Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells of the aerotolerant anaerobe Giardia lamblia respire in the presence of oxygen. Endogenous respiration is stimulated by glucose but not by other carbohydrates and Krebs cycle intermediates. Endogenous and glucose-stimulated respiration are insensitive to cyanide, malonate, and 2,4-dinitrophenol, but are inhibited by atabrin and iodoacetamide. G. lamblia produces ethanol, acetate and CO2 both aerobically and anaerobically either from endogenous reserves or exogenous glucose. Molecular hydrogen is not produced. The following enzyme activities were detected in homogenates: hexokinase, fructose-biphosphate aldolase, pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate dehydrogenase (decarboxylating), pyruvate synthase, acetyl-CoA synthetase, alcohol dehydrogenase (NADP+), NADH dehydrogenase, NADPH dehydrogenase, NADPH oxidoreductase and superoxide dismutase. The enzymes of energy and carbohydrate metabolism are nonsedimentable (109 000 x g for 30 min). Activities of lactate dehydrogenase, hydrogenase, phosphate acetyltransferase, acetate kinase, citrate synthase, succinate dehydrogenase, fumarate hydratase and catalase were below the limits of detection. The results suggest the occurrence of glycolysis, energy production by substrate level phosphorylation and a flavin, iron-sulfur protein mediated electron transport system as well as the absence of cytochrome mediated oxidative phosphorylation and functional Krebs cycle.
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PMID:Energy metabolism of the anaerobic protozoon Giardia lamblia. 610 7

Plasmid vectors for the acetic acid-producing strains of Acetobacter and Gluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochrome c, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene of A. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of an A. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of the Acetobacter strains was developed and applied to improve their properties.
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PMID:Genetic organization of Acetobacter for acetic acid fermentation. 809 54

In this paper, we demonstrate the ability of liquid-liquid partition chromatography (LLPC) to detect conformational alterations occurring in well-characterized enzymes. The conformational changes induced in dehydrogenases such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenases (LDH) and malate dehydrogenase (MDH) upon binding of ligand(s) were detectable by LLPC. The ligand-dependent equilibrium between two forms of citrate synthase (CS), glutamate-oxaloacetate transaminase (GOT), hexokinase (HK) and 3-phosphoglycerate kinase (PGK) could also be demonstrated. Furthermore, different conformational forms of some of the apoenzymes could also be detected and separated by LLPC. The results obtained here are discussed in relation to those obtained by other methods.
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PMID:Enzyme conformational alterations detected by partition column chromatography. 879 88

Protein disulfide-isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum. At high concentrations, it also serves as a chaperone and inhibits aggregation. However, at lower concentrations, PDI can display the unusual ability to facilitate aggregation, termed anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771). Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0. 3 microM) relative to the unfolded protein substrate, PDI facilitates aggregation of alcohol dehydrogenase (11 microM) that has been denatured thermally or chemically. But at higher concentrations (>0.8 microM), PDI inhibits aggregation under the same conditions. With denatured citrate synthase, PDI does not facilitate aggregation, but higher concentrations do inhibit aggregation. Anti-chaperone behavior is associated with the appearance of both PDI and substrate proteins in insoluble complexes, while chaperone behavior results in the formation of large (>500 kDa) but soluble complexes that contain both proteins. Physiological concentrations of calcium and magnesium specifically increase the apparent rate of PDI-dependent aggregation and shift the chaperone activity to higher PDI concentrations. However, calcium has no effect on the Km or Vmax for PDI-catalyzed oxidative folding, suggesting that the interactions that lead to chaperone/anti-chaperone behavior are distinct from those required for catalytic activity. To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multivalent interactions between PDI and a partially aggregated protein stimulate further aggregate formation by noncovalently cross-linking smaller aggregates. However, at high ratios of PDI to substrate, cross-linking may be inhibited by saturation of the sites with PDI. The effects of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retention in the endoplasmic reticulum.
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PMID:Facilitated protein aggregation. Effects of calcium on the chaperone and anti-chaperone activity of protein disulfide-isomerase. 896 36

The coding region of the mitochondrial citrate synthase gene (CIT1) from Saccharomyces cerevisiae was amplified by PCR and cloned into an expression vector (pAL4) downstream of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans to yield pALCS1. Transformation of A. nidulans A773 with this construct gave stable transformants, AYC#1 and AYC#2, that were phenotypically stable for several mitotic divisions. Southern blot analysis showed that the CIT1 gene was successfully integrated into the chromosomes of the transformants. Western blot analysis and enzymatic assay for citrate synthase revealed that the integrated yeast gene was subject to inducible expression controlled by alcA promoter, which can be induced by threonine.
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PMID:Inducible expression of yeast mitochondrial citrate synthase in Aspergillus nidulans. 933 92

The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known. Previous experiments provided support for the significant contribution of a high-molecular-mass protein complex to the extreme thermotolerance of P. occultum. This protein complex, the 'thermosome', is composed of two subunits, alpha and beta, which form a hexadecameric double ring complex. In order to obtain the thermosome in amounts sufficient for structural and functional investigations, we produced the two subunits jointly and separately in Escherichia coli BL21(DE3). In all three cases, we isolated soluble, high-molecular-mass double-ring complexes from E. coli BL21(DE3). On electron micrographs, the recombinant complexes were indistinguishable from each other and from the natural thermosome. To characterize the quaternary structure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation. Spectral analysis, using absorption, fluorescence emission and far-UV circular dichroism spectroscopy were applied to compare the three recombinant protein complexes with the natural thermosome from P. occultum. All three recombinant complex species exhibit ATPase activity. Furthermore, we could demonstrate that the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin. Thus, we conclude that the recombinant protein complexes exhibit a chaperone-like activity, interacting with non-native proteins; they do so at temperatures far below the lower physiological limit of growth.
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PMID:Recombinant homo- and hetero-oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro. 987 54

The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function. In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coili BL21 (DE3) cells. Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates. The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C.
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PMID:The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: in vitro analysis of its chaperone activity. 1006 37

Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.
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PMID:Nucleolar protein B23 has molecular chaperone activities. 1021 37

Short, strong (low barrier) hydrogen bonds occur when the pK values of the atoms sharing the proton are similar. The overall distance is 2.5 A or less, the deuterium fractionation factor is less than 0.5, the proton NMR chemical shift can approach 20 ppm, and deuterium or tritium substitution causes an up-field change in the chemical shift. Such bonds can have deltaH values of 25 kcal/mol in the gas phase, and at least half that in water or other high-dielectric medium. The strength of the hydrogen bond in an active site drops by approximately 1 kcal/mol for each pH unit mismatch in pKs. When a weak hydrogen bond in the initial enzyme-substrate complex is converted into a low-barrier one by alteration of the pK of the substrate or catalytic group so that the pKs match, the increase in hydrogen bond strength can be used to help catalyze the reaction. A well-established example of this is the reaction catalyzed by serine proteases. The pK of neutral histidine is 14, while that of aspartate is approximately 6. Proton transfer from serine to permit attack on bound substrate produces protonated histidine, with a pK now matching that of aspartate. Studies with trifluoromethyl ketone inhibitors that form tetrahedral adducts show up to five orders of magnitude in binding strength as the result of formation of a low-barrier hydrogen bond between aspartate and histidine. Other enzymes whose mechanisms appear to involve low-barrier hydrogen bonds include liver alcohol dehydrogenase, steroid isomerase, triose-P isomerase, aconitase, citrate synthase, and zinc proteases. It is likely that low-barrier hydrogen bonds form at the transition state of any reaction involving general-acid or general-base catalysis, as at that point the pKs of the catalytic group and reactant will be equal.
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PMID:Low-barrier hydrogen bonds and enzymatic catalysis. 1105 Oct 90

Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.
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PMID:Phe71 is essential for chaperone-like function in alpha A-crystallin. 1159 24


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