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At least 919 records · Page 51Linked to original sources
PubMed1991

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.

ATP Citrate (pro-S)-Lyase↗
PubMed2003

Regional citrate anticoagulation during simulated treatments of sustained low efficiency diafiltration.

Renal replacement therapy is frequently required for critically ill patients with a high risk of bleeding. Conventional heparinization strategies to prevent extracorporeal blood circuit clotting can cause significant haemorrhage in such patients because of systemic anticoagulation. Regional citrate anticoagulation (RCA) is a well-established technique that minimizes this complication by the decalcification of blood in the extracorporeal circuit such that it is incapable of clotting. To date, there are no reports on the use of RCA for sustained low-efficiency dialysis/diafiltration (SLED), a hybrid therapy that involves the use of conventional haemodialysis machinery to deliver lower solute clearances over prolonged periods of time. In preparation for clinical study, an in vitro simulation of SLED was devised (blood substitute flow 250 mL/min, dialysate flow 200 mL/min, predilution haemofiltration 100 mL/min). Blood substitute was decalcified by an infusion of 4% trisodium citrate (TSC) proximally into the extracorporeal blood circuit, with partial restoration of calcium homeostasis from dialysate containing ionized [Ca2+] at 0.9 mmol/L. This simulation was used to establish first the 4% TSC requirement for therapeutic decalcification, and second the associated changes in ionized [Ca2+] and [Mg2+] within the blood substitute from chelation with citrate and subsequent removal of the resulting divalent cation-citrate complex. Serial measurements of blood substitute [Ca2+] from strategic points along the extracorporeal circuit showed therapeutic decalcification was not achieved with 4% TSC infusion rates up to 400 mL/h, and extrapolation of experimental results suggests that 450 mL/h will be required. Under these conditions, ionized [Ca2+] and [Mg2+] in the blood substitute venous return and would be 0.42 and 0.2 mmol/L, respectively, with 0.35 mmol of citrate being returned per minute via the blood substitute venous return. These results were modelled for various changes in SLED operating parameters, and discussed in detail. An appropriate regimen for 4% TSC infusion and divalent cation replacement is proposed for clinical study in the future.

Acute Kidney Injury↗
PubMed2004

Effects of acute ingestion of sodium citrate on metabolism and 5-km running performance: a field study.

The purpose of the study was to (a) assess the effects of sodium citrate ingestion on metabolism and performance capacity in a 5-km competitive outdoor stadium run in trained male runners, and (b) elucidate the potential relationship between citrate-induced changes in plasma volume, body mass, and performance. Ten subjects (age 22.1 +/- 2.5 yrs, body mass 74.1 +/- 6.1 kg, height 180.1 +/- 5.7 cm, (.)VO(2)max 60.8 +/- 5.5 ml x kg(-1) x min(-1)) participated in the study. There was no effect of treatment on 5-km running time: 1100.0 +/- 79.1 and 1082.7 +/- 62.0 s in citrate (CIT) and in placebo (PLC) trials, respectively, p = 0.09. Blood pH increased from 7.34 +/- 0.07 to 7.49 +/- 0.07 (p = 0.002) as a result of administering sodium citrate in the amount of 0.5 g x kg(-1) body mass in 1.5 litres of solution but remained stable while the equal volume of placebo drink was consumed: 7.40 +/- 0.04 and 7.44 +/- 0.09. The relative change in plasma volume after administering the drink was -1.99 +/- 3.49% in the PLC and 9.75 +/- 6.51% in the CIT trial (p = 0.001). Body mass did not differ before drinking; however, before the start the subjects were heavier in the CIT trial (74.2 +/- 6.1 kg) vs. the PLC trial (73.4 +/- 6.2 kg, p = 0.048). The shifts in plasma volume and body mass were not related to changes in performance. The results suggest that ingestion of sodium citrate induces an increase in water retention, plasma volume, and blood pH before exercise but does not improve performance in a 5-km competitive run in field conditions in trained male runners.

Adult↗
PubMed2005

Regional citrate anticoagulation during hemodialysis: a simplified procedure using Duocart biofiltration.

BACKGROUND: Regional citrate anticoagulation during hemodialysis is promising, but its clinical implementation is routinely cumbersome because a continuous adjustment of calcium infusion at the dialyzer outlet is needed. Duocart biofiltration (DCB) is a new hemodialysis method using a calcium and magnesium-free dialysate containing only sodium chloride and bicarbonate combined with the infusion into the venous line of a solution containing the ionic complement (K, Ca, Mg) and glucose. Since the dialysate is calcium- and magnesium-free and infusion rate of the solution containing calcium is automatically determined by the dialysis delivery system according to the on-line measured value of ionic dialysance, DCB seems a technique especially suitable for citrate anticoagulation procedure. METHODS: Thirty DCB sessions were performed in 10 patients with increased risk of bleeding. A commercially available mixture of trisodium citrate, citric acid and glucose was infused into the arterial line at a rate equal to 3% of dialyzer blood flow. The ionic complement (K: 48 mM, Ca: 42 mM, Mg: 14 mM, glucose: 110 mM) was infused at a rate equal to 1/24 ionic dialysance value automatically determined each 15 min by the dialysis monitor. DCB sessions were compared to 21 conventional bicarbonate hemodialysis (BHD) sessions with low-molecular-weight heparin anticoagulation. RESULTS: Whole blood activated clotting time (WBACT) measured in the venous line (before infusion of ionic complement) was 200% of the WBACT value in the arterial line. Clotting and citrate-related adverse events were not observed. Postdialysis compression time of the arteriovenous access is significantly (p<0.001) shorter after DCB sessions (3.9+/-1.1 min) compared with BHD sessions (8.7+/-4.6 min). CONCLUSION: Citrate anticoagulation during Duocart biofiltration is effective, safe and suitable for routine use because calcium infusion rate is automatically adjusted without the need of monitoring degree of anticoagulation and level of ionized calcium.

Adult↗
PubMed2004

Effects of ingestion of bicarbonate, citrate, lactate, and chloride on sprint running.

PURPOSE: Ingestion of sodium bicarbonate is known to enhance sprint performance, probably via increased buffering of intracellular acidity. The goal was to compare the effect of ingestion of sodium bicarbonate with that of other potential buffering agents (sodium citrate and sodium lactate) and of a placebo (sodium chloride) on sprinting. METHODS: In a double-blind randomized crossover trial, 15 competitive male endurance runners performed a run to exhaustion 90 min after ingestion of each of the agents in the same osmolar dose relative to body mass (3.6 mosmol x kg) on separate days. The agents were packed in gelatin capsules and ingested with 750 mL of water over 90 min. During each treatment we assayed serial finger-prick blood samples for lactate and bicarbonate. A familiarization trial was used to set a treadmill speed for each runner's set of runs. We converted changes in run time between treatments into changes in a time trial of similar duration using the critical-power model, and we estimated likelihood of practical benefit using 0.5% as the smallest worthwhile change in time-trial performance. RESULTS: The mean run times to exhaustion for each treatment were: bicarbonate 82.3 s, lactate 80.2 s, citrate 78.2 s, and chloride 77.4 s. Relative to bicarbonate, the effects on equivalent time-trial time were lactate 1.0%, citrate 2.2%, and chloride 2.7% (90% likely limits +/- 2.1%). Ingested lactate and citrate both appeared to be converted to bicarbonate before the run. There were no substantial differences in gut discomfort between the buffer treatments. CONCLUSION: Bicarbonate is possibly more beneficial to sprint performance than lactate and probably more beneficial than citrate or chloride. We recommend ingestion of sodium bicarbonate to enhance sprint performance.

Adult↗
PubMed2005

Sodium bicarbonate and sodium citrate: ergogenic aids?

Numerous studies have used exogenous administration of sodium bicarbonate (NaHCO(3)) and sodium citrate (Na-citrate) in an attempt to enhance human performance. After ingestion of NaHCO(3) and Na-citrate, two observations have been made: (a) There was great individual variability in the ergogenic benefit reached, which can be attributed to the level of physical conditioning of the subjects and to their tolerance of the buffer substance; and (b) the subjects who had ingested NaHCO(3) and Na-citrate show higher levels of pH, bicarbonate, and lactate ions concentrations in their exercising blood than do the subjects who had ingested the placebo. A majority of the studies have suggested that the ingestion of both substances provides an ergogenic effect due to the establishment and maintenance of an elevated pH level during exercise. However, the exact mechanism by which the ergogenic effects occur has not been demonstrated conclusively. Sodium bicarbonate and Na-citrate seem to be effective in activities with a sufficient duration to generate a difference in the hydrogen ion gradient, characterized by a very high intensity and involving large muscular groups. However, in activities of equally high intensity, but with longer duration, the results obtained have been conflicting and inconclusive.

Citrates↗
PubMed2003

Effect of sodium citrate on structure-function relationships of Cheddar cheese.

The objective of this study was to determine the effect of sodium citrate on the structure and functionality of Cheddar cheese. The hypothesis was that citrate (sodium citrate) injection would affect cheese properties mainly through its effect on bound calcium (calculated as the difference between total calcium and the water-soluble calcium content of a cheese extract). A 9-kg block of Cheddar cheese was made, vacuum-packaged, and then stored for 2 wk at 4 degrees C. After storage, the cheese was cut into 0.5- to 0.6-kg blocks that were vacuum-packaged and stored for 1 wk at 4 degrees C prior to injection. Cheese blocks were then high-pressure injected with a buffer solution (pH 5.27) containing 40% (wt/ wt) citric acid trisodium dihydrate and 6.25% (wt/wt) anhydrous citric acid, from zero (control) to five times (successive injections performed 24 h apart). Increased citric acid content of cheese from 0.22 (uninjected) to 1.39% (after five injections) caused phosphate solubilization. Thus, the calculated bound phosphate content of cheese decreased from 0.54 to 0.45 mmol/g of protein. However, unexpectedly, the soluble calcium content decreased from 0.34 (control) to 0.28 mmol/g of protein (after five injections), whereas the bound calcium content remained unchanged (0.42 mmol/g of protein). The decrease in soluble calcium probably resulted from the formation and concentration of crystals in the cheese surface, which was not included in samples for analysis, and from the expulsion of serum from within the cheese. Higher concentration of solutes in the water phase of cheese would increase the volume of serum, but the cheese had limited holding capacity and serum was expelled. Citrate injection increased the sodium content of cheese from 0.63 to 0.93%, but it had no effect on cheese pH (5.2). After five injections, the protein matrix expanded, occupying an increased area of cheese matrix (83 vs. 78%). Even though citrate injection had no effect on bound calcium, and thus the rate and extent of cheese flow were unaffected, increased phosphate solubilization, and possibly decreased ionic calcium content, resulted in expansion of the protein matrix and increased cheese hardness.

Calcium↗
PubMed1975

Purification and properties of citrate lyase from Streptococcus diacetilactis.

Citrate lyase from Streptococcus diacetilactis has been purified to yield a protein that was homogeneous as judged by sedimentation velocity and sedimentation equilibrium experiments. The enzyme's sedimentation coefficient is 16.8 S and its molecular weight is around 585,000. It contains three nonidentical subunits of about 53,000, 34,000, and 10,000 daltons. The enzyme in its active form contains an acetyl group which turns over during the citrate cleavage reaction. Removal of the acetyl group inactivates the enzyme. The deacetyl enzyme can be partially reactivated by acetylation with acetic anhydride. The enzyme undergoes slow "reaction-inactivation." The rate of inactivation is first order and the rate constant of inactivation is much lower than that for a similar inactivation process of the citrate lyase from Klebsiella aerogenes. Like the latter enzyme it contains stoichiometric amounts of phosphopantothenate. The enzyme is inactivated at pH greater than 8.1 and the presence of citrate provides protection against this inactivation. Sedimentation studies of the enzyme at pH 8.7 indicate that the enzyme is dissociated, which may account for the inactivation. The enzyme is immunologically different from citrate lyases of K. aerogenes and Escherichia coli.

ATP Citrate (pro-S)-Lyase↗
PubMed1986

Renal function of patients in long-term treatment with lithium citrate alone or in combination with neuroleptics and antidepressant drugs.

Renal function was studied in patients given lithium citrate alone or in combination with neuroleptics or tricyclic antidepressants or both. No other drugs were given. None of the groups given lithium citrate with other drugs had lithium ion clearances that differed significantly from the groups given lithium citrate alone, nor was there any difference in the clearances of sodium, potassium, or creatinine between these groups. The 24-hour urine volume of patients receiving antidepressant drugs was similar to that of the patients receiving lithium citrate monotherapy but was significantly higher in patients given neuroleptics. The increase in urine volume could not be ascribed to alterations in glomerular filtration rate or proximal tubular resorption but could be accounted for entirely by lowered resorption of water in the distal tubules. We concluded that no change of lithium citrate dose is required when patients so treated are given additional neuroleptic or tricyclic antidepressant drugs.

Adult↗
PubMed2004

Different roles of electrostatics in heat and in cold: adaptation by citrate synthase.

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.

Adaptation, Physiological↗
PubMed1990

Fluoride bioavailability from slow-release sodium fluoride given with calcium citrate.

Clinical pharmacology of slow-release sodium fluoride given with calcium citrate was examined in acute and long-term studies. Following a single oral administration of 50 mg slow-release sodium fluoride, a peak serum fluoride concentration (Cmax) of 184 ng/ml was reached in 2 h; thereafter, serum fluoride concentration declined with a T1/2 of 5.9 h. The concurrent administration of calcium citrate (400 mg calcium) gave an equivalent Tmax (time required to attain Cmax) and T1/2, but a lower Cmax of 135 ng/ml. The coadministration of a meal with fluoride also reduced Cmax but increased Tmax. The area under the serum concentration curve of slow-release sodium fluoride was reduced 17-27% by a meal or calcium citrate. Thus, calcium citrate reduced fluoride absorption and peak fluoride concentration in serum of slow-release sodium fluoride but did not affect the time required to reach peak concentration or the rate of subsequent decline. The effect of a meal was similar, except for a longer period required to reach peak serum concentration. During long-term administration of 25 mg slow-release sodium fluoride coadministered with 400 mg calcium as calcium citrate on a twice daily schedule, the trough level of serum fluoride could be kept between 95 and 190 ng/ml, believed to be the therapeutic window.

Adult↗
PubMed1983

[Regulation of citrate synthase in facultative methylotrophic bacteria].

Commonly the TCA cycle fulfils an anabolic and a catabolic function in case of aerobic chemoorganoheterotrophic nutrition. In methylotrophic growth the TCA cycle is dispensable as a bioenergetic pathway. This is reflected by properties of citrate synthase in facultative methylotrophic bacteria. Two citrate synthases, a "chemoorganoheterotrophic" one, which is inhibited by NADH (or ATP in Acetobacter MB 58), and a "methylotrophic" one, which is not or less affected by energy indicators, were found in Pseudomonas oleovorans, Pseudomonas MS, Pseudomonas MA, and Acetobacter MB 58. The concentration of these citrate synthases depends on the manner of nutrition. Bacteria with ICL-negative-variant of the serine pathway and with ribulosebisphosphate pathway seem to possess only a "chemoorganoheterotrophic" citrate synthase. Possibly the anabolic function of this citrate synthase can be realized by metabolites.

Acetobacter↗
PubMed2006

Utilization of citrate and lactate through a lactate dehydrogenase and ATP-regulated pathway in boar spermatozoa.

Incubation of boar spermatozoa in Krebs-Ringer-Henseleit medium with either 10 mM lactate or 10 mM citrate induced a fast and robust increase in the intracellular levels of ATP in both cases, which reached a peak after 30 sec of incubation. Utilization of both citrate and lactate resulted in the export of CO(2) to the extracellular medium, indicating that both substrates were metabolized through the Krebs cycle. Incubation with citrate resulted in the generation of extracellular lactate, which was inhibited in the presence of phenylacetic acid. This indicates that lactate is produced through the pyruvate carboxylase step. In addition, there was also a significant increase in tyrosine phosphorylation induced by both citrate and lactate. Boar sperm has a sperm-specific isoform of lactate dehydrogenase (LDH), mainly located in the principal piece of the tail. Kinetic studies showed that boar sperm has at least two distinct LDH activities. The major activity (with an estimated Km of 0.51 mM) was located in the supernatants of sperm extracts. The minor LDH activity (with an estimated Km of 5.9 mM) was associated with the nonsoluble fraction of sperm extracts. Our results indicate that boar sperm efficiently metabolizes citrate and lactate through a metabolic pathway regulated by LDH.

Adenosine Triphosphate↗
PubMed1993

Localized proton MR spectroscopy of citrate in vitro and of the human prostate in vivo at 1.5 T.

Citrate is a secretory product of the normal prostate, a lack of citrate in prostate tissue is expected to be pathognomonic for adenocarcinoma. In the present study proton AB-signal characteristics of citrate at low field strength of 1.5 T were investigated by volume selective spectroscopic and theoretical methods. The nonappearance of fast phase modulation of the J-coupled system at low field strength in vivo is verified by measurements of sodium citrate solution. In vivo localized spectroscopy of small volume elements of (2 cm)3 with the double spin-echo method within the prostatic gland provides citrate signal reception even using a Helmholtz coil with 170-mm diameter. Volume selective proton spectra with different echo times are presented which are comparable to spectra acquired by former authors with endorectal coils.

Citrates↗
PubMed1996

Acetyl coenzyme A carboxylase activity in neonatal rat cardiac myocytes in culture: citrate dependence and effects of hypoxia.

The presence of a 280,000 M(r) isoform of acetyl-CoA carboxylase (ACC-280) in the cardiac myocyte suggests that heart muscle is capable of malonyl-CoA synthesis. Cellular factors which regulate activity of ACC-280 are unknown. We have employed a neonatal rat cardiac myocyte culture (where the majority of ACC is present as ACC-280) to examine the effects of hypoxia and decreased cellular ATP on the activity of ACC in the cells. The myocyte culture has the following advantages over similar studies in the intact rat heart: the presence of a pure population of myocytes and the ability to measure cytosolic ACC free from contamination by mitochondrial carboxylases. ACC activity in cultured cardiac myocytes is completely dependent on the presence of citrate (A0.5=3.8 mM). Under control conditions, the cytosolic citrate concentration in situ is determined to be less than 1 mM. With 5 h of hypoxia, cytosolic ATP decreases from 9.85 +/- 0.23 to 2.83 +/- 0.25 mM and cytosolic AMP increases from undetectable levels to 40 +/- 0.4 microM. With hypoxia, a significant portion of the total ACC activity is now expressed in the absence of citrate and the amount of activity which is stimulated by 10 mM citrate is significantly less (1,268 +/- 0.106 nmol/4 x 10(5) cells) than is seen under control conditions (3.042 +/- 0.048). There are no significant changes in the total amount of cellular protein on the plates after 5 h of hypoxia. Consistent with net ACC activation in hypoxia, malonyl-CoA levels increase in the cells by 7 h of hypoxia. Decreased radioactive phosphate content of immunopurified ACC-280 after 5 h of hypoxia is consistent with net dephosphorylation of ACC-280 and increased citrate-independent activity.

Acetyl-CoA Carboxylase↗
PubMed1993

Crystal structure of a ternary complex of Escherichia coli malate dehydrogenase citrate and NAD at 1.9 A resolution.

The structure of malate dehydrogenase from Escherichia coli complexed with the substrate analog, citrate and the cofactor NAD, has been determined by X-ray crystallography. A monoclinic crystal of the malate dehydrogenase, grown in citrate buffer, was soaked in 10 mM NAD solution and found to be isomorphous with the apo-form. The X-ray data extended to 1.9 A, nearly the same resolution limit as the apo-enzyme crystals. The ternary complex of malate dehydrogenase has very few conformational differences from that of the pseudo binary complex of enzyme with bound citrate. In addition, the NAD molecule has a very similar conformation to the NAD as found in the crystal structure of the cytosolic eukaryotic malate dehydrogenase. Similar hydrogen bond interactions are made by both enzymes from polar groups belonging to the NAD. Such interactions include hydrogen bonds from the ribose oxygens and the phosphate oxygens, to backbone amide and carbonyl atoms of the protein and to side-chains of a select few conserved hydrophilic residues. The only notable difference occurs in the active site region where the nicotinamide moiety is obstructed from further entering the active site by the C-6 carbonyl atoms of citrate. In this position there are no direct polar interactions between the protein and the nicotinamide moiety. Energy minimization of the structure with malate substituted for citrate in the active site shows that the nicotinamide moiety assumes the same position in the active site as the NAD in cytosolic malate dehydrogenase. The carboxamide atoms of the energy minimized model make significant hydrogen bond interactions with the catalytic residue, H177, and with the main-chain atoms of I117 and V146 in the vicinity of the active site, while the position of the rest of the cofactor remains unchanged.

Binding Sites↗
PubMed2000

Thermostability and thermoactivity of citrate synthases from the thermophilic and hyperthermophilic archaea, Thermoplasma acidophilum and Pyrococcus furiosus.

Citrate synthases from Thermoplasma acidophilum (optimal growth at 55 degrees C) and Pyrococcus furiosus (100 degrees C) are homo-dimeric enzymes that show a high degree of structural homology with each other, and thermostabilities commensurate with the environmental temperatures in which their host cells are found. A comparison of their atomic structures with citrate synthases from mesophilic and psychrophilic organisms has indicated the potential importance of inter-subunit contacts for thermostability, and here we report the construction and analysis of site-directed mutants of the two citrate synthases to investigate the contribution of these interactions. Three sets of mutants were made: (a) chimeric mutants where the large (inter-subunit contact) and small (catalytic) domains of the T. acidophilum and P. furiosus enzymes were swapped; (b) mutants of the P. furiosus citrate synthase where the inter-subunit ionic network is disrupted; and (c) P. furiosus citrate synthase mutants in which the C-terminal arms that wrap around their partner subunits have been deleted. All three sets of mutant enzymes were expressed as recombinant proteins in Escherichia coli and were found to be catalytically active. Kinetic parameters and the dependence of catalytic activity on temperature were determined, and the stability of each enzyme was analysed by irreversible thermal inactivation experiments. The chimeric mutants indicate that the thermostability of the whole enzyme is largely determined by the origin of the large, inter-subunit domain, whereas the dependence of catalytic activity on temperature is a function of the small domain. Disruption of the inter-subunit ionic network and prevention of the C-terminal interactions both generated enzymes that were substantially less thermostable. Taken together, these data demonstrate the crucial importance of the subunit contacts to the stability of these oligomeric enzymes. Additionally, they also provide a clear distinction between thermostability and thermoactivity, showing that stability is necessary for, but does not guarantee, catalytic activity at elevated temperatures.

Citrate (si)-Synthase↗
PubMed2000

Optimization of rat hepatocyte culture in citrated human plasma.

BACKGROUND: Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartificial liver assist systems. Sodium citrate is a common anticoagulant but has been shown to be cytotoxic to hepatocytes. We have tested the effect of various supplements on the viability and function of adult primary rat hepatocytes exposed to citrated plasma. MATERIALS AND METHODS: Freshly isolated rat hepatocytes were cultured in the collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic functions were evaluated. RESULTS: Hepatocytes exposed to unsupplemented citrated plasma lost significant viability and function within the first 2 days. Cells cultured in plasma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1. 2-2.1 micromol/day/10(6) cells) and albumin (51-62 microg/day/10(6) cells) synthesis rates equal to or higher than those of controls. Among the various components of the concentrated medium supplement, calcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenance of a high albumin synthesis rate also required the addition of hydrocortisone (7.5 microg/ml) and insulin (0.5 U/ml). CONCLUSIONS: Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems.

Albumins↗