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1.
The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000–150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent km of 166 μM and a Vmax of 5.3 μmol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex.  相似文献   

2.
The enzyme responsible for 15-lipoxygenation of arachidonic acid was purified to homogeneity from human eosinophil-enriched leukocytes using a combination of ammonium sulfate precipitation, hydrophobic interaction chromatography, and high pressure liquid chromatography on hydroxyapatite and cation-exchange columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein revealed a single major band (apparent Mr 70,000). Amino acid sequence analysis yielded a single N-terminal sequence. Comparison of the N-terminal 15 residues reveals 71% sequence identity to the rabbit reticulocyte lipoxygenase and 36% sequence identity to the rat basophilic leukemia 5-lipoxygenase. In contrast, sequence identity to the soybean lipoxygenase-1 is not observed. These results demonstrate that human 15-lipoxygenase can be isolated from eosinophil-enriched leukocytes and is accessible for direct sequence analysis. Furthermore, we present initial evidence that the mammalian lipoxygenases constitute an homologous family of enzymes. The availability of homogeneous human 15-lipoxygenase will play a key role in elucidating other relationships in this family of enzymes.  相似文献   

3.
Previous work showed that rabbit aorta metabolizes arachidonic acid via 15-lipoxygenase to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes an enzymatic rearrangement to 11-hydroxy-14,15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids (THETAs). Endothelial cells have several heme-containing enzymes including cytochromes P450 (CYP), nitric oxide synthase (eNOS), and prostacyclin (PGI(2)) synthase that catalyze the rearrangement of 15-HPETE to HEETAs. Incubation of arachidonic acid and 15-lipoxygenase, or 15-HPETE with rabbit aortic microsomes or rat liver microsomes, a rich source of CYP, resulted in the formation of a product that comigrated with THETAs and HEETAs on HPLC. Immunoblot analysis showed the presence of CYP2C8 and CYP2J2 in aortic tissue and when CYP2J2 or CYP2C8 was incubated with arachidonic acid and 15-lipoxygenase, the major products were 11,12,15- and 11,14,15-THETAs. Incubation of purified hematin, CYP2C11, eNOS or PGI(2) synthase enzymes with arachidonic acid and 15-lipoxygenase produced a different pattern of metabolites from rabbit aortic microsomes. Clotrimazole, a non-specific CYP inhibitor, and ebastine and terfenadone, specific CYP2J2 inhibitors, blocked the ability of aortic microsomes to produce THETAs while specific inhibitors of PGI(2) synthase, eNOS or CYP2C8/2C9 had no effect on THETA production. We suggest that a CYP, possibly CYP2J2, may function as the hydroperoxide isomerase converting 15-HPETE to HEETAs in rabbit vascular tissue. Further hydrolysis of the epoxy group of the HEETAs results in the formation of 11,12,15- and 11,14,15-THETAs. The HEETAs and THETAs are both vasodilators and may function as important regulators of vascular tone.  相似文献   

4.
Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.  相似文献   

5.
15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.  相似文献   

6.
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.  相似文献   

7.
We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.  相似文献   

8.
Two different proteins with arachidonate 15-lipoxygenase activity have been purified to near homogeneity from human leukocytes. Both have the same molecular mass (74 kDa) on SDS/PAGE and appear to be equally active with three different fatty acid substrates. The N-terminal amino acid sequences of both forms were identical to the sequence of human reticulocyte 15-lipoxygenase [Sigal, E., Craik, C.S., Highland, E., Grunberger, D., Costello, L.L., Dixon, R.A.F. & Nadel, J.A. (1988) Biochem. Biophys. Res. Commun. 157, 457-464]. The two forms of 15-lipoxygenase could be clearly separated by cation-exchange chromatography. Of particular interest, the relative amounts of the two forms differed markedly between leukocytes obtained from normal donors and leukocytes from an individual with eosinophilia.  相似文献   

9.
应用RT-PCR方法从人淋巴细胞中扩增出亲环素B(cyclophilinB,CyPB)基因,克隆入pET-28a载体中表达.表达产物以包涵体形式存在,占细菌可溶性蛋白的15%.经Ni-NTA树脂金属螯合亲和层析和SephadexG-50柱层析纯化,SDS-PAGE检测呈单一条带,毛细管电泳为单一色谱峰,纯度达95%.经复性处理,表达产物显示肽基脯氨基顺反异构酶活性  相似文献   

10.
Treatment of adipocytes with okadaic acid (a specific inhibitor of type 1 and 2a protein phosphatases) resulted in a rapid 8-10-fold stimulation of cell extract myelin basic protein (MBP) kinase activity (t1/2 = 10 min) and kinase activity toward a synthetic peptide RRLSSLRA (S6 peptide) (t1/2 = 5 min). Insulin brought about a smaller stimulation of these two activities (t1/2 = 2.5 min). MBP kinase activity from cells treated with okadaic acid or insulin was resolved by anion exchange chromatography into two well defined peaks; S6 peptide kinase activity was less well resolved. The two partially purified MBP kinases were inactivated by the protein tyrosine phosphatase CD45 or by protein phosphatase 2a (PP-2a). In contrast, partially purified S6 peptide kinase activity was inactivated only by PP-2a or protein phosphatase 1 (PP-1). Furthermore, a 38-kDa protein which co-eluted with one peak of MBP kinase and a 42-kDa protein which co-eluted with the other peak of MBP kinase were phosphorylated on tyrosine after treatment with okadaic acid. These findings illustrate several important points concerning regulation of MBP and S6 peptide kinases. First, these protein kinases are regulated by phosphorylation, and, second, in the absence of hormonal stimuli their activities are strongly suppressed by protein phosphatases. Lastly, the increased tyrosine phosphorylation accompanying the activation of MBP kinases following okadaic acid treatment suggests a role for PP-2a in events that are mediated by tyrosine phosphorylation.  相似文献   

11.
An enzyme, which catalyses the isomerisation of cis-3-enals to trans-2-enals, has been partially purified from cucumber fruit. The isomerase activity has been resolved from significant contamination by the related activities, lipoxygenase and hydroperoxide cleavage enzymes. An examination of the substrate specificity of the isomerase enzyme showed it to be specific for the cis-3-enals. The most efficient isomerisation was achieved with cis-3-hexenal and cis-3-nonenal which are, physiologically, the two most significant substrates. The trans-3-enal and cis-3-enol were not suitable substrates for the enzyme.  相似文献   

12.
[1-14C]Arachidonic acid was incubated with homogenates of the fungus, Saprolegnia parasitica. The products consisted of comparable amounts of two epoxy alcohols, 15-Ls-hydroxy-11,12-epoxy-5cis,8cis,13trans- eicosatrienoic acid and 15-hydroxy-13,14-epoxy-5cis,8cis,11cis-eicosatrienoic acid. Results of incubations carried out in the presence of nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid, p-hydroxymercuribenzoate as well as glutathione peroxidase plus reduced glutathione demonstrated that transformation of arachidonic acid into epoxy alcohols occurred with the formation of 15-Ls-hydroperoxy-5cis,8cis,11cis,13trans- eicosatetraenoic acid (15-HPETE) as an intermediate. The pathway involved a lipoxygenase catalyzing the oxygenation of arachidonic acid at the 15L position to produce 15-HPETE, and a hydroperoxide isomerase activity which catalyzed conversion of 15-HPETE into the two epoxy alcohols. Studies with 15-[18O2]HPETE demonstrated that both oxygens of 15-HPETE were retained in the epoxy alcohols. Furthermore, experiments with mixtures of 15-[18O2]-and 15-[16O2]HPETE showed that conversion of 15-HPETE into epoxy alcohols occurred by an intramolecular transfer of hydroperoxide oxygen.  相似文献   

13.
It was found that Pz-peptidase (assayed with 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys) and endopeptidase 24.15 (assayed with benzoyl-Gly-Ala-Ala-Phe-p-aminobenzoate) were co-purified from rat skeletal muscle, were co-eluted in high-resolution gel chromatography and co-existed in a homogeneous preparation of rat testis endopeptidase 24.15. The action of partially purified Pz-peptidase from rat testis on 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg was blocked by an inhibitor of endopeptidase 24.15, and also by a substrate of this enzyme. The partially purified enzyme hydrolysed two substrates of endopeptidase 24.15 with Km values similar to those published previously, and its action on 2,4-dinitrophenyl-Pro-Leu-Gly-Pro-Trp-D-Lys was inhibited by compounds that are considered specific for endopeptidase 24.15. We conclude that the activities previously attributed to two distinct enzymes are due to only one, and that the merging of the two literatures may lead to new lines of research.  相似文献   

14.
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.  相似文献   

15.
We examined the characteristics of an arachidonate 12-lipoxygenase in bovine tracheal epithelial cells in relation to the enzyme expressed in leukocytes and platelets. Homogenous preparations of intact or disrupted tracheal epithelial cells metabolized arachidonic acid predominantly to (12S)-hydroxyeicosatetraenoic acid, and subcellular fractionation by differential centrifugation demonstrated that the 12-lipoxygenase activity was localized predominantly to the 100,000 x g supernatant (cytosol fraction). Analysis of cytosolic enzymatic activity for pH dependence (maximum activity at pH 7.4-8.0), divalent cation effects (no dependence on cations), and kinetic characteristics (lag phase elimination by addition of hydroperoxide) exhibited similarity to leukocyte and platelet 12-lipoxygenases. Immunoprecipitation experiments demonstrated that the epithelial 12-lipoxygenase reacted with a monoclonal antibody (lox-2) directed against leukocyte 12-lipoxygenase but not with an antibody (HPLO-3) against the platelet enzyme. Immunoaffinity chromatography of the epithelial 100,000 x g supernatant fraction using lox-2 linked to Affi-Prep 10 yielded a single predominant protein band (Mr = 72,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identical in apparent mass to the bovine leukocyte lipoxygenase. Western blotting using a polyclonal antibody to leukocyte 12-lipoxygenase showed peroxidase staining of the same 72-kDa protein band. Activity assays of the purified enzymes demonstrated that substrate specificity for the epithelial 12-lipoxygenase was similar to that of the leukocyte enzyme, but the epithelial enzyme more efficiently converted 18-carbon fatty acids to the corresponding monohydroxylated conjugated dienes. We conclude that bovine tracheal epithelial cells express a 12-lipoxygenase that has immunological reactivity similar to leukocyte and distinct from platelet 12-lipoxygenase and possesses substrate specificity distinct from both enzymes. We further suggest that lipoxygenase heterogeneity may provide a basis for different functional roles for the enzyme in different cell types.  相似文献   

16.
Hydroperoxide isomerase: a new enzyme of lipid metabolism   总被引:16,自引:7,他引:9       下载免费PDF全文
An enzyme has been isolated from flaxseed (Linum usitatissimum) which utilizes the product of lipoxidase for its substrate. The enzyme, termed hydroperoxide isomerase, converts the conjugated diene hydroperoxide of linoleic acid to the corresponding monoenoic ketohydroxy fatty acid. The structure of the latter has been determined by ultraviolet, infrared, and nuclear magnetic resonance spectroscopy; periodate and permangate oxidation; gas chromatography; and thin layer chromatography. Hydroperoxide isomerase activity has also been demonstrated in crude extracts from barley (Hordeum vulgare), wheat germ (Triticum aestivum), mung beans (Phaseolus aureus), and corn (Zea mays) and from partially purified extracts of soybean (Glycine max).  相似文献   

17.
Arachidonate 15-lipoxygenase was purified from human eosinophil-enriched leukocytes after showing that 15-lipoxygenase activity was 100-fold greater in eosinophils than in neutrophils. Partial purification was achieved using ammonium sulfate precipitation, cation-exchange and hydrophobic-interaction chromatography. New evidence is presented suggesting that 15-lipoxygenase has electrostatic and hydrophobic properties distinct from 5-lipoxygenase. In addition, ATP is shown to inhibit, and phosphatidylcholine is shown to stimulate, 15-lipoxygenase, suggesting a regulatory role for these compounds in the lipoxygenation of arachidonic acid.  相似文献   

18.
The partially purified phospholipid hydroperoxide glutathione peroxidase (PHGPx) from A431 cells was used to systematically compare the inhibitory effect on the enzyme activity of various lipoxygenases and cyclooxygenases. Under the standard assay system, platelet 12-lipoxygenase, 15-lipoxygenase, and cyclooxygenase-2 were the most sensitive to the inhibition by PHGPx. 5-Lipoxygenase and cyclooxygenase-1 were less sensitive to the inhibition by PHGPx than platelet 12-lipoxygenase and cyclooxygenase-2, respectively, and the difference was approximately 10-fold. Reduction of 12(S)-hydroperoxyeicosatetraenoic acid to 12(S)-hydroxyeicosatetraenoic acid by PHGPx was observed in the presence of glutathione (GSH), and the inhibitory effect of PHGPx on 12-lipoxygenase-catalyzed arachidonate metabolism was reversed by the addition of exogenous lipid hydroperoxide. The results indicate that PHGPx directly reduced lipid hydroperoxides and then down-regulated the activity of arachidonate oxygenases. Moreover, a high-level expression of PHGPx mRNA and its 12-lipoxygenase-inhibitory activity was observed in cancer cells and endothelial cells, and these results suggest that PHGPx may play a significant role in the regulation of reactive oxygen species formation in these cells.  相似文献   

19.
We have purified two enzymic activities from flaxseed acetone powder: a lipoxygenase and a hydroperoxide dehydrase. The lipoxygenase activity belongs to an iron-containing protein having a molecular weight of 130 kDa which, upon incubation with alpha-linolenic acid, forms 13-hydroperoxy-9(Z), 11(E), 15(Z)- octadecatrienoic acid. The hydroperoxide dehydrase (a 55 kDa protein) metabolizes this hydroperoxide to an allene oxide which in turn is spontaneously hydrolyzed to alpha- and gamma-ketols. Relationships between these two enzymes were studied and results suggest an inhibition of the lipoxygenase by hydroperoxide dehydrase.  相似文献   

20.
Calcium-dependent protein kinase (CDPK) activities were detected both in the soluble and the membrane fraction of various tomato (Lycopersicon esculentum Mill.) organs, using a synthetic peptide mimicking the serine 11 phosphorylation site of a tomato sucrose synthase (SS, EC 2.4.1.13) isoform as substrate. The levels of membrane and soluble Ser-CDPK activities were differentially regulated during fruit development. The membrane Ser-CDPK activity was maximal in young fruit but decreased as the fruit developed, suggesting a specific role during fruit growth. Using an in gel assay with purified tomato SS as substrate, we showed that partially purified soluble and membrane Ser-CDPK preparations both contained a SS-kinase polypeptide of 55 kDa. The membrane and soluble Ser-CDPK activities were largely inactivated in the absence of calcium or when MgCl(2) was replaced by MnCl(2). Both soluble and membrane Ser-CDPK activities were very sensitive to staurosporine. Using Fe(III)-immobilized metal chromatography to determine the apparent phosphorylation status of the enzyme in vivo, we showed that soluble SS was largely dephosphorylated in fruits fed EGTA or staurosporine, compared to fruits fed water or sucrose. Moreover, the level of SS increased by about two-fold in the membrane fraction of fruits fed the Ser-CDPK inhibitors, compared to the control. The level of SS protein in the membrane and soluble fractions of tomato fruit was developmentally regulated, the membrane form being specifically detected in actively growing fruits. Together, our results suggest that a mechanism involving protein phosphorylation/dephosphorylation and/or calcium would in part control the association of SS isoforms with membranes in developing tomato fruit.  相似文献   

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