Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.     

cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium

Bovine corneal epithelium contains arachidonate 12- and 15-lipoxygenase activity, while human corneal epithelium contains only 15-lipoxygenase activity. Our purpose was to identify the corneal 12- and 15-lipoxygenase isozymes. We used cDNA cloning to isolate the amino acid coding nucleotide sequences of two bovine lipoxygenases. The translated sequence of one lipoxygenase was 82% identical with human 15-lipoxygenase type 2 and 75% identical with mouse 8-lipoxygenase, whereas the other translated nucleotide sequence was 87% identical with human 12-lipoxygenase of the platelet type. Expression of 15-lipoxygenase type 2 and platelet type 12-lipoxygenase mRNAs were detected by Northern analysis. In addition to these two lipoxygenases, 12-lipoxygenase of leukocyte (tracheal) type was detected by polymerase chain reaction (PCR), sequencing, and Northern analysis. Finally, PCR and sequencing suggested that human corneal epithelium contains 15-lipoxygenase types 1 and 2.     

cDNA cloning of 15-lipoxygenase type 2 and 12-lipoxygenases of bovine corneal epithelium1

Bovine corneal epithelium contains arachidonate 12- and 15-lipoxygenase activity, while human corneal epithelium contains only 15-lipoxygenase activity. Our purpose was to identify the corneal 12- and 15-lipoxygenase isozymes. We used cDNA cloning to isolate the amino acid coding nucleotide sequences of two bovine lipoxygenases. The translated sequence of one lipoxygenase was 82% identical with human 15-lipoxygenase type 2 and 75% identical with mouse 8-lipoxygenase, whereas the other translated nucleotide sequence was 87% identical with human 12-lipoxygenase of the platelet type. Expression of 15-lipoxygenase type 2 and platelet type 12-lipoxygenase mRNAs were detected by Northern analysis. In addition to these two lipoxygenases, 12-lipoxygenase of leukocyte (tracheal) type was detected by polymerase chain reaction (PCR), sequencing, and Northern analysis. Finally, PCR and sequencing suggested that human corneal epithelium contains 15-lipoxygenase types 1 and 2.     

Demonstration of a 12-lipoxygenase activity in bovine polymorphonuclear leukocytes

In this study we present evidence for the existence of an intrinsic 12-lipoxygenase in the bovine polymorphonuclear leukocyte which differs from the well-known platelet 12-lipoxygenase. Intact bovine polymorphonuclear leukocytes synthesize predominantly 5-lipoxygenase products. However, this 5-lipoxygenase activity disappears completely upon sonication of the cells, whereas a 12-lipoxygenase activity then becomes apparent. This 12-lipoxygenase resembles the platelet 12-lipoxygenase in metabolizing arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and in being independent of Ca2+ as well as of ATP. The most striking difference between the two 12-lipoxygenases is their behaviour towards linoleic acid. While the platelet 12-lipoxygenase does not convert linoleic acid, the 12-lipoxygenase from bovine polymorphonuclear leukocytes, apparent only in the cell-free system, converts linoleic acid into 13-hydroxyoctadecadienoic acid as efficiently as it converts arachidonic acid into 12-hydroxyeicosatetraenoic acid. This provides a convenient method to distinguish both 12-lipoxygenase activities. The fact that this new 12-lipoxygenase is able to metabolize linoleic acid into 13-hydroxyoctadecadienoic acid suggests that this enzyme, in contrast to platelet 12-lipoxygenase, resembles 5-lipoxygenases in showing a preference for hydrogen abstraction at a position which is determined by the distance to the carboxylic end of the fatty acid.     

Arachidonate 15-lipoxygenase in human corneal epithelium and 12- and 15-lipoxygenases in bovine corneal epithelium: Comparison with other bovine 12-lipoxygenase

Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-lipoxygenase and a 15-lipoxygenase. The 12-lipoxygenase was found in the microsomal fraction, while the 15-lipoxygenase was mainly present in the cytosol (100 000 × g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosa-tetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 μ M. IC₅₀ for the 12-lipoxygenase was 0.3 μM. The bovine corneal 12-lipoxygenase was compared with the 12-lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized ¹⁴C-labelled linoleic acid and α-linolenic acid poorly (5–16%) in comparison with [^l4C]arachidonic acid. [¹⁴C]Docosahexaenoic acid and [¹⁴C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyconal antibody against porcine leukocyte 12-lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [¹⁴C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-lipoxygenase and that bovine corneal epithelium contains both a 15-lipoxygenase and a 12-lipoxygenase. The corneal 12-lipoxygenase appears to differ catalytically from earlier described bovine 12-lipoxygenases.     

Two immunologically and catalytically distinct arachidonate 12-lipoxygenases of bovine platelets and leukocytes

12-Lipoxygenases were found in the cytosol fraction of bovine leukocytes and platelets. The bovine leukocyte enzyme was immunoprecipitable by a monoclonal antibody directed to 12-lipoxygenase of porcine leukocytes, but not by a monoclonal antibody against the human platelet enzyme. In contrast, the bovine platelet enzyme cross-reacted only with antibody against the human platelet enzyme. The leukocyte and platelet enzymes were partially purified to final specific enzyme activities of 1.1 and 0.3 mumol/min/mg protein, respectively, by immunoaffinity chromatography using each cross-reacting antibody as a ligand. The leukocyte enzyme reacted with various octadecapolyenoic acids as well as eicosapolyenoic and docosapolyenoic acids, whereas the platelet enzyme was almost inactive with octadecapolyenoic acids. Moreover, the two enzymes showed different heat-instabilities and reaction time courses. Thus, the 12-lipoxygenases of bovine leukocytes and platelets were immunologically and catalytically distinct enzymes.     

15-Lipoxygenation of leukotriene A(4). Studies Of 12- and 15-lipoxygenase efficiency to catalyze lipoxin formation

The unstable epoxide leukotriene (LT) A(4) is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA(4) to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100000xg supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100000xg supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K(m) for LTA(4) (9-27 microM), as compared to the other lipoxygenases tested (58-83 microM). The recombinant human 12-lipoxygenase demonstrated the lowest K(m) value for LTA(4) (9 microM) whereas the porcine leukocyte 12-lipoxygenase had the highest V(max). The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA(4) and 6S-LXA(4) together with the all-trans LXA(4) and LXB(4) isomers were isolated. Production of LXB(4) was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate was considerably more efficient to inhibit conversion of LTA(4) to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC(50) 1 and 50 microM, respectively) in the human platelet cytosolic fraction.     

Arachidonate 5-lipoxygenase of porcine pancreas: its localization in acinar cells

Arachidonate 5-lipoxygenase has been found so far in various types of leukocyte. When a homogenate of porcine pancreas was incubated with arachidonic acid, 5-hydroxy-6,8,11,14-eicosatetraenoic acid was predominantly produced concomitant with small amounts of compounds derived from leukotriene A4. After differential centrifugation of the homogenate, the 5-lipoxygenase activity was found predominantly in the 1000 x g pellet and 105,000 x g supernatant. When porcine pancreas was investigated immunohistochemically with anti-5-lipoxygenase antibody, Langerhans islets were unstained, and infiltration of 5-lipoxygenase-positive leukocytes was hardly observed. In contrast, acinar cells were positively stained. Immunoelectron microscopy demonstrated the localization of the enzyme along the nuclear membranes of the acinar cells.     

Localization of Arachidonate 12-Lipoxygenase in Canine Brain Tissues

The cytosol fraction from a thoroughly irrigated canine cerebrum was subjected to immunoaffinity chromatography using a monoclonal antibody against porcine leukocyte 12-lipoxygenase. Arachidonate 12-lipoxygenase eluted from the column with some retardation. The enzyme, with a specific activity of 9 nmol/min/mg of protein, converted arachidonic acid to 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid. The enzyme was active not only with arachidonic acid, but also with linoleic and alpha-linolenic acids. In contrast, 12-lipoxygenase of canine platelets was almost inactive with linoleic and alpha-linolenic acids, and the platelet enzyme was also distinguished from the cerebral enzyme in terms of reactivity with the anti-12-lipoxygenase antibody. 12-Lipoxygenase activity was also detected in the cytosol fractions of other parts of canine brain: basal ganglia, hippocampus, cerebellum, olfactory bulb, and medulla oblongata.     

Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.     

Purification of arachidonate 5-lipoxygenase from porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 5-lipoxygenase was purified to near homogeneity from the 105,000 X g supernatant of porcine leukocyte homogenate by immunoaffinity chromatography using a monoclonal anti-5-lipoxygenase antibody. Reaction of the purified enzyme with arachidonic acid produced predominantly 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid with concomitant formation of several more polar compounds in smaller amounts. These minor products were identified as the degradation products of leukotriene A4, namely, 6-trans-leukotriene B4 (epimeric at C-12) and an epimeric mixture of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids. These compounds were also produced by reaction of the enzyme with 5-hydroperoxy-eicosatetraenoic acid. Association of the 5-lipoxygenase and leukotriene A synthase activities was demonstrated by several experiments: heat inactivation of enzyme, effect of selective 5-lipoxygenase inhibitors, requirements of calcium ion and ATP, and self-catalyzed inactivation of enzyme. The enzyme was also active with 12- and 15-hydroperoxy-eicosatetraenoic acids producing (5S,12S)- and (5S,15S)-dihydroperoxy acids, respectively. Maximal velocities of the reactions with these hydroperoxy acids as compared with that of arachidonic acid (100%, 0.6 mumol/3 min/mg of protein) were as follows: 5-hydroperoxy acid, 3.5%, 12-hydroperoxy acid, 22%, and 15-hydroperoxy acid, 30%.     

Translocation of 5-lipoxygenase to the membrane in human leukocytes challenged with ionophore A23187

Challenge of human peripheral blood leukocytes with ionophore A23187 resulted in leukotriene (LT) synthesis, a decrease in total cellular 5-lipoxygenase activity, and a change in the subcellular localization of the enzyme. In homogenates from control cells, greater than 90% of the 5-lipoxygenase activity and protein was localized in the cytosol (100,000 X g supernatant). Ionophore challenge (2 microM) resulted in a loss of approximately 55% of the enzymatic activity and 35% of the enzyme protein from the cytosol. Concomitantly, there was an accumulation of inactive 5-lipoxygenase in the membrane (100,000 X g pellets) which accounted for at least 45% of the lost cytosolic protein. There was a good correlation between the quantities of LT synthesized and 5-lipoxygenase recovered in the membrane over an ionophore concentration range of 0.1-6 microM. The time course of the membrane association was similar to that of LT synthesis. Furthermore, although the pellet-associated enzyme recovered from ionophore-treated leukocytes was inactive, an irreversible, Ca2+-dependent membrane association of active 5-lipoxygenase could be demonstrated in cell-free systems. To determine whether ionophore treatment induced proteolytic degradation of 5-lipoxygenase, the total activity and protein content of 10,000 X g supernatants from control and ionophore-treated cells were examined. These supernatants, which included both cytosolic and membrane-associated enzyme, showed a 35% loss of 5-lipoxygenase activity but only an 8% loss of enzyme protein as a result of ionophore challenge (2 microM). Therefore, the majority of the loss of 5-lipoxygenase activity was most likely due to suicide inactivation during the LT synthesis, rather than to proteolytic degradation. Together these results are consistent with the hypothesis that ionophore treatment results in a Ca2+-dependent translocation of 5-lipoxygenase from the cytosol to a membrane-bound site, that the membrane-associated enzyme is preferentially utilized for LT synthesis, and that it is consequently inactivated. Thus, membrane translocation of 5-lipoxygenase may be an important initial step in the chain of events leading to full activation of this enzyme in the intact leukocyte.     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins

Arachidonate 12-lipoxygenases of porcine and bovine leukocytes were different in substrate specificity and immunogenicity from the enzyme of bovine platelets (Arch. Biochem. Biophys. (1988) 266, 613). In order to extend the comparative studies on the two types of 12-lipoxygenase, we purified the enzyme from the cytosol of human platelets by immunoaffinity chromatography to a specific activity of about 0.3 mumol/min per mg protein at 37 degrees C. The purified enzyme was active with eicosapolyenoic acids and docosahexaenoic acid. Linoleic and linolenic acids were poor substrates in contrast to the high reactivity of the leukocyte enzymes with these octadecapolyenoic acids. The finding that the human platelet enzyme catalyzed 15-oxygenation of 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, raised a question if lipoxins were produced by incubation of the enzyme with leukotriene A4. However, the leukotriene A4 was scarcely transformed to lipoxin isomers by 12-lipoxygenases of human and bovine platelets. In sharp contrast, the porcine and bovine leukocyte enzymes converted leukotriene A4 to various lipoxin isomers by the reaction rates of 3% and 2% of the arachidonate 12-oxygenation. Thus, 12-lipoxygenases of human and bovine platelets were catalytically distinct from the porcine and bovine leukocyte enzymes in terms of their reactivities not only with linoleic and linolenic acids, but also with leukotriene A4 as lipoxin precursor.     

Cloning and characterization of a murine macrophage lipoxygenase

We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxygenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.     

Characterization of 12-L-hydroperoxyeicosa-5,8,10,14-tetraenoic acid peroxidase in platelets by monoclonal antibody against glutathione peroxidase

In the present investigation, 12-L-hydroxyeicosa-5,8,14-tetraenoic acid (12-HPETE) peroxidase in the platelet 12-lipoxygenase pathway was characterized by using a monoclonal antibody to erythrocyte glutathione peroxidase. Pure glutathione peroxidase was used for the immunization of mice. Monoclonal antibody directed against the erythrocyte glutathione peroxidase was obtained from hybridomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a mouse immunized with the enzyme. The subclass of monoclonal antibody was immunoglobulin M with kappa-light chain. Enzyme activity assays using cumene hydroperoxide and [1-14C]12-HPETE as substrates were employed. The monoclonal antibody reacted with glutathione peroxidase in the cumene hydroperoxide assay. In order to see whether platelet 12-HPETE peroxidase reacts with the monoclonal antibody, platelet cytosol and glutathione peroxidase were incubated with the monoclonal antibody and the antibody was precipitated by goat anti-mouse immunoglobulin M. The activities of platelet 12-HPETE peroxidase and glutathione peroxidase remaining were then assayed by using [1-14C]12-HPETE as substrate. The ability of glutathione peroxidase to transform 12-HPETE to 12-HETE was removed by the monoclonal antibody; however, the activity of platelet cytosol was not removed by the antibody. The results indicated that the antigenic specificity of 12-HPETE peroxidase in the platelet 12-lipoxygenase pathway is different from that of erythrocyte glutathione peroxidase.     

Expression of arachidonate 12-lipoxygenase in rat tissues: a possible role in glucagon secretion.

There are three isoforms of arachidonate 12-lipoxygenase in mammals: platelet, leukocyte, and epidermal types. We found in this study that the leukocyte-type enzyme was present in rat pineal gland, lung, spleen, aorta, adrenal gland, spinal cord, and pancreas, as assessed by RT-PCR. Immunohistochemical analysis showed that the enzyme was localized in macrophages in lung and spleen, alpha-cells of pancreatic islet, zona glomerulosa cells of adrenal cortex, and neuronal cells of spinal cord and superior cervical ganglion. The presence of the 12-lipoxygenase in pancreatic alpha-cells was confirmed by glucagon staining in a consecutive section. We overexpressed the leukocyte-type 12-lipoxygenase cDNA in a glucagon-secreting alphaTC clone 6 cell line that had been established from a transgenic mouse. Glucagon secretion was stimulated by approximately twofold in the 12-lipoxygenase-expressing cells compared to the mock-transfected and original cells. The results suggest that the 12-lipoxygenase of the leukocyte type augments glucagon secretion from pancreatic islets.     

12-Lipoxygenase from bovine polymorphonuclear leukocytes, an enzyme with leukotriene A4-synthase activity

Bovine polymorphonuclear leukocytes exhibit a 12-lipoxygenase activity upon sonication. In contrast to bovine platelet 12-lipoxygenase and other 12-lipoxygenases, this enzyme is unable to convert 5(S)-HETE (5(S)-hydroxy,6-trans-8,11,14-cis-eicosatetraenoic acid) or 5(S)-HPETE (5(S)-hydroperoxy,6-trans-8,11,14-cis-eicosatetraenoic acid) into 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid. Surprisingly, the formation of leukotriene A4-derived products namely leukotriene B4 and the leukotriene B4-isomers 12-epi,6-trans- leukotriene B4 and 6-trans-leukotriene B4, was observed upon incubation of this enzyme with 5(S)-HPETE. Hence, the 12-lipoxygenase from bovine polymorphonuclear leukocytes possesses leukotriene A4-synthase activity.     

Purification and characterization of glutathione peroxidase from human blood platelets. Age-related changes in the enzyme

Platelet glutathione peroxidase (GPx) is known to play a pivotal role in controlling the level of lipid hydroperoxides, especially those resulting from the 12-lipoxygenase activity. GPx was purified fromm the cell cytosol by more than 700-fold using an exchange chromatography, FPLP, gel filtration and covalent fixation. Isoelectric focusing revealed a peak activity at pH 5.1. The molecular mass of enzyme was found between 90 and 100 kDa by gel filtration, and was approximating at 23kDa by SDS-PAGE. A polyclonal antibody raised against commercial bovine erthrocyte GPx recognized the human platelet enzyme. It is concluded that human platelet GPx is likely a homotetramer of 92 kDa as described for most sources. We have also found that the decreased platelet GPx activity observed in platelets from elederly people is associated with a lower content of the immunoreactive enzyme.     

Lipoxygenase activities of human platelets and their subcellular fractions: Comparison between lipoxygenase-deficient platelets and normal platelets

Lipoxygenase activities were estimated in washed platelets (intact platelets) and their subcellular fractions obtained from 7 patients with deficient platelet lipoxygenase activities and 9 normal subjects. From sonicated platelet preparations, 12,000 g supernatant (F-I), cytosol (F-II) and microsomal fractions (F-III) were prepared by differential centrifugation. When 12-hydroxyeicosatetraenoic acid (12-HETE) produced by the incubation of arachidonic acid with intact platelets or each of their subcellular fractions from normal subjects was measured by reversed-phase high-performance liquid chromatography analysis, the lipoxygenase activities of F-I, F-II and F-III were 87%, 31% and 17%, respectively, of the enzyme activity of intact platelets. One of the patients showed no detectable lipoxygenase activity in any preparation, while the other patients showed reduced enzyme activities in all preparations. The addition of CaCl2 significantly increased 12-HETE synthesis solely by F-I from these patients. In most of these patients, contrary to normal subjects, it appeared that the lipoxygenase activity was not fully expressed in intact platelets, since the F-I produced more 12-HETE than the intact platelets.