Uncatalyzed and amine catalyzed decarboxylation of acetoacetic acid: an examination in terms of No Barrier Theory

Rate constants for decarboxylation of acetoacetic acid, its anion, and its imine with aminoacetonitrile have been calculated from equilibrium constants and distortion energies using No Barrier Theory. The mechanisms of decarboxylation of both acetoacetic acid and its imine involve preequilibrium formation of the zwitterion.     

Fatty acid synthesis by isolated leucoplasts from developingBrassica seeds: Role of nucleoside triphosphates and DHAP-shuttle as the source of energy

Fatty acid synthesis in leucoplasts isolated from developing seeds ofBrassica campestris was absolutely dependent on external source of ATP. None of the other nucleoside triphosphates could replace ATP in the reaction mixture. Use of ADP alone also resulted in reduced rates of fatty acid synthesis. However, in combination with inorganic phosphate or inorganic pyrophosphate, it improved the rate of fatty acid synthesis, giving up to 50% of the ATP-control activity. Inorganic phosphate or inorganic pyrophosphate alone again did not serve as an energy source for fatty acid synthesis. AMP, alongwith inorganic pyrophosphate could promote fatty acid synthesis to up to 42% of the activity obtained with ATP. The three components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate of dihydroxy acetone phosphate-shuttle together could restore 50% of the activity obtained with ATP. Omission of any one of the components of this shuttle drastically reduced the rate of fatty acid synthesis to 15–24% of the ATP-control activity. Inclusion of ATP in reaction mixtures containing shuttle components enhanced the rate of synthesis over control. The optimum ratio of shuttle components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate determined was 1:1:2. Maximum rates of fatty acid synthesis were obtained when dihydroxy acetate phosphate was used as the shuttle triose. Glyceraldehyde-3-P, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate as shuttle trioses were around 35–60% as effective as dihydroxy acetone phosphate in promoting fatty acid synthesis. The results presented here indicate that although the isolated leucoplasts readily utilize exogenously supplied ATP for fatty acid synthesis, intraplastidic ATP could also arise from dihydroxy acetone phosphate shuttle components or other appropriate metabolites     

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V _max and K _m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg^?1 min^?1 and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K _i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml ^? resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

Simultaneous measurement of plasma concentrations and 13C-enrichment of short-chain fatty acids,lactic acid and ketone bodies by gas chromatography coupled to mass spectrometry

A new method has been developed for the simultaneous measurement, in a reduced plasma sample, of concentration and 13C-isotopic enrichment of acetic, propionic, butyric, lactic, acetoacetic and beta-hydroxybutyric acids by gas chromatography coupled to mass spectrometry. After plasma deproteinisation, a diethylic extraction and a N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide derivatisation were performed. Both diethyl extraction and derivatisation procedures were optimised using the central composite designs methodology. The optimised method provides good linearity, intra-day and within-day repeatability. Except for beta-hydroxybutyric (49 microM) and acetoacetic acid (5 microM), detection limits were ranging between 0.2 and 0.7 microM allowing uses of this method for colonic metabolism studies.     

Extraction of defatted rice bran with subcritical aqueous acetone

Defatted rice bran extracts were obtained by subcritical treatment using aqueous acetone as extractant. Treatment with 40% (v/v) acetone at 230 °C for 5 min yielded an extract with the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (0.274 mmol of ascorbic acid/g of bran), total carbohydrate (0.188 g/g of bran), protein (0.512 g/g of bran), and total phenolic contents (88.2 mg of gallic acid/g of bran). The effect of treatment temperature (70-230 °C) was investigated using 40% (v/v) acetone, and the extract under 230 °C treatment showed the highest levels of all the determinations described above. The extracts obtained with various concentrations of aqueous acetone were subjected to UV absorption spectra and HPLC analysis, and the results showed changes in composition and polarity. Antioxidative activity evaluated against oxidation of bulk linoleic acid of the extract obtained with 80% (v/v) acetone was higher than that not only of the extract from subcritical water treatment but also of that obtained 40% (v/v) acetone treatment.     

Degradation of 4-hydroxyphenylacetic acid by Trichosporon cutaneum.

Trichosporon cutaneum degraded 4-hydroxyphenylacetic acid to acetoacetic and malic acids. 3.4-Dihydroxyphenylacetic acid, an intermediate in the reaction sequence, underwent hydroxylation before the benzene ring was cleaved.     

Phenolic constituents from the lichen Parmotrema stuppeum (Nyl.) Hale and their antioxidant activity

Lichen, Parmotrema stuppeum (P. stuppeum) was successively extracted with benzene and acetone. Both the extracts were fractionated on 1% oxalic acid impregnated silica gel column to obtain four phenolic compounds. The structures of compounds were identified by 1H and 13C NMR spectra as methyl orsenillate, orsenillic acid, atranorin and lecanoric acid respectively. Antioxidant activity of benzene extract, acetone extract and isolated compounds were evaluated in a beta-carotene-linoleate model system. The pure compounds showed moderate antioxidant activity. This is the first report on the isolation and characterisation of compounds from the lichen P. stuppeum as well as on their antioxidant activity.     

Studies on an improved lead phosphate technique for the demonstration of non-specific acid phosphatase in non-deparaffinized organ and tissue sections

Summary The inconstant results and the aspecific reaction patterns obtained after use of the lead phosphate method for the demonstration of acid phosphatase cannot be attributed to destruction of the enzyme by acetone fixation and embedding in paraffin. The marked shrinking effect of pure acetone is partially avoided by fixation in 80% acetone at 0–4° C. The appearance of spotty gaps employing the lead phosphate method in non-deparaffinized sections is the result of the presence of by-products in the paraffin and not of an enzyme-inactivating action of the albumen-containing adhesive. Constant results in the localization of acid as well as of alkaline phosphatase can be obtained by pretreatment of non-deparaffinized sections with 80% acetone. Paraffin mixtures containing stearin and stearic acid are not suitable for this improved incubation technique. The conditions for the obtaining of reliable data as to the presence of non-specific acid phosphate activity are more favourable in non-deparaffinized sections of material fixed in cold acetone than in formaldehyde-fixed frozen sections. The effect of quantitative variations of the components of the substrate mixture on the result of the reaction is discussed. It has been clearly shown that the relative proportion of the concentrations of glycerophosphate ions and lead ions is of importance for optimal results. The distribution of non-specific acid phosphatase, histochemically demonstrable with the improved technique in a number of organs and tissues of the rabbit, rat and mouse is described.With 18 Figures in the Text     

Phototrophic Fe(II) oxidation promotes organic carbon acquisition by Rhodobacter capsulatus SB1003

Anoxygenic phototrophic Fe(II) oxidation is usually considered to be a lithoautotrophic metabolism that contributes to primary production in Fe-based ecosystems. In this study, we employed Rhodobacter capsulatus SB1003 as a model organism to test the hypothesis that phototrophic Fe(II) oxidation can be coupled to organic carbon acquisition. R. capsulatus SB1003 oxidized Fe(II) under anoxic conditions in a light-dependent manner, but it failed to grow lithoautotrophically on soluble Fe(II). When the strain was provided with Fe(II)-citrate, however, growth was observed that was dependent upon microbially catalyzed Fe(II) oxidation, resulting in the formation of Fe(III)-citrate. Subsequent photochemical breakdown of Fe(III)-citrate yielded acetoacetic acid that supported growth in the light but not the dark. The deletion of genes (RRC00247 and RRC00248) that encode homologs of atoA and atoD, required for acetoacetic acid utilization, severely impaired the ability of R. capsulatus SB1003 to grow on Fe(II)-citrate. The growth yield achieved by R. capsulatus SB1003 in the presence of citrate cannot be explained by lithoautotrophic growth on Fe(II) enabled by indirect effects of the ligand [such as altering the thermodynamics of Fe(II) oxidation or preventing cell encrustation]. Together, these results demonstrate that R. capsulatus SB1003 grows photoheterotrophically on Fe(II)-citrate. Nitrilotriacetic acid also supported light-dependent growth on Fe(II), suggesting that Fe(II) oxidation may be a general mechanism whereby some Fe(II)-oxidizing bacteria mine otherwise inaccessible organic carbon sources.     

Evaluation of the antimicrobial activity of the acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid, norstictic acid, and protocetraric acid constituents

The acetone extract of the lichen Ramalina farinacea and its (+)-usnic acid constituent showed antimicrobial activity against Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans, and Candida glabrata. Norstictic acid was active against Aeromonas hydrophila as well as the above microorganisms except Yersinia enterocolitica. Protocetraric acid showed activity only against the tested yeasts Candida albicans and Candida glabrata. The MIC values of the extract as well as of the three substances were determined. No antifungal activity of the acetone extract has been observed against ten filamentous fungi.     

Acid Protease Produced by Trametes sanguinea,a Wood-destroying Fungus

A wood-destroying fungus, Trametes sanguinea, produced a potent acid protease in a submerged culture. Maximum proteolytic activity of the culture was attained after 140-hours cultivation in a medium containing dextrin and corn steep liquor. The acid protease was obtained in crystalline form from the mycelium-free culture filtrate by the following successive treatments: acetone precipitation, ionexchange column chromatography, ammonium sulfate fractionation, dialysis, and crystallization by acetone. Throughout the over-all process, the acid protease was purified approximately 30-fold with about 8% recovery of the original activity.     

Hormones with adrenocorticotropic and opiate-like activities from the carp (Cyprinus carpio) pituitary

Carp (Cyprinus carpio) pituitary acetone powder was extracted with a mixture of water, hydrochloric acid and acetone. An acid acetone powder was formed by adding the pituitary extract into a large volume of chilled acetone and subsequently recovering the precipitate. The powder was subjected to ion exchange chromatography on CM cellulose. Fractions adsorbed on the ion exchanger exhibited ACTH-like activity as evidenced in the ability to stimulate lipolysis in isolated hamster adipocytes and corticosterone production in isolated rat adrenal decapsular cells and also in cross-reactivity in an ACTH-specific radioimmunoassay. A portion of the ACTH-like bioactivity and immunoactivity was unadsorbed on the ion exchanger. Opiate-like activity in opiate receptor binding assay, employing [3H]D-ala2-D-leu5 enkephalin or [3H]naloxone as ligand, also resided in fractions adsorbed on CM cellulose. The data indicate a separation of ACTH-like and opiate-like activities, and the presence of opiate-like molecules with different affinities of binding to mu and delta opiate receptors.     

Novel surface lipids of diapausing Manduca sexta pupae. Long chain oxoalcohol esters of acetoacetic, hydroxybutyric, and acetic acids

Ester components in the surface wax from diapausing tobacco hornworm pupae, Manduca sexta L., were separated by thin layer chromatography and gas-liquid chromatography, and characterized by infrared spectroscopy and gas-liquid chromatography-mass spectrometry. Three groups of esters were identified as natural derivatives of acetic acid, acetoacetic acid, and 3-hydroxybutyric acid. The major ester fraction was identified as a mixture of C26 (10%), C27 (5%), and C28 (85%) oxoalcohol esters of acetoacetic acid. The major homolog consisted of equal amounts of 11-oxooctacosanyl 3-oxobutanoate and 12-oxooctacosanyl 3-oxobutanoate. Lesser amounts of 11- and 12-oxooctacosanyl and n-octacosanyl esters of acetic and 3-hydroxybutyric acids were also identified. The chain length distributions of these C26, C27, and C28 oxoalcohol and n-primary alcohol ester moieties, as well as the isomeric ratios for the 11- and 12-oxoalcohol isomers, were similar to the oxoaldehydes and unesterified oxoalcohols previously identified by Buckner et al (Buckner, J. S., Nelson, D. R., Haak, H., and Pomonis, J. G. (1984) J. Biol. Chem. 259, 8452-8470) as lipid components of the surface wax of M. sexta pupae.     

Effects of Commercial Fatty Acids on Cutinase Release by Ascochyta pisi

The effect of a range of commercially available C₁₆ and C₁₈ fatty acids, on the release of cutinase by Ascochyta pisi, was studied. When juniperic acid was used as sole carbon source, cutinase activity was released in the culture medium, and was markedly enhanced by post-treatment of A. pisi cultures with acetone used as enzyme extractor. Without acetone, less cutinase was naturally released in culture medium containing juniperic acid at 0.5 %, than at 0.0l % or 0.05 %. Upon post-treatment with acetone, the same level of cutinase was released with all three concentrations, thus suggesting that the enzyme was induced, but not completely released in the presence of 0.5 % juniperic acid. When ricinelaidic or ricinoleic acids were supplemented at 0.5 % to cutin in the culture medium, they strongly inhibited the release of cutinase, even with acetone post-treatment. Comparable inhibition by ricinoleic acid was also observed when juniperic acid was used as cutinase inducer, thus suggesting that not only the release, but also the production of cutinase were inhibited.     

d-ASPARTATE OXIDASE ACTIVITY IN EXTRACTS OF MAMMALIAN CENTRAL NERVOUS TISSUE

Abstract— d -Aspartate oxidase activity has been measured in water extracts of acetone powders prepared from cat forebrain, cerebellum and spinal cord, rat brain, hog brain and sheep brain stem, and compared with that found in rabbit and cat kidney. The results suggest that the brain enzyme has very similar properties to the n-aspartate oxidase ( d -aspartate: oxygen oxidorcductase (deaminating), EC 1.4.3.1) of kidney. Crude extracts (ammonium sulphate fractions of water extracts of acetone powders) displayed little activity without added FAD. FMN could not replace FAD. With oxygen as electron acceptor, the enzyme oxidized d -aspartate much more rapidly than d -glutamate, and displayed quite high activities with N -substituted derivatives of d -aspartate as substrates. Those amino acids susceptible to oxidation by d -amino acid oxidase were not oxidized by the d -aspartate oxidase. The regional distribution of the d -aspartate oxidase activity within the CNS differed from that of d -amino acid oxidase. As has been previously observed for kidney d -aspartate oxidase activity, dicarboxylic acids competitively inhibited this enzymic activity in brain extracts, while sodium benzoate and sodium barbitone, inhibitors of d -amino acid oxidase, were without effect.     

Separation of the intermediates of leucine catabolism by high-performance liquid chromatography

A method has been developed for the separation of leucine, 2-ketoisocaproic acid, isovaleryl CoA, 3-methylcrotonyl CoA, 3-hydroxy-3-methylglutaryl CoA, 3-methylglutaconyl CoA, acetyl CoA, and acetoacetic acid by ion-exchange high-performance liquid chromatography. The analysis requires 180 min. Use of this method to assess the catabolism of radiolabeled leucine in normal cultured human skin fibroblasts shows that these cells do not accumulate CoA esters, but convert leucine mainly to 2-ketoisocaproic acid, glutamate, and hydroxyisovalerate. In the fibroblasts of a patient with maple syrup urine disease, only 2-ketoiscaproic acid is produced from leucine.     

Effect of starvation and acetone on the enzyme systems of biotransformation and toxicity of xenobiotics--CYP2E1 substrates in rats

In experiments on 205 rats it was fixed, that starvation during 2-3 days, as well as introduction of acetone (250 and 1000 mg/kg) considerably increases CYP2E1-dependent aniline and p-nitrophenol hydroxylase activity in the liver, kidneys, lungs and CYP3A dependent erythromycin N-demethylase activity, at the same time, suppress in a liver activity enzymes, dependent CYP2D, CYP1A2 and CYP2C as well as of activity UDP-glucuronosyl-transferase, sulfotransferase and glutathione-S-transferase. The starvation causes accumulation of KoA and increases activity of N-acetyltransferase in the liver. Starvation induces the change of enzymes activity and correlates with the intensifying of the processes of lipolysis, glycogenolysis, gluconeogenesis and, especially, ketogenesis which are appreciably initiated by introduction of acetone. The starvation and introduction of acetone increases metabolism of acetanilide and brombenzene, and, increasing the formation of toxic metabolites, raise its hepato-, nephro- and pulmotoxicity. The starvation attenuates elimination of indometacin from blood plasma, but intensifies conjugation of sulfadimidine with acetic acid.     

Utilization of kerosene by a hydrocarbon-assimilating strain of Fusarium oxisporum

Fusarium oxysporum IFO 6384 was grown in three different media, one containing glucose and two containing kerosene as carbon source. One of the kerosene media contained also Tween 60. The harvested dry mats were weighed at various stages of growth. Substitution of glucose with kerosene led to a sharp drop in the production of fungal mycelia. Analysis of the mat samples representing maximum yields indicated that the mycelia grown in the kerosene media were richer in free amino acid and poorer in protein as well as in polymeric and low-molecular weight carbohydrates than those grown in the glucose medium. Treatment of the culture filtrates with three volumes of acetone led to the separation of two fractions. In the first, acetone soluble fraction the free sugars and amino acids were determined, whereas the second, acetone precipitable one, was examined for its proteolytic activity. The gelatinase activity of the acetone precipitable material (APM) obtained from the kerosene media was lower than that from the glucose medium. The former activity, in contrast to the latter reached its maximum and minimum values at pHs 4 and 9, respectively.