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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DeWitt, Charles W. (The Upjohn Co., Kalamazoo, Mich.) and Janet A. Rowe. Sialic acids (N,7-O-diacetylneuraminic acid and N-acetylneuraminic acid) in Escherichia coli. I. Isolation and identification. J. Bacteriol. 82:838-848. 1961.-Two sialic acids, N-acetylneuraminic acid and N,7-O-diacetylneuraminic acid, were obtained in crude mixtures from whole cells of Escherichia coli and from its endotoxin by weak acid hydrolysis followed by anion exchange resin chromatography. Yields from whole cells were 0.1 to 0.2% (dry weight) with 50 to 60% purity. Identification of the sialic acids was by comparative paper chromatography and colorimetric assays using the acidic p-dimethylaminobenzaldehyde (direct Ehrlich), resorcinol and thiobarbituric acid reactions. The N-acetyl derivative was also shown to be susceptible to hydrolysis by clostridial N-acetylneuraminic aldolase and the end products identified, N-acetylamannosamine by paper chromatography and pyruvic acid by oxidation of DPNH with lactic acid dehydrogenase. The two sialic acids were separated on paper chromatograms, eluted, and assays for total and ester acyl groups showed the suspected N-acetyl derivative to contain 0.11 O-acyl and 1.16 N-acetyl groups per mole sialic acid and the diacetyl derivative to have 1.10 O-acyl and 0.93 N-acetyl groups per mole. The O-acyl group was identified as acetyl by preparation of the hydroxamate.
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PMID:Sialic acids (N,7-O-diacetylneuraminic acid and N-acetylneuraminic acid) in Escherichia coli. I. Isolation and identification. 1388 21

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates--nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD(+)-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I(50) (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.
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PMID:The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites. 1467 May 98

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5'-diphosphate (ADP) by this enzyme. ADP then reacts with phosphoenolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5'-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of L-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.
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PMID:Polynucleotide phosphorylase-based photometric assay for inorganic phosphate. 1505 37

The desaturation of myristoyl-CoA to myristoleoyl-CoA was measured in microsomal preparations of hen liver. The desaturation was maximal at pH 7.4. The enzymatic activity was linear with time up to 10 min and proportional to microsomal protein concentrations. The initial velocity was linear with substrate concentrations between 13 and up to 200 microM. A decrease in desaturation activity was observed at substrate concentrations greater than 266 microM. There was an absolute requirement for reduced pyridine nucleotide (NADH), while a maximum activity was observed at a myristoyl-CoA:NADH mole ratio of 1. Competitive inhibition studies of myristoyl-CoA desaturation suggest that the inhibitors, stearyl and oleyl-CoA, were more effective than palmitoyl-CoA. Free CoA did not inhibit the delta(9)-desaturase system. The desaturation of myristoyl-CoA was stimulated by bovine serum albumin and reduced by cytoplasmic proteins. The effect of cytoplasmic proteins on the enzymatic reaction was completely abolished by trypsin digestion and boiling for 30 min. On the basis of these data, it was concluded that 9,10-desaturation of acyl-CoA derivatives containing 14-18 carbon fatty acyl chains is catalyzed by the same enzyme.
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PMID:Desaturation of myristoyl-CoA to myristoleoyl-CoA by hen liver microsomal delta(9)-desaturase. 1521 74

The role of nitrate reduction to produce nitric oxide (NO) and its subsequent oxidation by oxyhaemoglobin as a mechanism to maintain plant cell energetics during hypoxia is examined. Nitrate reduction in hypoxic conditions can be considered as an alternative respiratory pathway, with nitrate as an intermediate electron acceptor, contributing to the oxidation of NADH. NO, produced in the reaction, does not accumulate due to the induction of hypoxia-induced (class 1) haemoglobins. These haemoglobins remain in the oxyhaemoglobin form, even at oxygen tensions two orders of magnitude lower than necessary to saturate cytochrome c oxidase. They act, probably in conjunction with a flavoprotein, as NO dioxygenases converting NO back to nitrate, consuming NAD(P)H in the process. The overall system oxidizes 2.5 moles of NADH per one mole of nitrate recycled during the reaction, leading to the maintenance of redox and energy status during hypoxia and resulting in the reduced production of ethanol and lactic acid.
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PMID:Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways. 1544 80

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.
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PMID:Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate. 1554 98

In mixed-acid fermentation, succinate synthesis requires one mole of phosphoenolpyruvate (PEP), one mole of CO2, and two moles of NADH for every mole of succinate to be formed. Different carbon sources with different properties were used to address these requirements. Sorbitol generates one more mole of NADH than glucose. Fermentation of sorbitol was shown in this study (and by others) to produce significantly more succinate than fermentation of glucose, due to increased NADH availability. Xylose fermentation conserves the intracellular PEP pool, since its transport does not require the phosphotransferase system normally used for glucose transport. The extra PEP can then be assimilated in the succinate pathway to improve production. In this study, fermentation of xylose did yield higher succinate production than glucose fermentation. Subsequent inactivation of the acetate and lactate pathways was performed to study metabolite redistribution and the effect on succinate production. With the acetate pathway inactivated, significant carbon flux shifted toward lactate rather than succinate. When both acetate and lactate pathways were inactivated, succinate yield ultimately increased with a concomitant increase in ethanol yield.
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PMID:Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli. 1577 May 11

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.
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PMID:Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant. 1580 71

Seven human disorders of postsqualene cholesterol biosynthesis have been described. One of these, congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome, results from mutations in the X-linked gene NADH sterol dehydrogenase-like (NSDHL) encoding a sterol dehydrogenase. A series of mutant alleles of the murine Nsdhl gene are carried by bare patches (Bpa) mice, with Bpa(1H) representing a null allele. Heterozygous Bpa(1H) females display skin and skeletal abnormalities in a distribution reflecting random X inactivation, whereas hemizygous male embryos die before embryonic day 10.5. To investigate the molecular basis of defects associated with perturbations in cholesterol biosynthesis, microarray analysis was performed comparing gene expression in embryonic fibroblasts expressing the Bpa(1H) allele versus wild-type (wt) cells. Labeled cDNAs from cells grown in normal serum or lipid-depleted serum (LDS) were hybridized to microarrays containing 22,000 mouse genes. Among 44 genes that showed higher expression in the Bpa(1H) versus wt cells grown in LDS, 11 function in cholesterol biosynthesis, 7 are involved in fatty acid synthesis, 3 (Srebp2, Insig1, and Orf11) encode sterol-regulatory proteins, and 2 (Ldlr and StarD4) are lipid transporters. Of the 21 remaining genes, 16 are known genes, some of which have been implicated previously in cholesterol homeostasis or lipid-mediated signaling, and 5 are uncharacterized cDNA clones.
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PMID:Changes in gene expression associated with loss of function of the NSDHL sterol dehydrogenase in mouse embryonic fibroblasts. 1580 45

A novel in vivo method of producing succinate has been developed. A genetically engineered Escherichia coli strain has been constructed to meet the NADH requirement and carbon demand to produce high quantities and yield of succinate by strategically implementing metabolic pathway alterations. Currently, the maximum theoretical succinate yield under strictly anaerobic conditions through the fermentative succinate biosynthesis pathway is limited to one mole per mole of glucose due to NADH limitation. The implemented strategic design involves the construction of a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has less NADH requirement). The synthesis of succinate uses a combination of the two pathways to balance the NADH. Consequently, experimental results indicated that these combined pathways gave the most efficient conversion of glucose to succinate with the highest yield using only 1.25 moles of NADH per mole of succinate in contrast to the sole fermentative pathway, which uses 2 moles of NADH per mole of succinate. A recombinant E. coli strain, SBS550MG, was created by deactivating adhE, ldhA and ack-pta from the central metabolic pathway and by activating the glyoxylate pathway through the inactivation of iclR, which encodes a transcriptional repressor protein of the glyoxylate bypass. The inactivation of these genes in SBS550MG increased the succinate yield from glucose to about 1.6 mol/mol with an average anaerobic productivity rate of 10 mM/h (approximately 0.64 mM/h-OD600). This strain is capable of fermenting high concentrations of glucose in less than 24 h. Additional derepression of the glyxoylate pathway by inactivation of arcA, leading to a strain designated as SBS660MG, did not significantly increase the succinate yield and it decreased glucose consumption by 80%. It was also observed that an adhE, ldhA and ack-pta mutant designated as SBS990MG, was able to achieve a high succinate yield similar to SBS550MG when expressing a Bacillus subtilis NADH-insensitive citrate synthase from a plasmid.
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PMID:Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. 1588 21


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