RESUMO
Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared. Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T. cruzi and A. thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism. In lipid particles of strains expressing the P. carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W. were detected. Microsomes from the three expressing strains retained some OSC activity. Affinity of enzymes from A. thaliana and T. cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment. A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles. Computer analysis supports the hypothesis of the structural difference since OSC from S. cerevisiae, A. thaliana, and T. cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P. carinii possesses six transmembrane domains. In the strain expressing cycloartenol synthase from A. thaliana, the accumulation of lipid particles largely exceeded that of the other strains.
Assuntos
Arabidopsis/enzimologia , Transferases Intramoleculares/metabolismo , Pneumocystis carinii/enzimologia , Saccharomyces cerevisiae/genética , Frações Subcelulares/enzimologia , Trypanosoma cruzi/enzimologia , Animais , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Transferases Intramoleculares/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoAssuntos
Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Genes de Protozoários , Oxirredutases/genética , Oxirredutases/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Esterol 14-Desmetilase , Esteróis/biossíntese , Trypanosoma brucei brucei/genéticaRESUMO
Levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis, was cloned and functionally characterized. A Ginkgo biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl diphosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Ginkgo biloba/enzimologia , Plantas Medicinais , Sequência de Aminoácidos , Ácido Aspártico/química , Catálise , Clonagem Molecular , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Deleção de Genes , Biblioteca Gênica , Modelos Químicos , Dados de Sequência Molecular , Fosfatos de Poli-Isoprenil/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
[see reaction]. Animals, fungi, and some protozoa convert oxidosqualene to lanosterol in the ring-forming reaction in sterol biosynthesis. The Trypanosoma cruzi lanosterol synthase has now been cloned. The sequence shares with the T. brucei lanosterol synthase a tyrosine substitution for the catalytically important active-site threonine found in animal and fungal lanosterol synthases.
Assuntos
Transferases Intramoleculares/metabolismo , Trypanosoma cruzi/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Domínio Catalítico , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The Arabidopsis chy1 mutant is resistant to indole-3-butyric acid, a naturally occurring form of the plant hormone auxin. Because the mutant also has defects in peroxisomal beta-oxidation, this resistance presumably results from a reduced conversion of indole-3-butyric acid to indole-3-acetic acid. We have cloned CHY1, which appears to encode a peroxisomal protein 43% identical to a mammalian valine catabolic enzyme that hydrolyzes beta-hydroxyisobutyryl-CoA. We demonstrated that a human beta-hydroxyisobutyryl-CoA hydrolase functionally complements chy1 when redirected from the mitochondria to the peroxisomes. We expressed CHY1 as a glutathione S-transferase (GST) fusion protein and demonstrated that purified GST-CHY1 hydrolyzes beta-hydroxyisobutyryl-CoA. Mutagenesis studies showed that a glutamate that is catalytically essential in homologous enoyl-CoA hydratases was also essential in CHY1. Mutating a residue that is differentially conserved between hydrolases and hydratases established that this position is relevant to the catalytic distinction between the enzyme classes. It is likely that CHY1 acts in peroxisomal valine catabolism and that accumulation of a toxic intermediate, methacrylyl-CoA, causes the altered beta-oxidation phenotypes of the chy1 mutant. Our results support the hypothesis that the energy-intensive sequence unique to valine catabolism, where an intermediate CoA ester is hydrolyzed and a new CoA ester is formed two steps later, avoids methacrylyl-CoA accumulation.
Assuntos
Arabidopsis/metabolismo , Ácidos Indolacéticos/genética , Peroxissomos/metabolismo , Tioléster Hidrolases/fisiologia , Valina/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Humanos , Ácidos Indolacéticos/química , Ácidos Indolacéticos/fisiologia , Dados de Sequência Molecular , Mutação , OxirreduçãoRESUMO
Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In mammals, both a non-sterol isoprenoid signal derived from farnesyl diphosphate (FPP) and a sterol-derived signal appear to act together to positively regulate the rate of HMGR degradation. Although the nature and number of sterol-derived signals are not clear, there is growing evidence that oxysterols can serve in this capacity. In yeast, a similar non-sterol isoprenoid signal generated from FPP acts to positively regulate HMGR degradation, but the existence of any sterol-derived signal has thus far not been revealed. We now demonstrate, through the use of genetic and pharmacological manipulation of oxidosqualene-lanosterol cyclase, that an oxysterol-derived signal positively regulated HMGR degradation in yeast. The oxysterol-derived signal acted by specifically modulating HMGR stability, not endoplasmic reticulum-associated degradation in general. Direct biochemical labeling of mevalonate pathway products confirmed that oxysterols were produced endogenously in yeast and that their levels varied appropriately in response to genetic or pharmacological manipulations that altered HMGR stability. Genetic manipulation of oxidosqualene-lanosterol cyclase did result in the buildup of detectable levels of 24,25-oxidolanosterol by gas chromatography, gas chromatography-mass spectroscopy, and NMR analyses, whereas no detectable amounts were observed in wild-type cells or cells with squalene epoxidase down-regulated. In contrast to mammalian cells, the yeast oxysterol-derived signal was not required for HMGR degradation in yeast. Rather, the function of this second signal was to enhance the ability of the FPP-derived signal to promote HMGR degradation. Thus, although differences do exist, both yeast and mammalian cells employ a similar strategy of multi-input regulation of HMGR degradation.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Regulação para Baixo , Hidrólise , Hidroximetilglutaril-CoA Redutases/genética , Transferases Intramoleculares/antagonistas & inibidores , Transferases Intramoleculares/metabolismo , Biossíntese de Proteínas , Transcrição Gênica , Ubiquitinas/metabolismoAssuntos
Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Southern Blotting , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Transferases Intramoleculares/química , Lanosterol/biossíntese , Dados de Sequência Molecular , Trypanosoma brucei brucei/genética , Leveduras/enzimologia , Leveduras/genéticaRESUMO
The Arabidopsis thaliana LUP1 gene encodes an enzyme that converts oxidosqualene to pentacyclic triterpenes. Lupeol and beta-amyrin were previously reported as LUP1 products. Further investigation described here uncovered the additional products germanicol, taraxasterol, psi-taraxasterol, and 3,20-dihydroxylupane. These results suggest that the 80 known C(30)H(50)O compounds that are structurally consistent with being oxidosqualene cyclase products may be derived from fewer than 80 enzymes and that some C(30)H(52)O(2) compounds may be direct cyclization products of oxidosqualene.
Assuntos
Arabidopsis/enzimologia , Transferases Intramoleculares/metabolismo , Esqualeno/análogos & derivados , Triterpenos/química , Triterpenos/metabolismo , Cromatografia Líquida de Alta Pressão , Ciclização , Triterpenos Pentacíclicos , Esqualeno/metabolismoRESUMO
Cycloartenol synthase converts oxidosqualene to the pentacyclic sterol precursor cycloartenol. An Arabidopsis thaliana cycloartenol synthase Ile481Val mutant was previously shown to produce lanosterol and parkeol in addition to its native product cycloartenol. Experiments are described here to construct Phe, Leu, Ala, and Gly mutants at position 481 and to determine their cyclization product profiles. The Phe mutant was inactive, and the Leu mutant produced cycloartenol and parkeol. The Ala and Gly mutants formed lanosterol, cycloartenol, parkeol, achilleol A, and camelliol C. Monocycles comprise most of the Gly mutant product, showing that an alternate cyclization route can be made the major pathway by a single nonpolar mutation.
Assuntos
Arabidopsis/enzimologia , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Substituição de Aminoácidos , Arabidopsis/genética , Ciclização , Cromatografia Gasosa-Espectrometria de Massas , Transferases Intramoleculares/genética , Lanosterol/análogos & derivados , Lanosterol/biossíntese , Lanosterol/metabolismo , Espectroscopia de Ressonância Magnética , Mutação , Fitosteróis/biossíntese , Fitosteróis/metabolismo , Prótons , Relação Estrutura-Atividade , TriterpenosRESUMO
Plants and certain protists use cycloeucalenol cycloisomerase (EC ) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein. We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Liases Intramoleculares/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Escherichia coli , Isomerases/genética , Dados de Sequência MolecularRESUMO
[reaction: see text] Lanosterol synthase converts oxidosqualene to the tetracyclic sterol precursor lanosterol. The mutation experiments described here show that an active-site valine residue in lanosterol synthase contributes to cyclization control through steric effects. Mutating to smaller alanine or glycine residues allows formation of the monocyclic achilleol A, whereas the leucine and isoleucine mutants make exclusively lanosterol. The phenylalanine mutant is inactive.
Assuntos
Transferases Intramoleculares/química , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , Sítios de Ligação , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Microssomos/enzimologia , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , PrótonsRESUMO
Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.
Assuntos
Dictyostelium/enzimologia , Transferases Intramoleculares/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Dictyostelium/genética , Humanos , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Mamíferos , Dados de Sequência Molecular , Plantas/enzimologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Holoprosencephaly (HPE) is the most common birth defect of the brain in humans. It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies. The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE. Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients. We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein. Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.
Assuntos
Holoprosencefalia/enzimologia , Holoprosencefalia/genética , Transferases Intramoleculares/genética , Sequência de Bases , Análise Mutacional de DNA , Primers do DNA/genética , DNA Complementar/genética , Éxons , Feminino , Teste de Complementação Genética , Humanos , Íntrons , Masculino , Mutação de Sentido Incorreto , Linhagem , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genéticaRESUMO
A cDNA library was prepared from Artemisia annua, and a 129-bp fragment was amplified from this library using primers corresponding to sequences conserved in known dicot sesquiterpene synthases. A 1641-bp open reading frame that encoded a predicted protein 35-38% identical to dicot sesquiterpene synthases was cloned using this fragment as a hybridization probe. The gene product expressed in Escherichia coli cyclized farnesyl diphosphate to a 96:4 mixture of (-)8-epicedrol and cedrol. Neither cedrol epimer was detected by GC-MS in an A. annua extract prepared from the same specimen as the cDNA.
Assuntos
Artemisininas , Asteraceae/genética , Carbono-Carbono Liases/genética , Plantas Medicinais/genética , Fosfatos de Poli-Isoprenil/metabolismo , Terpenos/síntese química , Sequência de Aminoácidos , Antimaláricos/metabolismo , Asteraceae/enzimologia , Carbono-Carbono Liases/metabolismo , Clonagem Molecular , DNA Complementar/genética , Escherichia coli/genética , Biblioteca Gênica , Genes de Plantas , Modelos Químicos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Plantas Medicinais/enzimologia , Sesquiterpenos Policíclicos , RNA de Plantas/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sesquiterpenos/metabolismo , Terpenos/químicaRESUMO
A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Genes de Plantas , Transferases Intramoleculares/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
A Schizosaccharomyces pombe cDNA encoding lanosterol synthase was cloned by complementing a Saccharomyces cerevisiae lanosterol synthase mutant. The predicted 83-kDa protein is 54-58% identical to other lanosterol synthases. The previously known lanosterol synthases contain 229 conserved residues, which should encompass the catalytically essential amino acids. This number is decreased dramatically by including the Sc. pombe lanosterol synthase in the analysis; 42 residues are no longer conserved and therefore are catalytically nonessential. We have begun mutagenic studies to identify catalytic residues from the remaining conserved residues. Mutant Sa. cerevisiae lanosterol synthase genes were generated in which phenylalanine was specifically substituted for conserved tryptophan residues. All of the resultant mutant enzymes retained the ability to complement the Sc. cerevisiae lanosterol synthase mutant, suggesting that these conserved tryptophan residues are not catalytically essential.
Assuntos
Transferases Intramoleculares , Isomerases/biossíntese , Isomerases/química , Schizosaccharomyces/enzimologia , Triptofano , Sequência de Aminoácidos , Animais , Sequência de Bases , Candida albicans/enzimologia , Clonagem Molecular , Sequência Conservada , DNA Complementar , Biblioteca Gênica , Humanos , Isomerases/genética , Dados de Sequência Molecular , Mutagênese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/genética , Homologia de Sequência de AminoácidosRESUMO
Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] catalyzes the cyclization of (S)-2,3-oxidosqualene to lanosterol in the reaction that forms the sterol nucleus. We report herein the cloning and characterization of the human gene (OSC) encoding lanosterol synthase, a predicted 83 kDa protein of 732 amino acids. The deduced amino acid sequence is 36-40% identical to known yeast and plant homologues and 83% identical to Rattus norvegicus lanosterol synthase. The new gene was shown to encode lanosterol synthase. The yeast lanosterol synthase deficient mutant SMY8 was complemented by the human gene, and a cell-free homogenate of SMY8 expressing the human gene was shown to convert 2,3-oxidosqualene to lanosterol.
Assuntos
Hominidae/genética , Transferases Intramoleculares , Isomerases/genética , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Arabidopsis/enzimologia , Sequência de Bases , Clonagem Molecular , DNA Complementar , Escherichia coli , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Ratos , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter.
Assuntos
Genes Fúngicos , Transferases Intramoleculares , Isomerases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Teste de Complementação Genética , Isomerases/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/genética , Genes de Plantas , Transferases Intramoleculares , Isomerases/biossíntese , Isomerases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA/isolamento & purificação , DNA/metabolismo , Expressão Gênica , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fitosteróis/biossíntese , Fitosteróis/química , Plasmídeos , RNA/isolamento & purificação , RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectroscopia de Infravermelho com Transformada de Fourier , TriterpenosRESUMO
In the presence of indomethacin, arachidonic acid relaxes precontracted rings of rat aorta only when the endothelium is intact. Arachidonate-induced, endothelium-dependent relaxation is potentiated by superoxide dismutase. In contrast, linoleic acid (LA) contracts endothelium-intact and -denuded rings. Arachidonate is metabolized in endothelial cells by both cyclo-oxygenase and 15-lipoxygenase. Therefore, we determined the vasodilatory effect of 15-lipoxygenase products. The products generated by soybean lipoxygenase (SLO) from arachidonate in the bioassay bath relax precontracted, de-endothelialized ring segments of rat aorta. This relaxation is potentiated by superoxide dismutase and is more prominent when high concentrations of SLO are used. The main metabolites recovered from the bioassay bath were 5,15-dihydroperoxyeicosatetraenoic acid and 8,15-dihydroperoxyeicosatetraenoic acid. At lower concentrations of SLO the degree of relaxation is less and the major product is 15-hydroperoxyeicosatetraenoic acid. LA is metabolized by SLO to 13-hydroperoxyoctadecadienoic acid. The relaxation induced by the incubation of LA with SLO in endothelium-denuded rings is less than that obtained with arachidonic acid. In endothelium-denuded rings that were precontracted with phenylephrine authentic 15-hydroperoxyeicosatetraenoic acid did not induce clear effect (at 40 microM 15-hydroperoxide caused relaxation, whereas at 15 microM induced small contraction) and 13-hydroperoxyoctadecadienoic acid of LA induced contraction. Neither 5,15-dihydroperoxyeicosatetraenoic acid nor 8,15-dihydroperoxyoctadecadienoic acid (1-15 microM) induced a well defined relaxation. This study indicates that arachidonic acid is metabolized by SLO to a vasodilatory compound(s) that is possibly derived from 15-hydroperoxyeicosatetraenoic acid.