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1.
The use of a fully active, synthetic analogue of coelenterate-type luciferin labeled in the carbonyl position with 14C and 18O was used to probe the mechanism of the luciferase catalyzed oxidative decarboxylation of this compound. In the presence of 17O 2, the CO 2 produced in this oxidation can be shown to contain approximately one 17O atom per CO 2 molecule. This result is consistent with a cyclic peroxide or dioxetanone-type mechanism. In the presence of luciferase, the oxygen in the luciferin carbonyl group is rapidly exchanged with solvent water prior to the production of CO 2. Thus, the CO 2 contains considerable oxygen derived from water, via exchange with the carbonyl group, and about one oxygen from O 2 via a cyclic peroxide. 相似文献
2.
Incorporation of 18O into CO 2 was measured under various buffer conditions when the bioluminescent oxidation of Cypridina luciferin, catalyzed by luciferase, was carried out either in H 216O medium with 18O 2 gas, or in H 218O medium with 16O 2 gas. The results indicate that (1) the exchange of oxygen between CO 2 and solvent H 2O is significantly influenced by the kind of buffer as well as by pH, (2) the exchange of oxygen between solvent H 2O and CO 2 produced from luciferin in a neutral buffer can be reasonably well estimated from the exchange that takes place when the same amount of CO 2 gas is introduced into the same buffer by the presently employed method, and (3) in the Cypridina bioluminescent reaction, one of two oxygens of O 2 is quantitatively incorporated into the product CO 2 prior to the exchange of oxygen between CO 2 and solvent H 2O. 相似文献
3.
The small Japanese “firefly squid,” Watasenia scintillans, emits a bluish luminescence from dermal photogenic organs distributed along the ventral aspects of the head, mantle, funnel, arms and eyes. The brightest light is emitted by a cluster of three tiny organs located at the tip of each of the fourth pair of arms. Studies of extracts of the arm organs show that the light is due to a luciferin-luciferase reaction in which the luciferase is membrane-bound. The other components of the reaction are coelenterazine disulfate (luciferin), ATP, Mg 2+, and molecular oxygen. Based on the results, a reaction scheme is proposed which involves a rapid base/luciferase-catalyzed enolization of the keto group of the C-3 carbon of luciferin, followed by an adenylation of the enol group by ATP. The AMP serves as a recognition moiety for docking the substrate molecule to a luciferase bound to membrane, after which AMP is cleaved and a four-membered dioxetanone intermediate is formed by the addition of molecular oxygen. The intermediate then spontaneously decomposes to yield CO 2 and coelenteramide disulfate (oxyluciferin) in the excited state, which serves as the light emitter in the reaction. 相似文献
4.
A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10 –5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10 –5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O 2 = oxyluciferin + H 2O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration. 相似文献
5.
Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H 218O showed incorporation of up to three 18O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H 2O. Toxin formed in the presence of 18O molecular oxygen incorporated up to six 18O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H 218O and 18O 2 labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen. 相似文献
6.
Bioluminescent oxidation of luciferin yields CO 2 besides oxyluciferin and light. The exchange of oxygen between the CO 2 and H 2O of the solvent becomes significant when less than approximately 1 μmol of luciferin is reacted in 4 ml of buffer solution, and the exchanged oxygen in CO 2 markedly increases by decreasing the amount of luciferin. Such an exchange is to be expected in any such system which produces CO 2 in aqueous solution, and must be taken into account in interpreting the results of experiments. 相似文献
7.
An advanced radiogasometric method for the study of plant leaf CO 2 exchange is presented. The method enables determination of the rates of CO 2 fixation, photorespiration and respiration in the light under steady‐state photosynthesis and discrimination between primary and stored photosynthates as substrates of photorespiratory and respiratory decarboxylations. The method is based on the analysis of the time curves of 14CO 2 evolution from labeled primary and stored photosynthates in leaves previously exposed to 14CO 2. The molar rates of different decarboxylation reactions are calculated from the initial slopes of the curves taking into account the specific radioactivity of CO 2 fed to leaves and/or evolved from leaves. To estimate the contribution of primary and stored photosynthates, the measurements of 14CO 2 evolution are performed after feeding plant leaves for different periods with 14CO 2. Photorespiration and respiration are distinguished on the basis of data obtained from measurements of 14CO 2 evolution under normal (210 ml l −1) and low (15 ml l −1) concentrations of oxygen. A principally new method for the determination of the rate of intracellular refixation of respiratory CO 2 has been developed. The method is based on the measurements of 14CO 2 evolution from leaves into the medium of very high concentrations (30 ml l −1) of 12CO 2, where the probability of refixation of 14CO 2 evolved inside the cell is close to zero. The results obtained were comparable with the data derived from parallel refixation measurements by means of gasometric methods. As an example of application, the data on CO 2 exchange in leaves of two contrasting groups of C 3‐species, differing in the ability of starch accumulation, are presented. 相似文献
8.
The bioluminescence-dependent oxidation of a long-chain fatty aldehyde catalyzed by luciferase from Photobacterium phosphoreum has been studied in 18O 2 experiments. The results show the incorporation of one atom of molecular oxygen into the product, the corresponding fatty acid. This incorporation is not the result of exchange of 18O 2 with the aldehyde prior to oxidation to the acid, thereby indicating that the bacterial luciferase catalyzes an aldehyde monooxygenase reaction which is coupled with bioluminescence. 相似文献
9.
The oxidation-reduction potential of the Cypridina luciferin-oxyluciferin system determined by a method of "bracketing" lies somewhere between that of anthraquinone 2-6-di Na sulfonate ( Eo
'' at pH of 7.7 = –.22) which reduces luciferin, and quinhydrone ( Eo
'' at pH of 7.7 = +.24), which oxidizes luciferin. Systems having an Eo
'' value between –.22 and +.24 volt neither reduce oxyluciferin nor oxidize luciferin. If the luciferin-oxyluciferin system were truly reversible considerable reduction and oxidation should occur between –.22 and +.24. The system appears to be an irreversible one, with both "apparent oxidation" and "apparent reduction potentials" in Conant''s sense. Hydrosulfites, sulfides, CrCl 2, TiCl 3, and nascent hydrogen reduce oxyluciferin readily in absence of oxygen but without luminescence. Luminescence only appears in water solution if luciferin is oxidized by dissolved oxygen in presence of luciferase. Rapid oxidation of luciferin by oxygen without luciferase or oxidation by K 3Fe(CN) 6 in presence of luciferase but without oxygen never gives luminescence. 相似文献
10.
A new method is presented for measurement of the CO 2/O 2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The [ 14C]3-phosphoglycerate (PGA) from the Rubisco carboxylase reaction and its dilution by the Rubisco oxygenase reaction was monitored by directly measuring the specific radioactivity of PGA. 14CO 2 fixation with Rubisco occurred under two reaction conditions: carboxylase with oxygenase with 40 micromolar CO 2 in O 2-saturated water and carboxylase only with 160 micromolar CO 2 under N 2. Detection of the specific radioactivity used the amount of PGA as obtained from the peak area, which was determined by pulsed amperometry following separation by high-performance anion exchange chromatography and the radioactive counts of the [ 14C]PGA in the same peak. The specificity factor of Rubisco from spinach ( Spinacia oleracea L.) (93 ± 4), from the green alga Chlamydomonas reinhardtii (66 ± 1), and from the photosynthetic bacterium Rhodospirillum rubrum (13) were comparable with the published values measured by different methods. 相似文献
11.
Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between 14CO 2 and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U- 14C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn 2+ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol 14CO 2 exchange · min -1 · mg -1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min -1 · mg -1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed. 相似文献
12.
The O-methyl substituents of aromatic compounds constitute a C 1 growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C 1 substrate was determined by 14C radiotracer techniques. O-[ methyl- 14C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C 1 substrate. The data showed that for every O-methyl carbon converted to [ 14C]acetate, two were oxidized to 14CO 2. Quantitation of the carbon recovered in the two products, acetate and CO 2, indicated that acetate was formed in part by the fixation of unlabeled CO 2. The specific activity of 14C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO 2 and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[ methyl- 14C]vanillate by strain TH-001 can be described as follows: 3 14CH 3OC 7H 5O 3 + CO 2 + 4H 2O → 14CH 3COOH + 2 14CO 2 + 10H + + 10e - + 3HOC 7H 5O 3. 相似文献
13.
Using a manometric method, photosynthetic oxygen evolution and 14CO 2 fixation have been determined for leaf tissue of Triticum aestivum L., Hordeum vulgare L., Phaseolus vulgaris L., and Lemna minor L. Approximately similar values in the range 0.2 to 0.4 millimoles grams fresh weight −1 hour −1 were obtained for both gases. In tissue subjected to vacuum infiltration, O 2 evolution and 14CO 2 fixation were barely measurable. It is considered that the elimination of photosynthetic gas exchange results from a decreased supply of CO 2 to the chloroplasts. Chopping wheat laminae also leads to a reduction in photosynthetic gas exchange, slices 1 millimeter or less giving only 10 to 20% of the value for whole tissue. Respiration is unaffected by either treatment. Carbonic anhydrase did not improve photosynthetic gas exchange in infiltrated tissue. The use of sliced or vacuum-infiltrated leaf tissue in photosynthetic studies is discussed. 相似文献
14.
Two-tier vessels, developed for culturing of microalgae and cyanobacteria at high cell density on a shaken platform, were assembled from a flat lower chamber to be filled with a CO 2 buffer and an upper flat sterile chamber for the culture that was separated from the lower chamber by a porous polypropylene membrane. Diffusive gas exchange with the atmosphere was controlled by the O 2 outlet channel. Referred to surface area, rates of CO 2 transfer to a shaken weakly alkaline buffer solution across the membrane were higher than those reached on the conventional pathway through the free upper liquid surface. Membrane-mediated CO 2 supply enabled rapid growth of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 up to ultrahigh cell density. The biomass (dry weight) concentration of Synechococcus cultures reached more than 30 g L ?1 on a buffered medium with adequate concentrations of mineral nutrients. An increase of 15 to 20 g L ?1 was observed during repeated two-day cycles. Separate pathways for CO 2 supply and oxygen outlet prevented significant loss of CO 2. Convective gas flow through the oxygen outlet channel enabled the estimation of the O 2 generation rate. The permeability of the channel for diffusive O 2/N 2 exchange limited the O 2 concentration to a moderate value. It is concluded that shaken flat cultures using CO 2 supply through a porous hydrophobic membrane and diffusive release of O 2 through a separate pathway are promising for research on microalgae and cyanobacteria. 相似文献
15.
Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% 18O 2 and 80% N 2 indicates that one atom of 18O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase. Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing 18O2 indicates that one atom of 18O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress. 相似文献
16.
Mass spectral analyses of the CO 2 liberated in the luciferin-luciferase and firefly luciferin-luciferase reactions run in the presence of 17O 2 and H 218O show that the product is predominantly C 18O 16O (mass 46) and not C 17O 16O (mass 45). Incorporation of 18O into medium CO 2 by exchange does not account for the observed results. These experiments provide evidence that the and firefly bioluminescence reactions proceed via a linear peroxide mechanism rather than the dioxetane mechanism and suggest that a common mechanism may underly many bioluminescence reactions. 相似文献
17.
Ghost crabs Ocypode ceratophthalmus were exercised in air and water to measure CO 2 and O 2 exchange rates using the method of instantaneous measurements of oxygen consumption rate (MO 2) where applicable. Average heart rate increased from 100 to nearly 400 pulses per minute after five minutes of exercise on a treadmill at a run rate of 0.133 m s ?1. It took less than a minute for oxygen taken up through the lung epithelium from the air inside the branchial cavity to reach the maximal oxygen consumption rate of 26.1 mmol O 2 kg ?1 h ?1. Resting MO 2 was 4.06 mmol O 2 kg ?1 h ?1 in air, but decreased to 3.37 mmol O 2 kg ?1 h ?1 in seawater. Radioactive CO 2 from injected l-lactate is released linearly by the lung. The percent accumulated 14-CO 2 in exhaled air, plotted against time, intersects zero time on the x -axis, indicating rapid gas exchange at the lung surface. The P 50 values for native haemocyanin of 4.89 mm Hg before exercise, and 8.99 mm Hg after exercise, are typical of a high-affinity haemocyanin usually associated with terrestrial crabs. The current notion that Ocypode ceratophthalmus drown when submerged in seawater was not substantiated by our experiments. MO 2 in seawater increased from 3.37 mmol O 2 kg ?1 h ?1 for resting crabs to 5.72 mmol O 2 kg ?1 h ?1 during exercise. When submerged by wave-seawater in the natural environment and during exercise in respirometer-seawater O. ceratophthalmus do not swim but, having a specific density of 1.044, float nearly weightless with a minimum of body movements. 相似文献
18.
Preincubation of illuminated tobacco ( Nicotiana tabacum L.) leaf disks in glycidate (2,3-epoxypropionate) or glyoxylate inhibited photorespiration by about 40% as determined by the ratio of 14CO 2 evolved into CO 2-free air in light and in darkness. However, under identical preincubation conditions used for the light/dark 14C assays, the compounds failed to reduce photorespiration or stimulate net photosynthesis in tobacco leaf disks based on other CO 2 exchange parameters, including the CO 2 compensation concentration in 21% O 2, the inhibitory effect of 21% O 2 on net photosynthesis in 360 microliters per liter of CO 2 and the rate of net photosynthetic 14CO 2 uptake in air. The effects of both glycidate and glyoxylate on the 14C assay are inconsistent with other measures of photorespiratory CO2 exchange in tobacco leaf disks, and thus these data question the validity of the light to dark ratio of 14CO2 efflux as an assay for relative rates of photorespiration (Zelitch 1968, Plant Physiol 43: 1829-1837). The results of this study specifically indicate that neither glycidate nor glyoxylate reduces photorespiration or stimulates net photosynthesis by tobacco leaf disks under physiological conditions of pO2 and pCO2, contrary to previous reports. 相似文献
19.
β-Carotene 15–15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O 2 and H 2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either 16O 2-H 218O or 18O 2-H 216O medium for 15 min at 37 °C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O 2 gas used. Together with the data from 18O-retinal-H 216O and 16O-retinal-H 218O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O 2 into retinal. Thus, BCO1 is a dioxygenase. 相似文献
20.
Glycine decarboxylase has been successfully solubilized from pea ( Pisum sativum) leaf mitochondria as an acetone powder. The enzyme was dependent on added dithiothreitol and pyridoxal phosphate for maximal activity. The enzyme preparation could catalyze the exchange of CO 2 into the carboxyl carbon of glycine, the reverse of the glycine decarboxylase reaction by converting serine, NH 4+, and CO 2 into glycine, and 14CO 2 release from [1- 14C]glycine. The half-maximal concentrations for the glycine-bicarbonate exchange reaction were 1.7 millimolar glycine, 16 millimolar NaH 14CO 2, and 0.006 millimolar pyridoxal phosphate. The enzyme (glycine-bicarbonate exchange reaction) was active in the assay conditions for 1 hour and could be stored for over 1 month. The enzymic mechanism appeared similar to that reported for the enzyme from animals and bacteria but some quantitative differences were noted. These included the tenacity of binding to the mitochondrial membrane, the concentration of pyridoxal phosphate needed for maximum activity, the requirement for dithiothreitol for maximum activity, and the total amount of activity present. Now that this enzyme has been solubilized, a more detailed understanding of this important step in photorespiration should be possible. 相似文献
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