scholarly journals The microbial metabolism of thiophen-2-carboxylate

1973 ◽  
Vol 134 (2) ◽  
pp. 353-366 ◽  
Author(s):  
Roger E. Cripps
Keyword(s):  
Methylene Blue ◽  
High Speed ◽  
Enrichment Culture ◽  
Specific Radioactivity ◽  
Microbial Metabolism ◽  
Sole Source ◽  
Cellular Protein ◽  
Sulphur Atom ◽  
Degradative Pathway ◽  
Anaerobic Reduction

1. An organism was isolated by enrichment culture that was capable of using thiophen-2-carboxylate as sole source of carbon, energy and sulphur for growth. 2. Analysis of the cellular protein after growth of the organism on thiophen-2-[14C]carboxylate showed that only glutamate, proline and arginine were labelled. All the radioactivity in the glutamate was confined to C-1. 3. In the presence of 2.1 mm-arsenite, suspensions of the organism converted thiophen-2-[14C]carboxylate into 14C-labelled 2-oxoglutarate which had the same specific radioactivity as the starting material. 4. Cell-free extracts of the organism catalysed the release of 14CO2 from thiophen-2-[14C]carboxylate. This activity was largely dependent on the presence of ATP and CoA and was stimulated by NAD+ and Mg2+. Inclusion of hydroxylamine resulted in the appearance of thiophen-2-carbohydroxamic acid, indicating that the ATP and CoA were involved in the formation of the CoA ester of thiophen-2-carboxylate. 5. High-speed centrifuging of cell-free extracts resulted in supernatants with decreased thiophen-2-carboxylate-degrading activity. Activity was restored by the addition of the high-speed pellet or by Methylene Blue. 6. The metabolism of the CoA ester of thiophen-2-carboxylate by cell-free extracts could be linked to the anaerobic reduction of Methylene Blue. 7. The sulphur atom of the thiophen nucleus was converted into sulphate by growing cultures and resting suspensions of the organism. 8. A degradative pathway is proposed involving the hydroxylation (at C-5) of the CoA ester of thiophen-2-carboxylate followed by further metabolism to 2-oxoglutarate and sulphate.

scholarly journals The metabolism of 2-furoic acid by Pseudomonas F2

1969 ◽  
Vol 113 (4) ◽  
pp. 577-587 ◽  
Author(s):  
P W Trudgill
Keyword(s):  
Methylene Blue ◽  
Oxygen Uptake ◽  
Magnesium Chloride ◽  
Metal Ion ◽  
Enrichment Culture ◽  
Furoic Acid ◽  
Substrate Analogues ◽  
Anaerobic Reduction ◽  
Dowex 1

1. Pseudomonas F2 isolated by enrichment culture on 2-furoic acid and grown with it as carbon source oxidized the compound with a Qo2 of 170μl./mg. dry wt./hr. and the overall consumption of 2·5μmoles of oxygen/μmole of substrate. 2. In the presence of 1mm-sodium arsenite, oxygen uptake was restricted to 0·54μmole/μmole of 2-furoate oxidized, with the formation of 0·86μmole of 2-oxoglutarate/μmole of 2-furoate. 3. Cell suspensions, disrupted in a French pressure cell and centrifuged at 27000g, yielded supernatants capable of catalysing the slow oxidation of 2-furoate (0·17μmole/mg. of protein/hr.). 4. Fractionation of 27000g supernatants at 200000g yielded a soluble enzyme fraction capable of catalysing the oxidation of 2-furoate only in the presence of added 200000g pellet or of Methylene Blue. 5. The 2-furoate-stimulated uptake of oxygen or the anaerobic reduction of Methylene Blue by dialysed 27000g supernatant required the addition of ATP and CoA, and the rate of oxygen uptake was further enhanced by the addition of magnesium chloride and NAD+. 6. The role of ATP and CoA in the formation of 2-furoyl-CoA was demonstrated by the accumulation of 2-furoylhydroxamic acid in the presence of hydroxylamine. 7. Dialysed 200000g supernatant, treated with Dowex 1, required the addition of ATP, CoA and Methylene Blue before it could oxidize 2-furoate to 2-oxoglutarate, which was trapped in unitary stoicheiometric yield as its phenylhydrazone. Magnesium chloride and NAD+ were not stimulatory in this system. The oxidation of 2-furoate to 2-oxoglutarate was not inhibited by substrate analogues, metal ion-chelating agents, thiol-active compounds or inhibitors of cytochrome-mediated electron transport. 8. No evidence was obtained for the intervention of 2,5-dioxovalerate as an intermediate in 2-oxoglutarate formation.

scholarly journals The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects

1984 ◽  
Vol 219 (2) ◽  
pp. 461-470 ◽  
Author(s):  
D D Patel ◽  
C R Pullinger ◽  
B L Knight
Keyword(s):  
Cholesterol Synthesis ◽  
Reductase Activity ◽  
Cholesterol Biosynthesis ◽  
Specific Radioactivity ◽  
Density Lipoprotein ◽  
Cellular Protein ◽  
True Rate ◽  
Hmg Coa Reductase ◽  
Coa Reductase ◽  
In Cells

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.

scholarly journals Measurement of protein synthesis in rat lungs perfused in situ

1980 ◽  
Vol 188 (1) ◽  
pp. 269-278 ◽  
Author(s):  
Clyde A. Watkins ◽  
D. Eugene Rannels
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Free Amino ◽  
Plasma Concentrations ◽  
Specific Radioactivity ◽  
Sole Source ◽  
Bicarbonate Buffer ◽  
Normal Plasma ◽  
Rat Lungs

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-14C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O2/CO2 (19:1) or O2/N2/CO2 (4:15:1)]. [U-14C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [14C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [14C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.

Biochemical pathway and degradation of phthalate ester isomers by bacteria

2005 ◽  
Vol 52 (8) ◽  
pp. 241-248 ◽  
Author(s):  
J.-D. Gu ◽  
J. Li ◽  
Y. Wang
Keyword(s):  
Aromatic Ring ◽  
Enrichment Culture ◽  
Klebsiella Oxytoca ◽  
Sole Source ◽  
Phthalate Ester ◽  
Ring Cleavage ◽  
Mangrove Sediment ◽  
Individual Species ◽  
Dimethyl Phthalate ◽  
Dimethyl Phthalate Ester

Degradation of dimethyl isophthalate (DMI) and dimethyl phthalate ester (DMPE) was investigated using microorganisms isolated from mangrove sediment of Hong Kong Mai Po Nature Reserve. One enrichment culture was capable of utilizing DMI as the sole source of carbon and energy, but none of the bacteria in the enrichment culture was capable of degrading DMI alone. In co-culture of two bacteria, degradation was observed proceeding through monomethyl isophthalate (MMI) ester and isophthalic acid (IPA) before the aromatic ring opening. Using DMI as the sole carbon and energy source, Klebsiella oxytoca Sc and Methylobacterium mesophilicum Sr degraded DMI through the biochemical cooperation. The initial hydrolytic reaction of the ester bond was by K. oxytoca Sc and the next step of transformation was by M. mesophilicum Sr, and IPA was degraded by both of them. In another investigation, a novel bacterium, strain MPsc, was isolated for degradation of dimethyl phthalate ester (DMPE) also from the mangrove sediment. On the basis of phenotypic, biochemical and 16S rDNA gene sequence analyses, the strain MPsc should be considered as a new bacterium at the genus level (8% differences). This strain, together with a Rhodococcus zopfii isolated from the same mangrove sediment, was able to degrade DMPE aerobically. The consortium consisting of the two species degraded 450mg/l DMPE within 3 days as the sole source of carbon and energy, but none of the individual species alone was able to transform DMPE. Furthermore, the biochemical degradation pathway proceeded through monomethyl phthalate (MMP), phthalic acid (PA) and then protocatechuate before aromatic ring cleavage. Our results suggest that degradation of complex organic compounds including DMI and DMPE may be carried out by several members of microorganisms working together in the natural environments.

scholarly journals Anaerobic reduction of ethene to ethane in an enrichment culture

1998 ◽  
Vol 25 (3) ◽  
pp. 251-256 ◽  
Author(s):  
Francis H.M Koene-Cottaar ◽  
Gosse Schraa
Keyword(s):  
Enrichment Culture ◽  
Anaerobic Reduction

Metabolism of a monoterpene alcohol, linalool, by a soil pseudomonad

1977 ◽  
Vol 23 (3) ◽  
pp. 230-239 ◽  
Author(s):  
K. M. Madyastha ◽  
P. K. Bhattacharyya ◽  
C. S. Vaidyanathan
Keyword(s):  
Mineral Salt Medium ◽  
Enrichment Culture ◽  
Sole Source ◽  
Mineral Salt ◽  
Salt Medium ◽  
Culture Techniques ◽  
Unsaturated Lactone ◽  
Linalool Oxide

A microorganism of the genus Pseudomonas has been isolated from the soil by enrichment culture techniques with linalool(I) as the sole source of carbon and energy. The organism is also capable of utilizing limonene, citronellol, and geraniol as substrates but fails to grow on citral, citranellal, and 1,8-cineole. Fermentation of linalool by this bacterium in a mineral salt medium results in the formation of 10-hydroxylinalool(II), 10-carboxylinalool(III), oleuropeic acid(IX), 2-vinyl-2-methyl-5-hydroxyisopropyl-tetrahydrofuran(linalool oxide, V), 2-vinyl-2-methyl-tetrahydrofuran-5-one(unsaturated lactone, VI), and few unidentified minor metabolites. Probable pathways for the biodegradation of linalool are presented.

A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins

2011 ◽  
Vol 400 (3) ◽  
pp. 767-775 ◽  
Author(s):  
Evelyn Png ◽  
WanWen Lan ◽  
Melisa Lazaroo ◽  
Silin Chen ◽  
Lei Zhou ◽  
...  
Keyword(s):  
Protein Separation ◽  
High Speed ◽  
Cellular Protein ◽  
New Method ◽  
Subcellular Compartmentalization ◽  
Insight Into

Degradation of (–)-Ephedrine by Pseudomonas putida Detection of (–)-Ephedrine: NAD+-oxidoreductase from Arthrobacter globiformis

1980 ◽  
Vol 35 (1-2) ◽  
pp. 80-87 ◽  
Author(s):  
E. Klamann ◽  
F. Lingens
Keyword(s):  
Pseudomonas Putida ◽  
Enrichment Culture ◽  
Culture Fluid ◽  
Sole Source ◽  
Culture Technique ◽  
Arthrobacter Globiformis ◽  
Muconic Acid ◽  
Ammonium Sulphate Precipitation ◽  
Wide Range ◽  
Nad Oxidoreductase

Abstract A bacterium utilizing the alkaloid (-)-ephedrine as its sole source of carbon was isolated by an enrichment-culture technique from soil supplemented with 4-benzoyl-1,3-oxazolidinon-(2). The bacterium was identified as Pseudomonasputida by morphological and physiological studies. The following metabolites were isolated from the culture fluid: methylamine, formaldehyde, methyl- benzoylcarbinol (2-hydroxy-1-oxo-1-phenylpropane), benzoid acid, pyrocatechol and cis, cis- muconic acid. A pathway for the degradation of (-)-ephedrine by Pseudomonas putida is proposed and compared with the degradative pathway in Arthrobacter globiformis.The enzyme, which is responsible for the first step in the catabolism of (-)-ephedrine could be demonstrated in extracts from Arthrobacter globiformis. This enzyme catalyses the dehydrogena- tion of (-)-ephedrine yielding phenylacetylcarbinol/methylbenzoylcarbinol and methylamine. It requires NAD+ as cofactor and exhibits optimal activity at pH 11 in 0.1 m glycine/NaOH buffer. The Km value for (-)-ephedrine is 0.02 mM and for NAD+ 0.11 mм, respectively. No remarkable loss of activity is observed following treatment with EDTA. The enzyme has been shown to react with a wide range of ethanolamines. A slight enrichment was obtained by ammonium sulphate precipitation. The name (-)-ephedrine: NAD+-oxidoreductase (deaminating) is proposed.

scholarly journals DISTRIBUTION OF NEWLY SYNTHESIZED AMYLASE IN MICROSOMAL SUBFRACTIONS OF GUINEA PIG PANCREAS

1966 ◽  
Vol 30 (3) ◽  
pp. 519-530 ◽  
Author(s):  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
High Speed ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Pellet Fraction ◽  
Light Region ◽  
Centrifuge Tube ◽  
In Vivo Labeling

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).1 Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD260 absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C14, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

scholarly journals Metabolism of 2-Methylpropene (Isobutylene) by the Aerobic Bacterium Mycobacterium sp. Strain ELW1

2015 ◽  
Vol 81 (6) ◽  
pp. 1966-1976 ◽  
Author(s):  
Samanthi Kottegoda ◽  
Elizabeth Waligora ◽  
Michael Hyman
Keyword(s):  
Dry Weight ◽  
Microbial Metabolism ◽  
Sole Source ◽  
Butyl Alcohol ◽  
Lag Phase ◽  
Aerobic Bacterium ◽  
Butyl Ether ◽  
Content Type ◽  
Cobalt Ions ◽  
Alkene Oxidation

ABSTRACTAn aerobic bacterium (Mycobacteriumsp. strain ELW1) that utilizes 2-methylpropene (isobutylene) as a sole source of carbon and energy was isolated and characterized. Strain ELW1 grew on 2-methylpropene (growth rate = 0.05 h−1) with a yield of 0.38 mg (dry weight) mg 2-methylpropene−1. Strain ELW1 also grew more slowly on bothcis- andtrans-2-butene but did not grow on any other C2to C5straight-chain, branched, or chlorinated alkenes tested. Resting 2-methylpropene-grown cells consumed ethene, propene, and 1-butene without a lag phase. Epoxyethane accumulated as the only detected product of ethene oxidation. Both alkene consumption and epoxyethane production were fully inhibited in cells exposed to 1-octyne, suggesting that alkene oxidation is initiated by an alkyne-sensitive, epoxide-generating monooxygenase. Kinetic analyses indicated that 1,2-epoxy-2-methylpropane is rapidly consumed during 2-methylpropene degradation, while 2-methyl-2-propen-1-ol is not a significant metabolite of 2-methylpropene catabolism. Degradation of 1,2-epoxy-2-methylpropane by 2-methylpropene-grown cells led to the accumulation and further degradation of 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate, two sequential metabolites previously identified in the aerobic microbial metabolism of methyltert-butyl ether (MTBE) andtert-butyl alcohol (TBA). Growth of strain ELW1 on 2-methylpropene, 1,2-epoxy-2-methylpropane, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate was fully inhibited when cobalt ions were omitted from the growth medium, while growth on 3-hydroxybutyrate and other substrates was unaffected by the absence of added cobalt ions. Our results suggest that, like aerobic MTBE- and TBA-metabolizing bacteria, strain ELW1 utilizes a cobalt/cobalamin-dependent mutase to transform 2-hydroxyisobutyrate. Our results have been interpreted in terms of their impact on our understanding of the microbial metabolism of alkenes and ether oxygenates.

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