265 publications

References 43 publications

Methionine sulfoxide formation: the cause of self‐inactivation of reticulocyte lipoxygenase

Methionine sulfoxide formation: the cause of self‐inactivation of reticulocyte lipoxygenase

Self‐Inactivation by 13‐Hydroperoxylinoleic Acid and Lipohydroperoxidase Activity of the Reticulocyte Lipoxygenase

Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes*

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