RESUMEN
Considerable evidence indicates that acetaldehyde is released from the leaves of a variety of plants. The conventional explanation for this is that ethanol formed in the roots is transported to the leaves where it is converted to acetaldehyde by the alcohol dehydrogenase (ADH) found in the leaves. It is possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase (PDC), an enzyme with an uncertain metabolic role, which has been detected, but not characterized, in cottonwood leaves. We have found that leaf PDC is present in leaf veins and petioles, as well as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the range of 2mM. The leaf vein form of the enzyme was purified approximately 143-fold, and, at the optimum pH of 5.6, the K(m) value for pyruvate was 42 microM. This K(m) is lower than the typical millimolar range seen for PDCs from other sources. The purified leaf PDC also decarboxylates 2-ketobutyric acid (K(m)=2.2mM). We conclude that there are several possible sources of acetaldehyde production in cottonwood leaves: the well-characterized root-derived ethanol oxidation by ADH in leaves, and the decarboxylation of pyruvate by PDC in leaf veins, petioles, and other leaf tissues. Significantly, the leaf vein form of PDC with its high affinity for pyruvate, could function to shunt pyruvate carbon to the pyruvate dehydrogenase by-pass and thus protect the metabolically active vascular bundle cells from the effects of oxygen deprivation.
Asunto(s)
Acetaldehído/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Populus/enzimología , Populus/metabolismo , Piruvato Descarboxilasa/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Several recent studies have suggested that control of isoprene emission rate is in part exerted by supply of extrachloroplastic phosphoenolpyruvate to the chloroplast. To test this hypothesis, we altered PEP supply by differential induction of cytosolic nitrate reductase (NR) and PEP carboxylase (PEPC) in plants of Populus deltoides grown with NO3- or NH4+ as the sole nitrogen source. Growth with 8 mM NH4+ produced a high leaf nitrogen concentration, compared with 8 mM NO3-, as well as slightly elevated rates of photosynthesis and significantly enhanced rates of isoprene emission and content of dimethylallyl diphosphate (DMAPP, a precursor to isoprene biosynthesis), chlorophyll (a+b) and carotenoids. Growth with 8 mM NO3- resulted in parallel reductions in both leaf isoprene emission rate and DMAPP. The differential effects of growth with NH4+ or NO3- were not observed when plants were grown with 4 mM nitrogen. The effects of reduced DMAPP availability were specific to isoprene emission and were not propagated to higher isoprenoids, as the correlations between nitrogen content and either leaf chlorophyll (a+b) or total carotenoids were unaffected by nitrogen source. Biochemical analysis revealed significantly higher levels of NR and PEPC activity in leaves of 8 mM NO3- -grown plants, consistent with their fundamental roles in nitrate assimilation. Taken together, these results support the hypothesis that foliar assimilation of NO3- reduces isoprene emission rate by competing for carbon skeletons (mediated by PEPC) within the cytosol and possibly reductant within the chloroplast. Cytosolic competition for PEP is a major regulator of chloroplast DMAPP supply, and we offer a new "safety valve" hypothesis to explain why plants emit isoprene.
Asunto(s)
Hemiterpenos/biosíntesis , Nitrato Reductasas/biosíntesis , Populus/metabolismo , Butadienos , Carbono/metabolismo , Cloroplastos/metabolismo , Citosol/metabolismo , Inducción Enzimática , Hemiterpenos/metabolismo , Cinética , Modelos Biológicos , Nitrato-Reductasa , Nitrógeno/metabolismo , Compuestos Organofosforados/metabolismo , Pentanos , Fosfoenolpiruvato/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Populus/enzimologíaRESUMEN
Large releases of 1-penten-3-ol (pentenol) and 1-penten-3-one (pentenone) were recently observed from a variety of leaves subjected to freeze-thaw damage in the presence of oxygen. In order to understand the biochemical origins of these volatiles, soybean leaf extracts were used to determine if the formation of pentenol and pentenone can be explained by known O(2)-dependent lipoxygenase (LOX) reactions. Enzymatic formation of these C5 volatiles was found to be dependent on alpha-linolenic acid or the 13(S)-hydroperoxide of alpha-linolenic acid [13(S)-HPOT] and blocked by LOX inhibitors. Five soybean leaf LOX isozyme genes (VLXA, VLXB, VLXC, VLXD, and VLXE) were then expressed in Escherichia coli and used in in vitro incubations with 13(S)-HPOT to test for volatile formation. Each of the LOX isozymes catalyzed the formation of low levels of pentenol, but not pentenone. It therefore seems likely that the C5,13-cleavage activity of LOX is the direct source of abundant pentenol and the indirect source of pentenone observed upon leaf wounding.
Asunto(s)
Glycine max/enzimología , Pentanoles/metabolismo , Pentanonas/metabolismo , Hojas de la Planta/metabolismo , Congelación , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Lipooxigenasa/metabolismo , Oxígeno , Glycine max/metabolismo , Especificidad por Sustrato , Ácido alfa-Linolénico/análogos & derivados , Ácido alfa-Linolénico/metabolismoRESUMEN
Isoprene (2-methyl-1,3-butadiene) is the most abundant biogenic hydrocarbon released from vegetation, and there is continuing interest in understanding its biosynthesis from photosynthetic precursors in leaf chloroplasts. We used on-line proton-transfer-reaction mass spectrometry (PTR-MS) to observe the kinetics of (13)C-labeling of isoprene following exposure to (13)CO(2) and then the loss of (13)C after a return to normal (12)CO(2) in oak ( Quercus agrifolia Nee) and cottonwood (Populus deltoides Barr.) leaves. Assignments of labeled isoprene species were verified by gas chromatography-mass spectrometry. For the first time, it was possible to observe the half-lives of individually (13)C-labeled isoprene species during these transitions, and to trace some of the label to a C3 fragment that contained the two isoprene carbons derived from pyruvate via the deoxyxylulose-5-phosphate (DOXP) pathway. At steady state (under (13)CO(2)), approximately 80% of isoprene carbon was labeled, with fully labeled isoprene as the major species (approx. 60%). The source of the unlabeled C is suggested to be extrachloroplastic, but not from photorespiratory carbon. After a transfer to (12)CO(2), (13)C-labeling persisted in one isoprene carbon for several hours; this persistence was much more pronounced in (i) leaves inhibited by fosmidomycin, a specific inhibitor of the DOXP pathway, and (ii) in sun leaves which have higher ratios of soluble sugars to starch. From the mass 41-44 fragment data, and labeling predicted from the DOXP pathway in chloroplasts, precursors may arise from cytosolic pyruvate/phospho enolpyruvate equivalents transported into the chloroplast; this idea was supported by an indirect measure of pyruvate labeling. Other sources of cytosolic isoprene precursors (i.e. dimethylallyl diphosphate or pentose phosphate) could not be excluded. The data obtained shed light on the half-lives of photosynthetic metabolites, exchanges of carbon between cellular pools, and suggest multiple origins of isoprene precursors in leaves.
Asunto(s)
Butadienos/metabolismo , Dióxido de Carbono/metabolismo , Fosfomicina/análogos & derivados , Hemiterpenos , Pentanos , Hojas de la Planta/metabolismo , Algoritmos , Butadienos/antagonistas & inhibidores , Isótopos de Carbono , Cloroplastos/metabolismo , Citosol/metabolismo , Fosfomicina/farmacología , Cromatografía de Gases y Espectrometría de Masas , Semivida , Luz , Lovastatina/farmacología , Modelos Biológicos , Oxígeno/farmacología , Consumo de Oxígeno/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Populus/metabolismo , Ácido Pirúvico/metabolismo , Quercus/metabolismoRESUMEN
We measured the release of volatile organic compounds (VOCs) resulting from lawn mowing during continuous ambient air measurements in July and August 1998 in the outskirts of Innsbruck, Austria. These measurements were made with a proton-transfer-reaction mass spectrometry system, which allowed simultaneous, on-line monitoring of VOCs in the pptv range. We observed the emission of C6 wound compounds, including (Z)-3-hexenal, (E)-2-hexenal, hexenol plus hexanal, and acetaldehyde immediately following lawn mowing, and a rise in background levels of C6 wound compounds that lasted for several hours. Peak levels of biogenic VOCs following mowing were in the same concentration range (20-60 ppbv) as those originating from combustion engines of lawn mowers, and integrated biogenic emissions were much greater in the drying grass clippings. Additional emissions of acetone and other VOCs resulted from rainfall on these clippings. Since the estimated atmospheric chemical reactivity of VOCs resulting from lawn mowing is of the same order of magnitude as unburned hydrocarbons released during the mowing by gasoline-powered lawn mowers, these biogenic VOCs should be considered in urban air-quality control strategies.
Asunto(s)
Contaminantes Atmosféricos/análisis , Emisiones de Vehículos/análisis , Espectrometría de Masas , Compuestos Orgánicos/análisis , Poaceae , VolatilizaciónRESUMEN
Numerous publications have described measurements of breath isoprene in humans, and there has been a hope that breath isoprene analyses could be a noninvasive diagnostic tool to assess blood cholesterol levels or cholesterol synthesis rate. However, significant analytic problems in breath isoprene analysis and variability in isoprene levels with age, exercise, diet, etc., have limited the usefulness of these measurements. Here, we have applied proton transfer reaction-mass spectrometry to this problem, allowing on-line detection of breath isoprene. We show that breath isoprene concentration increases within a few seconds after exercise is started as a result of a rapid increase in heart rate and then reaches a lower steady state when breath rate stabilizes. Additional experiments demonstrated that increases in heart rate associated with standing after reclining or sleeping are associated with increased breath isoprene concentrations. An isoprene gas-exchange model was developed and shows excellent fit to breath isoprene levels measured during exercise. In a preliminary experiment, we demonstrated that atorvastatin therapy leads to a decrease in serum cholesterol and low-density-lipoprotein levels and a parallel decrease in breath isoprene levels. This work suggests that there is constant endogenous production of isoprene during the day and night and reaffirms the possibility that breath isoprene can be a noninvasive marker of cholesterologenesis if care is taken to measure breath isoprene under standard conditions at constant heart rate.
Asunto(s)
Butadienos/análisis , Colesterol/sangre , Frecuencia Cardíaca/fisiología , Hemiterpenos , Pentanos , Anticolesterolemiantes/uso terapéutico , Atorvastatina , Pruebas Respiratorias , Ejercicio Físico/fisiología , Ácidos Heptanoicos/uso terapéutico , Humanos , Lipoproteínas LDL/sangre , Espectrometría de Masas/métodos , Modelos Biológicos , Monitoreo Fisiológico/métodos , Esfuerzo Físico/fisiología , Postura , Pirroles/uso terapéutico , Mecánica Respiratoria/fisiología , Sueño/fisiología , Factores de TiempoRESUMEN
A sensitive, nonradioactive method was developed to measure cellular levels of dimethylallyl diphosphate (DMAPP), a central intermediate of isoprenoid metabolism in nature. The assay is based on the hydrolysis of DMAPP in acid to the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene), with subsequent analysis of isoprene by headspace gas chromatography with reduction gas detection. In the assay, cell samples are directly acidified with 4 M H(2)SO(4) in sealed reaction vials. Therefore, there is no need to extract metabolites, purify them, and keep them stable prior to analysis, and degradative enzymatic activities are destroyed. DMAPP levels of 23 +/- 4 nmol (g fresh weight)(-1) [ca. 85 nmol (g dry weight)(-1)] and 80 +/- 14 nmol (g fresh weight)(-1) [ca. 296 nmol (g dry weight)(-1)] were measured in dark- and light-adapted leaves of Populus deltoides (Eastern cottonwood), respectively. Evidence is presented to show that DMAPP is the major leaf metabolite giving rise to isoprene following acid hydrolysis. DMAPP levels in Bacillus subtilis and Saccharomyces cerevisiae were determined to be 40.8 +/- 16.7 pmol (OD(600))(-1) [ca. 638 pmol (mg dry weight)(-1)] and 6.3 +/- 3.7 pmol (OD(600))(-1) [ca. 139 pmol (mg dry weight)(-1)], respectively. The method should be suitable for any cell or tissue type and isolated cellular organelles.
Asunto(s)
Bacillus subtilis/química , Hemiterpenos , Compuestos Organofosforados/análisis , Pentanos , Hojas de la Planta/química , Saccharomyces cerevisiae/química , Bacillus subtilis/metabolismo , Butadienos/análisis , Butadienos/metabolismo , Cromatografía de Gases , Cromatografía por Intercambio Iónico , Hidrólisis , Cinética , Magnoliopsida/química , Compuestos Organofosforados/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Fosfatos de Poliisoprenilo/análisis , Fosfatos de Poliisoprenilo/metabolismo , Trazadores Radiactivos , Saccharomyces cerevisiae/metabolismo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
During enrichments to search for soil bacteria that form the volatile ketones, acetone and butanone, we isolated pure cultures of Pseudomonas aureofaciens, P. fluorescens, and P. putida that excrete the beta-keto-acid, 3-oxopentanoate (3-OPA), when grown on heptanoic acid as carbon source. Analysis of 3-OPA used enzymatic decarboxylation by acetoacetate decarboxylase to yield butanone, which was detected by headspace gas chromatography. The formation of 3-OPA was strongly dependent on heptanoic acid concentration, the level of oxygen, and the state of growth, and was not seen with even-chain or other odd-chain fatty acids. Uptake of 3-OPA during stationary phase of growth is probably related to polyhydroxyalkanoate (PHA) formation in these isolates. A model for formation and release of 3-OPA is proposed.
Asunto(s)
Acetona/metabolismo , Butanonas/metabolismo , Ácidos Heptanoicos/metabolismo , Ácidos Pentanoicos/metabolismo , Pseudomonas/metabolismo , Microbiología del Suelo , Carboxiliasas/metabolismo , Cromatografía de Gases , Oxígeno/metabolismo , Pseudomonas/crecimiento & desarrollo , Especificidad por SustratoRESUMEN
Methylbutenol (2-methyl-3-buten-2-ol) is an abundant volatile organic compound released from Western U.S. pines. To understand the mechanism of methylbutenol formation, we developed a sensitive gas chromatographic assay for its detection and determined that needles of gray pine (Pinus sabiniana) contain an enzyme that catalyzes the synthesis of methylbutenol from dimethylallyl diphosphate (DMAPP). The methylbutenol synthase activity was partially purified; its pH optimum was 7-8, and, like other prenyl diphosphate utilizing enzymes, it was dependent on the presence of a divalent cation, preferably Mn2+. The enzyme also required K+ or NH4+ for activity. The Km values for DMAPP and Mn2+ were about 4.8 and 6 mM, respectively. Geranyl diphosphate was not a substrate for the enzyme, so it is distinct from linalool synthase, a plant enzyme that catalyzes an analogous reaction. The methylbutenol synthase reaction may be responsible for the majority of light-dependent methylbutenol production by many pine species in the Western United States.
Asunto(s)
Butanoles/metabolismo , Difosfatos/metabolismo , ÁrbolesRESUMEN
Isoprene (2-methyl-1,3-butadiene), an abundant natural product of unknown function in plants, has recently been found to be one of the major volatiles formed by Bacillus subtilis. To understand the metabolic origins of isoprene in B. subtilis, we used (13)C- and (2)H-labeling methods with GC-MS analysis of released isoprene. The results indicate that, in this bacterium, isoprene is not formed by the mevalonate pathway or from catabolism of leucine, but, as in plant systems, it is a product of the methylerythritol phosphate pathway of isoprenoid synthesis. This work supports the idea that B. subtilis could be used as a microbial model for studying the biochemistry of isoprene formation.
Asunto(s)
Bacillus subtilis/metabolismo , Butadienos/metabolismo , Eritritol/análogos & derivados , Hemiterpenos , Pentanos , Fosfatos de Azúcar/metabolismo , Eritritol/metabolismo , Cromatografía de Gases y Espectrometría de MasasRESUMEN
There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of L-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. L-Leucine, but not D-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of L-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only alpha-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d(7))-L-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.
Asunto(s)
Acetona/metabolismo , Leucina/metabolismo , Vibrionaceae/metabolismo , Cinética , Modelos Biológicos , Modelos Químicos , Photobacterium/metabolismo , Vibrio/metabolismoRESUMEN
During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1, 3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.
Asunto(s)
Bacillus subtilis/fisiología , Butadienos/metabolismo , Hemiterpenos , Pentanos , Acetoína/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Medios de Cultivo , Glucosa/metabolismo , Cinética , Esporas Bacterianas/fisiología , Factores de TiempoRESUMEN
Benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus (AC-BADH) and TOL plasmid-encoded benzyl alcohol dehydrogenase from Pseudomonas putida (TOL-BADH) have previously been shown to oxidize a variety of aromatic alcohols but not aliphatic substrates. Here, we have expressed the genes for AC-BADH and TOL-BADH in Escherichia coli, purified the resulting over-expressed enzymes, and shown that each is an effective catalyst of both benzylic and allylic alcohol oxidation, but not of oxidation of nonallylic analogs. Enzyme specificity (kcat/Km) for both enzymes was higher with an aliphatic, allylic alcohol (3-methyl-2-buten-1-ol) than with benzyl alcohol. These results suggest that bacterial benzyl alcohol dehydrogenases use the resonance stabilization provided by allylic and benzylic alcohols to promote catalysis.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Acinetobacter calcoaceticus/enzimología , Oxidorreductasas de Alcohol/genética , Alcohol Bencilo/metabolismo , Catálisis , Cinética , Estructura Molecular , Oxidación-Reducción , Propanoles/metabolismo , Pseudomonas putida/enzimología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
We have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-MB), which is produced and emitted by certain pines. To this end we have isolated the soil bacterium Pseudomonas putida MB-1, which uses 232-MB as a sole carbon source. Strain MB-1 contains inducible 3-methyl-2-buten-1-ol (321-MB) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-MB is metabolized by isomerization to 321-MB followed by oxidation. 321-MB dehydrogenase was purified to near-homogeneity and found to be a tetramer (151 kDa) with a subunit mass of 37,700 Da. It catalyzes NAD+-dependent, reversible oxidation of 321-MB to 3-methyl-2-buten-1-al. The optimum pH for the oxidation reaction was 10.0, while that for the reduction reaction was 5.4. 321-MB dehydrogenase oxidized a wide variety of aliphatic and aromatic alcohols but exhibited the highest catalytic specificity with allylic or benzylic substrates, including 321-MB, 3-chloro-2-buten-1-ol, and 3-aminobenzyl alcohol. The N-terminal sequence of the enzyme contained a region of 64% identity with the TOL plasmid-encoded benzyl alcohol dehydrogenase of P. putida. The latter enzyme and the chromosomally encoded benzyl alcohol dehydrogenase of Acinetobacter calcoaceticus were also found to catalyze 321-MB oxidation. These findings suggest that 321-MB dehydrogenase and other bacterial benzyl alcohol dehydrogenases are broad-specificity allylic and benzylic alcohol dehydrogenases that, in conjunction with a 232-MB isomerase, might be useful in an enzyme-linked assay for 232-MB.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Pentanoles/metabolismo , Pseudomonas putida/enzimología , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/aislamiento & purificación , Secuencia de Aminoácidos , Inducción Enzimática , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Peso Molecular , Oxidación-Reducción , Pseudomonas putida/crecimiento & desarrollo , Pseudomonas putida/aislamiento & purificación , Microbiología del Suelo , Especificidad por SustratoRESUMEN
In laboratory cultures of ice nucleation-active (Ice+) Erwinia herbicola isolates, it has been difficult to achieve high-level expression of ice nuclei, especially nuclei active at temperatures warmer than -5 degrees C (i.e., type 1 ice nuclei). Here we demonstrate that starvation for phosphate and exposure to low temperature triggers expression of ice nuclei in E. herbicola cultures. Starvation for nitrogen, sulfur, or iron was less effective. Under optimal conditions with two different strains, essentially all cells produced ice nuclei active at -10 degrees C or warmer, with an average of 22% containing type 1 ice nuclei within 1 h of a low-temperature shift. These conditions did not greatly enhance the shedding of ice nucleation-active membrane vesicles that are known to be produced by Ice+ E. herbicola isolates. These results support the theory that the Ice+ phenotype may allow nutrient-limited epiphytes to trigger freezing damage, releasing nutrients from host plants.
Asunto(s)
Erwinia/fisiología , Hielo , Fosfatos/metabolismo , TemperaturaRESUMEN
Isoprene synthase is the enzyme responsible for the foliar emission of the hydrocarbon isoprene (2-methyl-1,3-butadiene) from many C3 plants. Previously, thylakoid-bound and soluble forms of isoprene synthase had been isolated separately, each from different plant species using different procedures. Here we describe the isolation of thylakoid-bound and soluble isoprene synthases from a single willow (Salix discolor L.) leaf-fractionation protocol. Willow leaf isoprene synthase appears to be plastidic, with whole-leaf and intact chloroplast fractionations yielding approximately equal soluble (i.e. stromal) and thylakoid-bound isoprene synthase activities. Although thylakoid-bound isoprene synthase is tightly bound to the thylakoid membrane (M.C. Wildermuth, R. Fall [1996] Plant Physiol 112: 171-182), it can be solubilized by pH 10.0 treatment. The solubilized thylakoid-bound and stromal isoprene synthases exhibit similar catalytic properties, and contain essential cysteine, histidine, and arginine residues, as do other isoprenoid synthases. In addition, two regulators of foliar isoprene emission, leaf age and light, do not alter the percentage of isoprene synthase activity in the bound or soluble form. The relationship between the isoprene synthase isoforms and the implications for function and regulation of isoprene production are discussed.
RESUMEN
A new analytical method using proton-transfer-reaction mass spectrometry (PTRMS) is described for the determination of trace constituents in human breath. PTRMS is sufficiently sensitive and specific that it does not require preconcentration or separation. At its present stage of development it is capable of detecting trace constituents present in air at the part-per-billion level. These capabilities are illustrated for isoprene, one of the most abundant endogenous hydrocarbons. Our results confirm recent observations of a diurnal level variation associated with sleep or wakefulness; a new finding is that young children have much lower levels of isoprene in breath than adults. To address the metabolic origin of human isoprene, we used PTRMS to analyze expired air for allylic C5 alcohols that have been proposed to be non-enzymatic precursors of isoprene. The lack of correlation between peak breath isoprene and these alcohols suggests that the hydrocarbon is formed by some other mechanism.
Asunto(s)
Butadienos/análisis , Hemiterpenos , Pentanos , Adulto , Anciano , Biomarcadores , Pruebas Respiratorias/instrumentación , Butadienos/metabolismo , Niño , Preescolar , Ritmo Circadiano/fisiología , Humanos , Indicadores y Reactivos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , ProtonesRESUMEN
Isoprene synthase is an enzyme that is responsible for the production of the volatile C5 hydrocarbon, isoprene, in plant leaves. Isoprene formation in numerous C3 plants is interesting because (a) large quantities of isoprene are emitted, 5 x 1014 g of C annually, (b) a plant may release 1 to 8% of its fixed C as isoprene, and (c) the function of plant isoprene production is unknown. Because of the dependence of foliar isoprene emission on light, the existence of a plastidic isoprene synthase has been postulated. To pursue this idea, a method to isolate chloroplasts from Salix discolor was developed and shows a plastidic isoprene synthase that is tightly bound to the thylakoid membrane and accessible to trypsin inactivation. The thylakoid-bound isoprene synthase has catalytic properties similar to known soluble isoprene synthases; however, the relationship between these enzymes is unknown. The discovery of a thylakoid-bound isoprene synthase with a stromal-facing domain places it in the chloroplast, where it may be subject to numerous direct and indirect light-mediated effects. Implications for the light-dependent regulation of foliar isoprene production and its function are presented.