Evaluation of unbound free heme in plant cells by differential acetone extraction

Heme functions not only as a prosthetic group of hemoproteins but also as a regulatory molecule, suggesting the presence of 'free' heme. Classically, total non-covalently bound heme is extracted from plant samples with acidic acetone after removal of pigments with basic and neutral acetone. Earlier work proposed that free heme can be selectively extracted into basic acetone. Using authentic hemoproteins, we confirmed that acidic acetone can quantitatively extract heme, while no heme was extracted into neutral acetone. Meanwhile, a certain amount of heme was extracted into basic acetone from hemoglobin and myoglobin. Moreover, basic acetone extracted loosely bound heme from bovine serum albumin, implying that the nature of hemoproteins largely influences heme extraction into basic acetone. Using a highly sensitive heme assay, we found that basic and neutral acetone can extract low levels of heme from plant samples. In addition, neutral acetone quantitatively extracted free heme when it was externally added to plant homogenates. Furthermore, the level of neutral acetone-extractable heme remained unchanged by precursor (5-aminolevulinic acid) feeding, while increased by norflurazon treatment which abolishes chloroplast biogenesis. However, changes in these heme levels did not correlate to genomes uncoupled phenotypes, suggesting that the level of unbound free heme would not affect retrograde signaling from plastids to the nucleus. The present data demonstrate that the combination of single-step acetone extraction following a sensitive heme assay is the ideal method for determining total and free heme in plants.     

Acetone formation in the Vibrio family: a new pathway for bacterial leucine catabolism

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.     

Measuring airway exchange of endogenous acetone using a single-exhalation breathing maneuver.

Exhaled acetone is measured to estimate exposure or monitor diabetes and congestive heart failure. Interpreting this measurement depends critically on where acetone exchanges in the lung. Health professionals assume exhaled acetone originates from alveolar gas exchange, but experimental data and theoretical predictions suggest that acetone comes predominantly from airway gas exchange. We measured endogenous acetone in the exhaled breath to evaluate acetone exchange in the lung. The acetone concentration in the exhalate of healthy human subjects was measured dynamically with a quadrupole mass spectrometer and was plotted against exhaled volume. Each subject performed a series of breathing maneuvers in which the steady exhaled flow rate was the only variable. Acetone phase III had a positive slope (0.054+/-0.016 liter-1) that was statistically independent of flow rate. Exhaled acetone concentration was normalized by acetone concentration in the alveolar air, as estimated by isothermal rebreathing. Acetone concentration in the rebreathed breath ranged from 0.8 to 2.0 parts per million. Normalized end-exhaled acetone concentration was dependent on flow and was 0.79+/-0.04 and 0.85+/-0.04 for the slow and fast exhalation rates, respectively. A mathematical model of airway and alveolar gas exchange was used to evaluate acetone transport in the lung. By doubling the connective tissue (epithelium+mucosal tissue) thickness, this model predicted accurately (R2=0.94+/-0.05) the experimentally measured expirograms and demonstrated that most acetone exchange occurred in the airways of the lung. Therefore, assays using exhaled acetone measurements need to be reevaluated because they may underestimate blood levels.     

葎草不同溶剂提取物和萃取物的杀虫活性

对律草[Humulus scandens(Lour.) Merr.]全草干粉的不同溶剂提取物以及丙酮提取物的不同溶剂萃取物的杀虫活性进行了研究.在葎草的石油醚、苯、三氯甲烷、乙酸乙酯、丙酮和乙醇提取物(333 g·L-1)中,丙酮和乙醇提取物对小菜蛾(Plutella xylostella L.)的触杀作用较强,其中丙酮提取物的触杀作用最强,试虫的48h校正死亡率达到86.67％.6种溶剂提取物(100g·L-1)对小菜蛾的拒食活性均较弱,24和48 h拒食率仅为9.65％～ 20.45％.6种溶剂提取物对棉蚜(Aphis gossypii Glover)均有较强的触杀效果.其中丙酮提取物的触杀作用最强,经50g·L-1丙酮提取物处理后棉蚜24和48 h的校正死亡率分别为76.78％和85.64％,而经100g·L-1丙酮提取物处理后棉蚜24和48 h的校正死亡率分别为82.63％和92.53％.丙酮提取物的石油醚、三氯甲烷和乙酸乙酯萃取物对小菜蛾均有一定的触杀活性,且随萃取物浓度提高及处理时间的延长触杀活性增强;其中石油醚萃取物的触杀作用最强,经25.0g·L-1石油醚萃取物处理后小菜蛾24和48 h校正死亡率分别达到80.00％和96.67％ 研究结果表明:葎草丙酮提取物对小菜蛾和棉蚜的杀虫活性均最强,其主要有效杀虫活性成分存在于丙酮提取物的石油醚萃取物中.     

Gluconeogenesis from acetone in diabetic rats

Previous investigations have demonstrated that acetone is a true, if minor precursor of glucose in vivo. In diabetic rats 1.30% of the carbon atoms of circulating glucose arises from acetone, whereas 0.67% does in normal 3-day fasted animals. Calculated from these fractions and the turnover rate of glucose, 48 micrograms/kg. min acetone-carbon is converted to glucose-carbon in diabetic and 16 micrograms/kg. min in normal rats. In both groups of rats the labelling of plasma lactate was stronger than that of glucose. In view of these results we conclude that: the transfer of C-atoms from acetone to glucose increases in diabetes; acetone remains a minor source of glucose even in ketonemic diabetic rats.     

Effects of acetone in heparin on the multiple inert gas elimination technique

We have detected acetone in several brands of heparin. If uncorrected, this leads to errors in measuring acetone in blood collected in heparinized syringes, as in the multiple inert gas elimination technique for measuring ventilation-perfusion ratio (VA/Q) distributions. Error for acetone retention [R = arterial partial pressure-to-mixed venous partial pressure (P-V) ratio] is usually small, because R is normally near 1.0, and the error is similar in arterial and mixed venous samples. However, acetone excretion [E = mixed expired partial pressure (P-E)-to-P-V ratio] will appear erroneously low, because P-E is accurately measured in dry syringes, but P-V is overestimated. A physical model of a homogeneous alveolar lung at room temperature and without dead space shows: the magnitude of acetone E error depends upon the ratio of blood sample to heparinized saline volumes and acetone partial pressures, without correction, acetone E can be less than that of less soluble gases like ether, a situation incompatible with conventional gas exchange theory, and acetone R and E can be correctly calculated using the principle of mass balance if the acetone partial pressure in heparinized saline is known. Published data from multiple inert gas elimination experiments with acetone-free heparin, in our labs and others, are within the limits of experimental error. Thus the hypothesis that acetone E is anomalously low because of physiological mechanisms involving dead space tissue capacitance for acetone remains to be tested.     

Pathways of acetone's metabolism in the rat

Distributions of 14C were different from those of 13C in glucoses formed by livers of rats in diabetic ketosis and perfused with [2-14C]acetone and [2-13C]lactate. There was 32-73% of the 14C and 8-12% of the 13C in carbons 3 and 4 of the glucoses with the remaining 14C and 13C distributed about equally in the other carbons. Incorporations of 14C from [2-14C]acetone (14-39%) also exceeded those from [2-14C]pyruvate (8-10%) into carbons 3 and 4 of glucoses formed by hepatocytes from rats fed acetone or fasted. [2-14C]Acetone and [2-14C]pyruvate were infused into rats that were fed, fasted, given acetone in their drinking water, or in diabetic ketosis. Thirty-seven to 52% of the 14C in the glucoses formed was in their carbons 3 and 4 when the acetone was infused and 8 to 14% when the pyruvate was infused. [1,3-14C]Hydroxybutyrate was formed by the rats in diabetic ketosis given [2-14C]acetone. It is concluded that acetone is metabolized in rats to a large extent by a pathway in which lactate or its metabolic equivalent is not an intermediate and that pathway is via acetyl-CoA. via acetyl-CoA.     

Lutein Dispersed in Acetone Aqueous Solution; Spectroscopic Analysis and Electron Microscope Observation

The sizes and shapes of lutein aggregates in acetone aqueous solution were examined by spectroscopic analyses and electron microscopic observation, since lutein dispersed into acetone aqueous solution and provided a much simpler system than others. This system is suitable for basic research on lutein aggregates. The lutein aggregate gave maximum molar ellipticity in acetone concentrations from 35 to 45% aqueous solution. When the acetone concentration increased, CD spectra were reversed in 45% concentration from a negative exciton chirality to the positive. Since the aggregates were most slender in these acetone concentrations, it was found that the length of the lutein aggregate was closely related to the molar ellipticity. The apparent widths of the aggregates became large with increases of the acetone concentration after the reversion of CD spectra, where the molar ellipticities were diminished. These results are similar to those for the lutein aggregates in dilute surfactant solution, and this system is clearer and simpler for electron microscopic observation than those in surfactant solutions.     

Designing and creating a modularized synthetic pathway in cyanobacterium Synechocystis enables production of acetone from carbon dioxide

Ketones are a class of important organic compounds. As the simplest ketone, acetone is widely used as solvents or precursors for industrial chemicals. Presently, million tonnes of acetone is produced worldwide annually, from petrochemical processes. Here we report a biotechnological process that can produce acetone from CO(2), by designing and creating a modularized synthetic pathway in engineered cyanobacterium Synechocystis sp. PCC 6803. The engineered Synechocystis cells are able to produce acetone (36.0 mgl(-1) culture medium) using CO(2) as the sole carbon source, thus opens the gateway for biosynthesis of ketones from CO(2).     

Net glucose production from acetone in isolated murine hepatocytes. The effect of different pretreatments of mice

• 1.1. To evaluate the condition under which net glucose production from acetone, added as sole substrate, occurs different pretreatments of mice, in combination with starvation, were used; (i) acetone pretreatment (acetone is a known inducer of cytochrome P-450 isozymes involved in this pathway), (ii) fructose pretreatment (to induce NADPH + H⁺ generating enzymes) or (iii) their combination.
• 2.2. There was net glucose formation from acetone only in that case, when the cells were prepared from 48 hr fasted animals pretreated with both acetone and fructose. However, using 2-¹⁴C-acetone, incorporation of ¹⁴C-carbon into glucose could be detected in all the cases and, at the same time, acetone was without any effect on protein synthesis.
• 3.3. The addition of acetone increased gluconeogenesis from alanine in almost all the cases. The only exception from this general rule was that the case, when hepatocytes were prepared from acetone pretreated 48 hr starved mice where, instead of the elevation of glucose formation, a decrease of that was caused by acetone.
• 4.4. Acetone decreased ¹⁴C-carbon incorporation into glucose from ¹⁴C-(U)-alanine added at saturating concentration in hepatocytes prepared from starved mice.
• 5.5. Similarly to acetone there was no net glucose formation from acetol either when added alone, however, it enhanced gluconeogenesis from alanine at non-saturating concentrations of the amino acid.
• 6.6. Methylglyoxal proved gluconeogenic in all the cases.
• 7.7. It is concluded that net glucose formation from acetone as sole substrate occurs only under those conditions which are far from a physiological situation, however, when gluconeogenesis from another substrate takes place, acetone can contribute to net glucose formation in hepatocytes prepared from fasted mice.

     

Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2.

The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO2-dependent pathway of acetone metabolism for this bacterium. The depletion of CO2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO2 and was prevented by the depletion of CO2. The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14CO2 into acid-stable products, while the degradation of glucose or beta-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO2- and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO2-dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.     

Biofiltration of toluene and acetone mixtures by a trickle-bed air biofilter

Toluene and acetone mixtures are commonly encountered from the manufacture of semi-conductor or opto-electronic apparatus. This study attempts to employ a trickle-bed air biofilter (TBAB) for treating toluene and acetone mixtures under different gas flow rates and influent concentrations. In the pseudo-steady-states, the elimination capacities of toluene and acetone increased but the removal efficiencies decreased with the increase of influent carbon loading. The removal efficiencies of toluene were higher than those of acetone, indicating that toluene is a preferred substrate in the mixtures. Greater than 90% removal efficiencies were achieved with influent carbon loadings of toluene and acetone below 125 and 15 g/m³ h, respectively. The TBAB appears efficient for controlling toluene and acetone mixture with medium toluene and low acetone loadings. Applicable operating conditions of TBAB for treating mixed toluene and acetone emission are suggested.     

Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113

Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG‐CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG‐CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l^?1 ethanol and 296 mmol l^?1 acetate and started producing 1·8 mol l^?1 acetone in single‐stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl‐CoA to acetone via a non‐naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.     

Determinants in the pathways followed by the carbons of acetone in their conversion to glucose

[2-14C]Acetone was infused into rats that were fed or fasted. Each was infused with either a trace quantity of acetone or a large quantity that resulted in a blood concentration of acetone of at least 4 mM. The distribution of 14C in the carbons of glucose from each rat was determined. Two of the rats were given acetone in their drinking water and one was diabetic. Whether a rat was chronically exposed to acetone, fed or fasted, normal or diabetic, if given the trace dose, over 80% of the 14C in the glucose it formed was in carbons 1, 2, 5, and 6 of the glucose. If a rat was given the large dose, about 50% was in carbons 3 and 4. Thus, the major determinant of the pathways followed by acetone when it is metabolized is its concentration and not the prior dietary state of the animal or its previous exposure to acetone. Incorporation into carbons 1, 2, 5, and 6 occurs in the conversion of the carbons of [2-14C]lactate into glucose, whereas incorporation into carbons 3 and 4 occurs in the conversion of the carbons of [1-14C]acetate into glucose. Therefore, at high acetone concentration, the pathway that has been proposed for acetone's metabolism via acetate predominates, and via acetate there can be no net synthesis of glucose from acetone. When rats were given cyanamide and then the large dose of acetone, 74% of the 14C in the glucose they formed was in carbons 3 and 4 of the glucoses. Thus, the relative contribution of the pathway to lactate, or its metabolic equivalent, that has been proposed appears to be lessened by the administration of an aldehyde dehydrogenase inhibitor.     

Bacterial acetone carboxylase is a manganese-dependent metalloenzyme

Bacterial acetone carboxylase catalyzes the ATP-dependent carboxylation of acetone to acetoacetate with the concomitant production of AMP and two inorganic phosphates. The importance of manganese in Rhodobacter capsulatus acetone carboxylase has been established through a combination of physiological, biochemical, and spectroscopic studies. Depletion of manganese from the R. capsulatus growth medium resulted in inhibition of acetone-dependent but not malate-dependent cell growth. Under normal growth conditions (0.5 microm Mn2+ in medium), growth with acetone as the carbon source resulted in a 4-fold increase in intracellular protein-bound manganese over malate-grown cells and the appearance of a Mn2+ EPR signal centered at g = 2 that was absent in malate-grown cells. Acetone carboxylase purified from cells grown with 50 microm Mn2+ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 microm Mn2+, consistently yielding a stoichiometry of 1.9 manganese/alpha2beta2gamma2 multimer, or 0.95 manganese/alphabetagamma protomer. Manganese in acetone carboxylase was tightly bound and not removed upon dialysis against various metal ion chelators. The addition of acetone to malate-grown cells grown in medium depleted of manganese resulted in the high level synthesis of acetone carboxylase (15-20% soluble protein), which, upon purification, exhibited 7% of the activity and 6% of the manganese content of the enzyme purified from acetone-grown cells. EPR analysis of purified acetone carboxylase indicates the presence of a mononuclear Mn2+ center, with possible spin coupling of two mononuclear sites. The addition of Mg.ATP or Mg.AMP resulted in EPR spectral changes, whereas the addition of acetone, CO2, inorganic phosphate, and acetoacetate did not perturb the EPR. These studies demonstrate that manganese is essential for acetone carboxylation and suggest a role for manganese in nucleotide binding and activation.     

Gluconeogenesis from acetone in starved rats.

To non-anaesthetized rats starved for 3 days, [U-14C]acetone, NaH14CO3, L-[U-14C]lactate, [2-14C]acetate or D-[U-14C]- plus D-[3-3H]-glucose was injected intravenously. From the change in the plasma concentration of labelled acetone versus time after the injection, the metabolic clearance rate of acetone was calculated as 2.25 ml/min per kg body wt., and its rate of turnover as 0.74 mumol/min per kg. The extent and time course of the labelling of plasma glucose, lactate, urea and acetoacetate were followed and compared with those observed after the injection of labelled lactate, acetate and NaHCO3. The labelling of plasma lactate was rapid and extensive. Some 1.37% of the 14C atoms of circulating glucose originated from plasma acetone, compared with 44% originating from lactate. By deconvolution of the Unit Impulse Response Function of glucose, it was shown that the flux of C atoms from acetone to glucose reached a peak at about 100 min after injection of labelled acetone. In comparable experiments the transfer from lactate reached a peak at 14 min after the injection of labelled lactate. It was concluded that acetone is converted into lactate to a degree sufficient to account for the labelling of plasma glucose and is thus a true, albeit minor, substrate of glucose synthesis in starved rats.     

Analysis of the inter-molecular interactions between amino acids and acetone by THz spectroscopy

The THz spectra of amino acids after application of spots of acetone were measured. The 0.6 THz band was commonly observed in many amino acids that formed the intra-molecular salt structure. The band can be attributed to the interaction vibration from the common structural configuration of amino acids and acetone molecules. The evidence suggests that the vibration between the amino acids with intra-molecular salt structure and acetone has a peak at 0.6 THz. A model of the interaction vibration of acetone and the functional groups of amino acids is proposed.     

PROTEIN COAGULATION AND ITS REVERSAL : GLOBIN

1. The globin prepared from hemoglobin by the acid acetone method is denatured globin. 2. The denaturation and coagulation of globin by acid acetone are reversible. 3. Soluble globin can be obtained from the acid acetone globin even if the globin is first precipitated by trichloracetic acid or heated to 100°C. 4. Hill and Holden''s theory that they separated native globin from hemoglobin without any intermediate denaturation is not proven by their experiments.     

Acetone and butanone metabolism of the denitrifying bacterium "Aromatoleum aromaticum" demonstrates novel biochemical properties of an ATP-dependent aliphatic ketone carboxylase

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium "Aromatoleum aromaticum" is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)(2) composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 P(i). The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content.