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)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of proteins and peptides was performed on samples deposited onto non-porous ether-type polyurethane (PU) membranes. Spectra obtained using PU membranes showed that mass resolution and accuracy were equivalent to values observed using a metal target, and superior to those obtained using poly(vinylidene difluoride) (PVDF) membranes. A small apparent increase in the mass of proteins and also loss of resolution were observed at very high laser irradiance due to charging, but were not observed under normal conditions. Analysis of NaCl-doped standards demonstrated that PU membranes yielded better results than a metallic target for salt-containing solutions. Relatively strong hydrophobic interactions between the proteins and peptides and the PU membrane allowed the incorporation of a washing step. This step allowed for the removal of salts and buffer components and thus provided an increase in resolution and mass accuracy. Digestion of citrate synthase (a protein of molecular weight 47,886) with trypsin was performed directly on the surface of the membrane for variable periods of time, and characteristic peptide fragments were observed by MALDI-TOFMS. Delayed extraction was used to increase the resolution and to permit more accurate mass assignments for those fragments. The use of PU membranes for MALDI-TOFMS analysis of proteins with higher molecular weights is also demonstrated.
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PMID:Use of a non-porous polyurethane membrane as a sample support for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins. 936 98

Enzymes from extreme halophiles have potential as catalysts in biotransformations. We have developed methods for the expression in Escherichia coli and purification of two enzymes from Haloferax volcanii: dihydrolipoamide dehydrogenase and citrate synthase. Both enzymes were expressed in E. coli using the cytoplasmic expression vectors, pET3a and pET3d. Citrate synthase was soluble and inactive, whereas dihydrolipoamide dehydrogenase was expressed as inclusion bodies. Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 microM FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH. Maximal activity was obtained after 3 days incubation at 4 degrees C. Purification of the two active enzymes was carried out using high-resolution methods. Dihydrolipoamide dehydrogenase was purified using copper-based metal ion affinity chromatography in the presence of 2 M KCl. Citrate synthase was recovered using dye-affinity chromatography in the presence of salt. A high yield of active enzyme was obtained in both cases. Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependence were comparable to those of the native enzymes. Expression of active protein was attempted both by growth of E. coli in the presence of salt and betaine, and also by using periplasmic expression vectors in combination with a high salt growth media. Neither strategy was successful.
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PMID:Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli. 1039 37

The Arabidopsis thaliana gene CUT1 encodes a very-long-chain fatty acid-condensing enzyme required for the production of epicuticular wax in bolting stems. We have examined the expression pattern of CUT1 in Arabidopsis at different developmental stages and under different environmental conditions. RNA blot analysis showed that CUT1 was highly expressed in shoots, but not in roots. CUT1 expression was detectable throughout development. Light was required for CUT1 expression, and expression was increased by salt and drought treatments. The promoter region of the CUT1 gene was cloned, and 1.2 kb of the sequence 5' to the translation start codon was used to direct beta-glucuronidase (GUS) expression in transgenic plants. Histochemical and fluorometric (quantitative) GUS assays confirmed that the CUT1 promoter directed epidermal-specific expression and was highly active in Arabidopsis and in tobacco. A construct using the CUT1 promoter to drive CUT1 expression (CUT1p-CUT1) was used to transform Arabidopsis. Transgenic plants which had somewhat increased (overexpression) or greatly reduced (co-suppression) wax loads were recovered. Thus, the CUT1 promoter should be useful for genetic engineering applications that require epidermis-specific expression of genes.
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PMID:Expression of the wax-specific condensing enzyme CUT1 in Arabidopsis. 1117 Nov 58

Six, 2 ns molecular dynamics simulations have been performed on the homodimeric enzyme citrate synthase. In three, both monomers were started from the open, unliganded X-ray conformation. In the remaining three, both monomers started from a closed, liganded X-ray conformation, with the ligands removed. Projecting the motion from the simulations onto the experimental domain motion revealed that the free-energy profile is rather flat around the open conformation, with steep sides. The most closed conformations correspond to hinge-bending angles of 12-14 compared to the 20 degrees that occurs upon the binding of oxaloacetate. It is also found that the open, unliganded X-ray conformation is situated at the edge of the steep rise in free energy, although conformations that are about 5 degrees more open were sampled. A rigid-body essential dynamics analysis of the combined open trajectories has shown that domain motions in the direction of the closed X-ray conformation are compatible with the natural domain motion of the unliganded protein, which has just two main degrees of freedom. The simulations starting from the closed conformation suggest a free-energy profile with a small barrier in going from the closed to open conformation. A combined essential dynamics and hinge-bending analysis of a trajectory that spontaneously converts from the closed to open state shows an almost exact correspondence to the experimental transition that occurs upon ligand binding. The simulations support the conclusion from an earlier analysis of the experimental transition that the beta-hairpin acts as a mechanical hinge by attaching the small domain to the large domain through a conserved main-chain hydrogen bond and salt-bridges, and allowing rotation to occur via its two flexible termini. The results point to a mechanism of domain closure in citrate synthase that has analogy to the process of closing a door.
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PMID:Investigation of the mechanism of domain closure in citrate synthase by molecular dynamics simulation. 1150 94

In a previous report, we observed an altered proportion of fiber types and a reduction of capillary per fiber ratio in extensor digitorus longus (EDL) and soleus (SOL) muscles of deoxicorticosterone acetate (DOCA)-salt hypertensive rats when compared with controls. The aim of the present study was to ascertain various carbohydrate and lipid enzyme activities and substrates that may be involved in the morphological changes reported. In the SOL muscle of hypertensive rats, glucose, glycogen and triglycerides (TG) levels were increased, citrate synthase (CS) and beta-hydroxy-acyl-CoA dehydrogenase (HAD) activities were reduced, while hexokinase (HK) and lipoprotein lipase (LPL), LPL mass, lactate and free fatty acids (FFA) levels were unchanged. In EDL muscles of hypertensive rats, glycogen levels and LPL mass were higher than in controls, while CS, HAD, HK, and LPL activities and glucose, lactate, FFA and TG levels were unmodified. Serum levels of insulin, TG, cholesterol and FFA were increased while glucose levels were decreased and high-density lipoprotein-cholesterol levels were similar in hypertensive rats when compared with controls. In conclusion, hypertensive rats showed increased glycogen in both EDL and SOL muscles, with hyperinsulinemia and reduced glycemia. Hyperinsulinemia might have been a compensatory response to insulin resistance. The oxidative capacity of SOL muscle was reduced indicating that glucose uptake was conduced via non-oxidative metabolism. TG, FFA and cholesterol were increased in serum and TG in SOL muscle.
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PMID:Metabolic changes in DOCA-salt hypertensive rats. 1191 12

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in <10 days). Despite changes in LV mass, specific activities of bioenergetic (cytochrome-c oxidase, citrate synthase, lactate dehydrogenase) and reactive oxygen species (ROS) (total cellular superoxide dismutase) enzymes were actively maintained within relatively narrow ranges regardless of treatment duration, organismal age, or transmural region. Although enalapril led to parallel declines in mitochondrial enzyme content and ventricular mass, total ventricular mtDNA content was unaffected. Altered enzymatic content occurred without significant changes in relevant mRNA and protein levels. Transcript levels of gene products involved in mtDNA maintenance (Tfam), mitochondrial protein degradation (LON protease), fusion (fuzzy onion homolog), and fission (dynamin-like protein, synaptojanin-2alpha) were also unchanged. In contrast, enalapril-mediated ventricular and mitochondrial remodeling was accompanied by a twofold increase in specific activity of catalase, an indicator of oxidative stress, suggesting that rapid cardiac adaptation is accompanied by tight regulation of mitochondrial enzyme activities and increased ROS production.
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PMID:Bioenergetic remodeling of heart during treatment of spontaneously hypertensive rats with enalapril. 1212 99

Electrostatics plays a major role in heat adaptation by thermophilic proteins. Here we ask whether electrostatics similarly contributes to cold adaptation in psychrophilic proteins. We compare the sequences and structures of citrate synthases from the psychrophile Arthobacter Ds2-3R, from chicken, and from the hyperthermophile Pyrococcus furiosus. The three enzymes share similar packing, burial of nonpolar surface area, and main-chain hydrogen bonding. However, both psychrophilic and hyperthermophilic citrate synthases contain more charged residues, salt bridges, and salt-bridge networks than the mesophile. The electrostatic free-energy contributions toward protein stability by individual charged residues show greater variabilities in the psychrophilic citrate synthase than in the hyperthermophilic enzyme. The charged residues in the active-site regions of the psychrophile are more destabilizing than those in the active-site regions of the hyperthermophile. In the hyperthermophilic enzyme, salt bridges and their networks largely cluster in the active-site regions and at the dimer interface. In contrast, in the psychrophile, they are more dispersed throughout the structure. On average, salt bridges and their networks provide greater electrostatic stabilization to the thermophilic citrate synthase at 100 degrees C than to the psychrophilic enzyme at 0 degrees C. Electrostatics appears to play an important role in both heat and cold adaptation of citrate synthase. However, remarkably, the role may be different in the two types of enzyme: In the hyperthermophile, it may contribute to the integrity of both the protein dimer and the active site by possibly countering conformational disorder at high temperatures. On the other hand, in the psychrophile at low temperatures, electrostatics may contribute to enhance protein solvation and to ensure active-site flexibility.
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PMID:Different roles of electrostatics in heat and in cold: adaptation by citrate synthase. 1499 20

The effects of dietary fat intake on the development of left ventricular hypertrophy and accompanying structural and molecular remodeling in response to hypertension are not understood. The present study compared the effects of a high-fat versus a low-fat diet on development of left ventricular hypertrophy, remodeling, contractile dysfunction, and induction of molecular markers of hypertrophy (ie, expression of mRNA for atrial natriuretic factor and myosin heavy chain beta). Dahl salt-sensitive rats were fed either a low-fat (10% of total energy from fat) or a high-fat (60% of total energy from fat) diet on either low-salt or high-salt (6% NaCl) chow for 12 weeks. Hearts were analyzed for mRNA markers of ventricular remodeling and activities of the mitochondrial enzymes citrate synthase and medium chain acyl-coenzyme A dehydrogenase. Similar levels of hypertension were achieved with high-salt feeding in both diet groups (systolic pressure of approximately 190 mm Hg). In hypertensive rats fed low-fat chow, left ventricular mass, myocyte cross-sectional area, and end-diastolic volume were increased, and ejection fraction was decreased; however, these effects were not observed with the high-fat diet. Hypertensive animals on low-fat chow had increased atrial natriuretic factor mRNA, myosin heavy chain isoform switching (alpha to beta), and decreased activity of citrate synthase and medium chain acyl-coenzyme A dehydrogenase, which were all attenuated by high-fat feeding. In conclusion, increased dietary lipid intake can reduce cardiac growth, left ventricular remodeling, contractile dysfunction, and alterations in gene expression in response to hypertension.
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PMID:Low carbohydrate/high-fat diet attenuates cardiac hypertrophy, remodeling, and altered gene expression in hypertension. 1706 May 11

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.
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PMID:Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)? 1841 54

Ninety-six female and male pigs were assigned to one of three treatments, 'confined' (C),'trained'(T) or 'free' (F) allowing for different levels of physical activity during the growth interval from 30 to 100kg. Treatment C consisted of individual housing in pens of 2.5 m(2); treatment T of individual housing and regular treadmill training and treatment F of housing in pens of 36 m(2) (40 pigs/pen). In m. biceps femoris (BF), the activity of lactate dehydrogenase (LDH) was decreased between 9 and 12% by training (treatment T vs C). Likewise, in BF from female pigs, training increased the activity of citrate synthase (CS) and of 3-OH-acyl-CoA-dehydrogenase (HAD) by 18 and 21%, respectively. Spontaneous activity (treatment F) reduced the activity of LDH for five muscles between 10 and 16% when compared with treatment C. Around the time of slaughter, glycogenolysis of BF was less for treatment F (6-17%) than for C and T (33-38%). Moreover, in BF from female pigs in treatment F, the initial but not the ultimate pH was increased when compared with treatment C. In comparison to C and T, treatment F improved juiciness in BF from male pigs and increased the amount of salt soluble protein in m. longissimus dorsi.
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PMID:Muscle metabolic traits, post mortem-pH-decline and meat quality in pigs subjected to regular physical training and spontaneous activity. 2206 26


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