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PubMed1990

81mKr equilibrium radionuclide ventriculography for the assessment of right heart function.

Right heart 81mKr equilibrium radionuclide ventriculography was used to derive right ventricular ejection fraction (RVEF) in 12 healthy male volunteers. Anatomical lung subtraction using 99mTc-MAA perfusion scintigraphy was compared with conventional background correction and the effect of imaging projection on the techniques of image analysis evaluated. Both intra and inter observer variability were reduced by anatomical lung subtraction when compared to conventional background correction. In the right anterior oblique (RAO) projection, background corrected RVEF was lower than lung subtracted RVEF - 0.544 +/- 0.05 and 0.612 +/- 0.08 (mean +/- SD) (P less than 0.02). Lung subtracted RVEF in the anterior projection was lower than that with background correction (P less than 0.05) and lower than lung subtracted RVEF in the RAO projection (P less than 0.001). We conclude that optimal separation of right heart structures is achieved in the RAO projection and that reproducibility of the analytical technique is improved by anatomical lung subtraction.

Adult↗
PubMed2004

Carbon and energy fluxes during haloadaptation of Halomonas sp. EF11 growing on phenol.

The haloalkaliphile Halomonas sp. EF11 can grow on phenol as sole source for carbon and energy, while maintaining an osmotic equilibrium predominantly by adjusting levels of a certain compatible solute. To determine the energy costs of haloadaptation and the fate of substrate-carbon, the strain was grown continuously in an isothermal compensation calorimeter, keeping all conditions constant except salinity. As salinity increased, slight linear reductions in exothermic heat flow and biomass formation occurred, and 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) synthesis increased linearly. However, beyond a certain salinity threshold the stationary phenol concentration increased exponentially, while heat flow fell sharply, indicating intoxication or wash-out. The clear transition point between the phases, where ectoine formation peaked, suggests that calorimetric measurements could be used to control the conversion of growth-inhibiting substrates (like phenol) into ectoine and to optimize the process. Enthalpy balance and chemical determinations revealed acetate and formate were formed as side products when the C/N ratio in the feed was low, while 2-muconic acid semialdehyde and formate were produced when the ratio was high. These findings indicate that phenol assimilation occurs via the meta pathway. However, enzyme assays implied that assimilation occurs via the ortho and meta pathways at a low C/N ratio and exclusively via the meta pathway at a high C/N ratio.

Adaptation, Physiological↗
PubMed1975

Partial purification and properties of thiamine pyrophosphokinase from pig brain.

Pig brain thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) was purified 260-fold over extracts of brain acetone powder. A direct, radiometric assay was used to follow the purification. By isoelectric focusing, the purified enzyme appeared to have an isoionic point of approx. pH 4.2, but these preparations were still not homogeneous by disc-gel electrophoresis nor by analytical ultracentrifugation. The purified enzyme has a broad pH optimum extending from pH 8.3 to 9.3 in 0.028 M phosphate/glycylglycine buffers. For optimal enzymatic activity, the ratio of magnesium to ATP must be fixed at 0.6, which suggests that for this ATP-pyrophosphoryl transfer reaction, the enzymatically preferred reactant may be Mg(ATP)6-/2. A preliminary study of the kinetics of the reaction reveals that the enzyme may function via a partial "ping-pong" mechanism; on this basis, dissociation constants for ATPt and for thiamine were evaluated. Pyrithiamine, butylthiamine, ethylthiamine, and oxythiamine appeared to be competitive inhibitors with respect to thiamine as the variable substrate, and their inhibitor dissociation constants were calculated. The relatively poor affinity of oxythiamine to the enzyme emphasizes the 4-amino group in the pyrimidine ring as one of the specificity requirements for thiamine pyrophosphokinase. Preliminary values for the apparent equilibrium coefficient of the thiamine pyrophosphokinase-catalyzed reaction, in terms of total species, has been approximated at several initial concentrations of reactants: e.g. K'eq,app = (see article) 9.66 - 10(-3) M; and [Th]initial - 1 - 10(-6) and 2 - 10(-6) M, respectively, where TDP, Th, t and eq represent thiamine diphosphate, thiamine, total concentration and equilibrium concentration, respectively.

Animals↗
PubMed2006

The effect of operating variables on chelant-assisted remediation of contaminated dredged sediment.

The paper shows the results from a number of lab-scale washing treatments using the four chelating agents EDTA, NTA, citric acid and [S,S]-EDDS aiming at the remediation of a real heavy metal-contaminated sediment. Investigation of the influence of chelant type and concentration as well as solution pH was the major focus of the work. The analysis of speciation of metals and chelating agents in solution was carried out through geochemical speciation modelling in order to identify the optimal conditions for the washing process as well as to evaluate the competition phenomena of metal-chelant complexes in solution. The major competing cations were found to be Ca above all and Mg under specific conditions. Among the investigated chelating agents, EDDS appeared to be less affected by competition by major cations while ensuring adequate heavy metal extraction efficiencies. For a 1:1 chelant/metal ratio, the following ranking was observed: EDDS>Cit>NTA>EDTA for As, EDDS>NTA congruent withEDTA>>Cit for Cu, EDDS congruent withEDTA congruent withNTA>Cit for Zn, EDTA>NTA>EDDS>Cit for Pb at pH 5 and EDTA congruent withEDDS congruent withNTA>>Cit for Pb at pH 8. For a 10:1 chelant/metal ratio geochemical modelling indicated that at the equilibrium the extracting solutions were dominated by the free form of the chelating agents, indicating the inability of such species to complex trace metals due the strong interactions existing between heavy metal ions and sediment constituents.

Arsenic↗
PubMed2006

Adsorption of malachite green on groundnut shell waste based powdered activated carbon.

In the present technologically fast changing situation related to waste management practices, it is desirable that disposal of plant waste should be done in a scientific manner by keeping in view economic and pollution considerations. This is only possible when the plant waste has the potential to be used as raw material for some useful product. In the present study, groundnut shell, an agricultural waste, was used for the preparation of an adsorbent by chemical activation using ZnCl2 under optimized conditions and its comparative characterisation was conducted with commercially available powdered activated carbon (CPAC) for its physical, chemical and adsorption properties. The groundnut shell based powdered activated carbon (GSPAC) has a higher surface area, iodine and methylene blue number compared to CPAC. Both of the carbons were used for the removal of malachite green dye from aqueous solution and the effect of various operating variables, viz. adsorbent dose (0.1-1 g l(-1)), contact time (5-120 min) and adsorbate concentrations (100-200 mg l(-1)) on the removal of dye, has been studied. The experimental results indicate that at a dose of 0.5 g l(-1) and initial concentration of 100 mg l(-1), GSPAC showed 94.5% removal of the dye in 30 min equilibrium time, while CPAC removed 96% of the dye in 15 min. The experimental isotherm data were analyzed using the linearized forms of Freundlich, Langmuir and BET equations to determine maximum adsorptive capacities. The equilibrium data fit well to the Freundlich isotherm, although the BET isotherm also showed higher correlation for both of the carbons. The results of comparative adsorption capacity of both carbons indicate that groundnut shell can be used as a low-cost alternative to commercial powdered activated carbon in aqueous solution for dye removal.

Adsorption↗
PubMed2000

Identification of single nucleotide polymorphisms in human DNA repair genes.

Variation in gene coding sequence represents a significant factor in predisposition to disease, including cancer. Variants of some DNA repair genes (e.g. MLH1, MSH2 and MSH6) are known to predispose to cancer. We identified single nucleotide polymorphisms (SNPs) in five DNA repair genes in 142 healthy individuals using a DNA sequencing protocol optimized for the direct detection of single nucleotide polymorphisms. This approach, called the heterozygote sequencing protocol (HSP), enables moderate-scale population surveys of SNPs. HSP uses fluorescently tagged primers and exploits the large dynamic range and low background of automated fluorescent sequencing. HSP may be used for any sequence that can be amplified by PCR. A total of 12 SNP variants in MGMT, ERCC1, CDK7, CCNH and XRCC4 were identified, 11 at polymorphic frequencies, with an average frequency of 0.22 (95% confidence interval 0.20-0.24). Among the 82 individuals for whom complete SNP profiles were available, no one person carried the GenBank reference sequence for all five genes. The extensive heterogeneity observed in these five genes is intriguing. All variants are in Hardy-Weinberg equilibrium, although the meaning of this equilibrium is unclear. Using this approach, possible associations of sequence variation, and hence of variation in DNA repair, with disease risk can be assessed.

Adolescent↗
PubMedDate not supplied

[Nutritional support for the large burn patient].

Large burn patients make up a subgroup of critical patients in whom the nutro-metabolic support reaches its maximum importance, due to the fact thermal aggression induces a hypermetabolic response which is prolonged until the wounds heal. In fact, there are few deubt with regard to the importance of nutritional support tin the management of these patients for reducing the complications and facilitating the closing of the wounds and the recovery of the patients. Thermal trauma induces the release of counter-regulatory hormones and of other mediators which favor proteineic catabolism, mainly muscular, lipolysis, and gluconeogenesis; as well, there is an alteration of thermoregulation, raising the equilibrium point. The estimate of the energetic requirements may be done by means of predictive equations, although these tend to over-estimate it; indirect calorimetry is the most exact method and this permits monitorization of the evolution, which is very variable in time, it gives the metabolic response to the thermal aggression, at the same time as permitting the analysis of the use of the administrated substrates. Its use has meant a dramatic decrease in the supply of calories administered to burn patients, with the present recommendation being 35-40 kcal/kg/d. The supply of non-proteineic calories has also been modified: It is recommended that at least 60-70% of the calories administered, be in the form of carbohydrates, without surpassing 1600 kcal/kg/d. The optimal relation of non-proteineic kcal:nitrogen, is 150:1. The administration route of the artificial nutrition support should be individualized in each patient, with the enteral route being the route of choice, as this is the most physiologic, the cheapest, and the safest; its use prevents the appearance of certain complications (Curling ulcer, cholecystitis, bacterial translocation); however, if his does not cover nutritional requirements of the burn patient, parenteral nutrition should be associated to this.

Burns↗
PubMed2002

Ab initio conformational studies on diols and binary diol-water systems using DFT methods. Intramolecular hydrogen bonding and 1:1 complex formation with water.

Studies on the conformational equilibrium for the following diols, ethane-1,2-diol (12EG, CAS 107-21-1), 2R-D-(-)-propane-1,2-diol (12PG, CAS 4254-14-2), (2S,3S)-L-(+)-butane-2,3-diol (L23BD, CAS 19132-06-0), and (2S,3R)-meso-butane-2,3-diol (m23BD, CAS 5341-95-7), are described using Gaussian ab initio calculations involving density functional theory (DFT) methods. We also report in this article results on the stability and conformation for the 1:1 water-diol complex formed by ethane-1,2-diol, propane-1,2-diol, and L- and meso-butane-2,3-diol. The relative stability of the intramolecular (internal) hydrogen bond in a range of diols (n = 2 to 6), based on ab initio geometry optimization and determination of the -O...H- distance, dOH, and -O-H...O- angle, theta, increases through the sequence 1,2 approximately equals 2,3 < 1,3 < 1,4 approximately equals 1,5 approximately equals 1,6, as judged from the bond linearity and -O...H- separation. Quantum mechanical and topological analysis of possible intramolecular hydrogen bonding in this complete series of diols provides convincing evidence for this in diols in which the hydroxyl groups are separated by three or more carbon atoms, that is, in (n, n+m) diols for m > or = 2, but not for ethane-1,2-diol or other vicinal diols, which do not satisfy Popelier's topological and electron density criteria based on the AIM theory of Bader. Based on these criteria it is unlikely that vicinal diols are in fact capable of forming an intramolecular hydrogen bond, in spite of geometric and spectroscopic data in the literature suggesting otherwise.

Journal Article↗
PubMed1998

A two-patch model of Gambian sleeping sickness: application to vector control strategies in a village and plantations.

A compartmental model is described for the spread of Gambian sleeping sickness in a spatially heterogeneous environment in which vector and human populations migrate between two "patches": the village and the plantations. The number of equilibrium points depends on two "summary parameters": gr the proportion removed among human infections, and R0, the basic reproduction number. The origin is stable for R0 < 1 and unstable for R0 > 1. Control strategies are assessed by studying the mix of vector control between the two patches that bring R0 below 1. The results demonstrate the importance of vector control in the plantations. For example if 20 percent of flies are in the village and the blood meal rate in the village is 10 percent, then a 20 percent added vector mortality in the village must be combined with a 9 percent added mortality in the plantations in order to bring R0 below 1. The results are quite insensitive to the blood meal rate in the village. Optimal strategies (that minimize the total number of flies trapped in both patches) are briefly discussed.

Communicable Disease Control↗
PubMed2002

Monitoring and kinetic parameter estimation for the binding process of berberine hydrochloride to bovine serum albumin with piezoelectric quartz crystal impedance analysis.

A new method for monitoring, in real time, the drug-binding process to protein with piezoelectric quartz crystal impedance (PQCI) is proposed. The method was used to monitor the binding process of berberine hydrochloride to bovine serum albumin (BSA). BSA was immobilized on the silver electrode surface of a piezoelectric quartz crystal and the optimized experimental conditions were established. The BSA-coated piezoelectric sensor was in contact with berberine solution. The time courses of the resonant frequency and equivalent circuit parameters of the sensor during the protein-drug binding were simultaneously obtained. On the basis of the analysis of the multidimensional information provided by PQCI, it was concluded that the observed frequency decrease was mainly ascribed to the mass increase of the sensor surface resulting from the binding. According to the frequency decrease with time, the kinetics of the binding process were quantitatively studied. A piezoelectric response model for the binding was theoretically derived. Fitting the experimental data to the model, the kinetic parameters, such as the binding and dissociation rate constants (k(1) and k(-1)) and the binding equilibrium constant (K(a)), were determined. The k(1), k(-1), and K(a) values obtained at 25 degrees C were 67.5 (+/-0.1) (mol liter(-1))(-1) s(-1), 1.7 (+/- 0.1) x 10(-3) s(-1), and 3.97 (+/- 0.06) x10(4) (mol liter(-1))(-1), respectively.

Animals↗
PubMed2005

Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding.

A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.

Binding Sites↗
PubMed1996

Mechanism of membrane permeabilization by sticholysin I, a cytolysin isolated from the venom of the sea anemone Stichodactyla helianthus.

Actinaria cytolysins are very potent basic toxins isolated from the venom of sea anemones, which are supposed to exert their toxic activity through formation of oligomeric pores in the host plasma membrane. To gain insight into their mechanism of action, the interaction of Stichodactyla helianthus sticholysin I (St-I) with lipid bilayers was studied. St-I increased the permeability of calcein-loaded lipid vesicles composed of different phospholipids. The rate of permeabilization improved when sphingomyelin (SM) was introduced into phosphatidylcholine (PC) vesicles, reaching an optimum value at equimolar concentrations of these two phospholipids. It was also a function of the pH, showing a local maximum of activity between pH 8 and 9 and a marked decrease at pH 10 and 11. Under optimal conditions (e.g., PC:SM 1:1, pH 8, toxin to vesicle ratio < 200), most of the toxin is bound to the lipid phase. The reduced toxin effect at low and high SM content, or at high pH, is principally due to a decreased toxin binding. From the dose dependence of the permeabilization, at constant lipid concentration, it was inferred that St-I increases membrane permeability by forming oligomeric pores comprising at least three cytolysin monomers. The involvement of oligomers was also suggested by the dependence of calcein release on the vesicle concentration at constant toxin dose. In fact, the time course of dye release was well described under all circumstances by a kinetic model which assumes that trimerization leads to a conductive pore. All the relevant equilibrium and rate constants were derived. Addition of St-I to one side of a planar lipid membrane increased the conductivity of the film in discrete steps of defined amplitude, indicating the formation of ion channels. The dose dependence of this effect was the same as with LUV. The channel was cation-selective and its conductance suggested a functional radius of about 1.0 nm, consistent with the size of the lesion previously observed in red blood cells. Pores exhibited rectification and voltage-dependent gating.

Animals↗
PubMed1998

Comparison of the DNA binding characteristics of the related zinc finger proteins WT1 and EGR1.

The interactions of the related zinc finger proteins WT1 and EGR1 with DNA have been investigated using a quantitative binding assay. A recombinant peptide containing the four zinc fingers of WT1 binds to the dodecamer DNA sequence GCG-TGG-GCG-TGT with an apparent dissociation constant (Kd) of (1.14 +/- 0.09) x 10(-9) M under conditions of 0.1 M KCl, pH 7.5, at 22 degrees C. Under the same conditions, a recombinant peptide containing the three zinc fingers of EGR1 binds to the dodecamer sequence, the first nine bases comprising the EGR consensus binding site, with an apparent Kd of (3.55 +/- 0.24) x 10(-9) M. The nature of the equilibrium binding of each peptide to DNA was investigated as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1 with DNA is an entropy-driven process, while the formation of the EGR1-DNA complex is favored by enthalpy and entropy. The DNA binding activities of both proteins have broad pH optima centered at pH 8.0. The binding of both proteins to DNA shows similar sensitivity to ionic strength, with approximately 7.7 +/- 0.8 ion pairs formed in the EGR1-DNA complex and 9.2 +/- 1.8 ion pairs formed in the WT1-DNA complex. Results of measuring the effects of point mutations in the DNA binding site on the affinity of WT1 and EGR1 indicates a significant difference in the optimal binding sites: for EGR1, the highest affinity binding site has the sequence GNG-(T/G)GG-G(T/C)G, while for WT1 the highest affinity binding site has the sequence G(T/C)G-(T/G)GG-GAG-(T/C)G(T/C).

Amino Acid Sequence↗
PubMed2004

Emission of phthalates from PVC and other materials.

The main objective of this study was to generate quantitative and qualitative emission data on phthalates from different materials. To achieve this the existing (Chamber for Laboratory Investigations of Materials, Pollution and Air Quality) Climpaq-based procedure for simplified measurements of emissions of plasticizer from PVC and other plasticized materials was modified. It was applied to a range of products. Some of them were suspected of contributing to the indoor concentration of plasticizers. The emissions from PVC flooring, polyolefine flooring, a refrigerator list, two electric cables, PVC skirting and floor wax were studied in separate Climpaqs. The emission from the PVC flooring in the Climpaq was compared with results from the ultra-small chamber Field and Laboratory Emission Cell (FLEC). Sampling and analysis methods were optimized to measure plasticizers. Samples were taken in exhaust air from the chambers after 6, 35, 62, 105, and 150 days from the start of the experiment. PVC flooring was tested for an additional 100 days. Polyolefine covered with wax resulted in an air concentration of 22 microg/m3 of dibutylphthalate (DBP), which is two orders of magnitude larger than any other materials, but did not emit di(2-ethylhexyl)phthalate (DEHP). The other materials resulted in max concentration of approximately 1 microg/m3 of DEHP and low emissions of DBP. The concentration of DEHP in each chamber increased slowly to a rather stable level which was reached after 150 days. DBP concentrations in the chambers with PVC skirting, PVC flooring, polyolefine and floor wax reached their quasi-static equilibrium after 60 days. The modified method did not create sufficient data for the calculation of emission rates. Adsorption of emission on chamber surfaces made it impossible to use the first part of the experiment for emission rate calculation. When the concentration had stabilized, it was found to be almost identical and independent of chamber and ventilation rate. Emission rates were reduced at high concentrations probably because the concentration in the material was near equilibrium with the concentration in the chamber air.

Air Pollution, Indoor↗
PubMed1974

Lipase Activities in Castor Bean Endosperm during Germination.

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal "ghosts" at equilibrium density 1.21 g/cm(3) on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.

Journal Article↗
PubMed1975

Ontogeny of mouse T-lymphocyte function.

The development of lymphocytes within the fetal and neonatal BALB/c mouse thymus is reviewed with particular emphasis on the maturity of immunologic functions. Fetal thymocytes respond by vigorous proliferation to stimulation by allogeneic lymphoid cells or by phytohemagglutinin. Such reactivity is much diminished in neonatal thymus or thymic-derived (T) cells in neonatal spleen. Splenic T cells seem to mature more slowly than immunoglobulin-bearing B lymphocytes in the neonatal spleen, but the finding is confounded by the presence of large numbers of "suppressor" T cells in the neonatal spleen. For example, the in vitro antibody response to the T-independent antigen dinitrophenyl-lysine-Ficoll is optimal by 2 or 3 weeks of age, but the in vitro response to T-dependent sheep erythrocytes does not reach adult levels until 6 weeks of age, suggesting a deficiency in T "helper cells." The response of neonatal spleen cells to sheep erythrocytes cannot be reconstituted by adult T cells however, unless neonatal splenic T cells are first depleted by anti-Thy 1 serum and complement. The target of this T suppressor cell seems to be only B cells, and not other T cells. The overall sequence of T lymphocyte maturation in the mouse seems to start with large numbers of reactive T cells as well as some functionally active helper or effector T cells in early neonatal life, and finally to achieve a stable equilibrium between T cell subpopulations between 5 and 6 weeks of age.

Animals↗
PubMed1987

Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum.

Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.

Animals↗
PubMed1996

How to derive flux control coefficients from the rate equations of classical enzyme kinetics.

A recent report by Brown and Cooper demonstrated the usefulness of calculating "flux control coefficients" for each of the rate constants involved in the assumed kinetic mechanism of a single enzyme. The calculations of Brown and Cooper involved numerical differentiation. The present article substantiates this report by showing that the numerical results of Brown and Cooper can also be obtained in an explicit form. The analytical equations given establish the relationship between rigorously specified overall rate processes and "elementary rate constants," both being defined by the rate equations of classical enzyme kinetics. It is shown that analytical flux control coefficients can be obtained for all types of rate processes considered in classical enzyme kinetics, including, "initial rates," equilibrium exchange reactions, and reactions at limiting levels of substrate (and/or product) saturation. By restricting the discussion to strictly consecutive (ordered, unbranched, linear) mechanisms, the line of reasoning can be presented in a relatively simple form. The main conclusions are the following: (a) It is advantageous to carry out the analysis in terms of paired (conjugated) control coefficients. (b) Flux control analysis of "elementary rate constants" does not require any extra kinetic argument. (c) Neither the immediate aim nor the results of the presented type of analysis are directly relevant to theories of metabolic control. On the contrary, the type of control analysis considered completes classical enzyme kinetics with a new facet. (d) For illustrating its usefulness, the concept of flux control coefficients is applied to the problem of optimization of enzyme activity.

Binding Sites↗