Journal articles on the topic 'Substrate exchange kinetics'
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1
Stein, Allan R. "The ion-pair mechanism and bimolecular displacement at saturated carbon. VI. Racemization and radio-bromide exchange for substituted 1-phenylbromoethanes; solvent effects." Canadian Journal of Chemistry 65, no. 2 (February 1, 1987): 363–71. http://dx.doi.org/10.1139/v87-062.
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Racemization and radio-bromide exchange kinetics for 1-phenylbromoethanes in acetonitrile and in nitromethane using tetrabutylammonium bromide are reported. The results, together with those previously reported for acetone solutions, provide direct empirical support for the ion-pair mechanism for nucleophilic substitution at saturated carbon. Changing the substituents on the phenyl from the 4-nitro through to the 3,4-dimethyl substrate and the solvent from acetone to the more polar acetonitrile and nitromethane shifts the transition state for bromide substitution from an early to a late stage of the equilibria series substrate [Formula: see text] intimate ion pair [Formula: see text] various solvated ion pairs [Formula: see text] free or dissociated ions. For all the substrates in acetone and, for the species giving the less stable carbocations, in acetonitrile and nitromethane, both racemizations and exchanges are bimolecular. In the latter solvents, the substrates giving the more stable carbocations show mixed kinetics.
2
Hasenhuetl, Peter S., Shreyas Bhat, Felix P. Mayer, Harald H. Sitte, Michael Freissmuth, and Walter Sandtner. "A kinetic account for amphetamine-induced monoamine release." Journal of General Physiology 150, no. 3 (February 9, 2018): 431–51. http://dx.doi.org/10.1085/jgp.201711915.
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The plasmalemmal monoamine transporters for dopamine, norepinephrine, and serotonin (SERT) are targets for amphetamines. In vivo, amphetamines elicit most, if not all, of their actions by triggering monoamine efflux. This is thought to be accomplished by an amphetamine-induced switch from the forward-transport to the substrate-exchange mode. The mechanism underlying this switch has remained elusive; available kinetic models posit that substrates and cosubstrate Na+ ions bind either in a random or in a sequential order. Neither can account for all reported experimental observations. We used electrophysiological recordings to interrogate crucial conformational transitions associated with the binding of five different substrates (serotonin, para-chloroamphetamine, and the high-affinity naphthyl-propan-amines PAL-287, PAL-1045, and PAL-1046) to human SERT expressed in HEK293 cells; specifically, we determined the relaxation kinetics of SERT from a substrate-loaded to a substrate-free state at various intracellular and extracellular Na+ concentrations. These rates and their dependence on intracellular and extracellular Na+ concentrations differed considerably between substrates. We also examined the effect of K+ on substrate affinity and found that K+ enhanced substrate dissociation. A kinetic model was developed, which allowed for random, but cooperative, binding of substrate and Na+ (or K+). The synthetic data generated by this model recapitulated the experimental observations. More importantly, the cooperative binding model accounted for the releasing action of amphetamines without any digression from alternating access. To the best of our knowledge, this model is the first to provide a mechanistic framework for amphetamine-induced monoamine release and to account for the findings that some substrates are less efficacious than others in promoting the substrate-exchange mode.
3
Restrepo, D., D. J. Kozody, L. J. Spinelli, and P. A. Knauf. "Cl-Cl exchange in promyelocytic HL-60 cells follows simultaneous rather than ping-pong kinetics." American Journal of Physiology-Cell Physiology 257, no. 3 (September 1, 1989): C520—C527. http://dx.doi.org/10.1152/ajpcell.1989.257.3.c520.
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The intra- and extracellular chloride concentration dependencies of the rate of Cl-Cl exchange in human promyelocytic leukemic HL-60 cells were studied by means of radioactive isotope (36Cl) efflux measurements. Efflux of isotope from cells follows an exponential time course. The Cl-Cl exchange flux follows Michaelis-Menten kinetics as a function of both intra- and extracellular chloride concentrations. The ratio of the maximum exchange velocity to the apparent Michaelis constant for both extracellular and intracellular substrate increases as a function of trans Cl concentration, indicating that Cl-Cl exchange in the HL-60 cell does not follow ping-pong kinetics. A kinetic scheme in which extracellular and intracellular chloride ions bind in random order to the transporter and are then translocated simultaneously can adequately model the experimental data.
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Kosa, Nicolas M., Kevin M. Pham, and Michael D. Burkart. "Chemoenzymatic exchange of phosphopantetheine on protein and peptide." Chem. Sci. 5, no. 3 (2014): 1179–86. http://dx.doi.org/10.1039/c3sc53154f.
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Deshmukh, Lalit, Vitali Tugarinov, John M. Louis, and G. Marius Clore. "Binding kinetics and substrate selectivity in HIV-1 protease−Gag interactions probed at atomic resolution by chemical exchange NMR." Proceedings of the National Academy of Sciences 114, no. 46 (October 30, 2017): E9855—E9862. http://dx.doi.org/10.1073/pnas.1716098114.
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The conversion of immature noninfectious HIV-1 particles to infectious virions is dependent upon the sequential cleavage of the precursor group-specific antigen (Gag) polyprotein by HIV-1 protease. The precise mechanism whereby protease recognizes distinct Gag cleavage sites, located in the intrinsically disordered linkers connecting the globular domains of Gag, remains unclear. Here, we probe the dynamics of the interaction of large fragments of Gag and various variants of protease (including a drug resistant construct) using Carr−Purcell−Meiboom−Gill relaxation dispersion and chemical exchange saturation transfer NMR experiments. We show that the conformational dynamics within the flaps of HIV-1 protease that form the lid over the catalytic cleft play a significant role in substrate specificity and ordered Gag processing. Rapid interconversion between closed and open protease flap conformations facilitates the formation of a transient, sparsely populated productive complex between protease and Gag substrates. Flap closure traps the Gag cleavage sites within the catalytic cleft of protease. Modulation of flap opening through protease−Gag interactions fine-tunes the lifetime of the productive complex and hence the likelihood of Gag proteolysis. A productive complex can also be formed in the presence of a noncognate substrate but is short-lived owing to lack of optimal complementarity between the active site cleft of protease and the substrate, resulting in rapid flap opening and substrate release, thereby allowing protease to differentiate between cognate and noncognate substrates.
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Oldfield, C. "Evaluation of steady-state kinetic parameters for enzymes solubilized in water-in-oil microemulsion systems." Biochemical Journal 272, no. 1 (November 15, 1990): 15–22. http://dx.doi.org/10.1042/bj2720015.
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1. Equations are derived for the steady-state kinetics of substrate conversion by enzymes confined within the water-droplets of water-in-oil microemulsion systems. 2. Water-soluble substrates initially confined within droplets that do not contain enzyme are assumed to be converted into product only after they enter enzyme-containing droplets via the inter-droplet exchange process. 3. Hyperbolic (Michaelis-Menten) kinetics are predicted when the substrate concentration is varied in microemulsions of fixed composition. Both kcat. and Km are predicted to be dependent on the size and concentration of the water-droplets in the microemulsion. 4. The predicted behaviour is shown to be supported by published experimental data. A physical interpretation of the form of the rate equation is presented. 5. The rate equation for an oil-soluble substrate was derived assuming a pseudo-two-phase (oil & water) model for the microemulsion. Both kcat. and Km are shown to be independent of phi aq. Km is larger than the aqueous solution value by a factor approximately equal to the oil/water partition coefficient of the substrate. The validity of the rate equation is confirmed by published data.
7
Goward, C. R., R. Hartwell, T. Atkinson, and M. D. Scawen. "The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus." Biochemical Journal 237, no. 2 (July 15, 1986): 415–20. http://dx.doi.org/10.1042/bj2370415.
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Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.
8
Knickelbein, R. G., P. S. Aronson, and J. W. Dobbins. "Characterization of Na(+)-H+ exchangers on villus cells in rabbit ileum." American Journal of Physiology-Gastrointestinal and Liver Physiology 259, no. 5 (November 1, 1990): G802—G806. http://dx.doi.org/10.1152/ajpgi.1990.259.5.g802.
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The presence of Na(+)-H+ exchange activity is demonstrated on both the brush-border membrane (BBM) and the basolateral membrane (BLM) of villus cells from rabbit ileum. The possibility that the Na(+)-H+ exchange activity on the BLM represents HCO3- cotransport is excluded. The two Na(+)-H+ exchangers are then compared in terms of kinetics and substrate and inhibitor specificity. The most striking difference between the two exchangers was sensitivity to amiloride and K+. The IC50 for amiloride on the BLM was 10-fold lower than the BBM (11.2 +/- 2.1 vs. 103 +/- 20.9 microM; P less than 0.02). External K+, in concentrations as low as 10 mM, inhibited Na(+)-H+ exchange on the BBM but not on the BLM. The Na+ Km and proton Km were twice as high on the BLM exchanger (46.3 +/- 3.4 vs. 28.8 +/- 2.3 mM and 468 +/- 9 vs. 232 +/- 45 nM, respectively). Proton Vmax was similar, whereas Na+ Vmax was higher on the BLM. Inhibition by Li+ was similar on both membranes. These results indicate distinct differences between the two Na(+)-H+ exchangers. Whether these differences are due to the two different gene products or are the result of posttranslational modification of a single gene product remains to be determined.
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Krupka, R. M. "Testing transport models and transport data by means of kinetic rejection criteria." Biochemical Journal 260, no. 3 (June 15, 1989): 885–91. http://dx.doi.org/10.1042/bj2600885.
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In the case of a transport system obeying Michaelis-Menten kinetics, completely general relationships are shown to exist between the final ratio of internal and external substrate concentrations, alpha, and the V/Km ratios found in zero-trans-entry, zero-trans-exit and equilibrium-exchange experiments (where V is a maximum substrate flux and Km a substrate half-saturation constant). The proof depends on a new method of derivation proceeding from the form of the experimental data rather than, as has been the practice in kinetic analysis, from a hypothetical reaction scheme. These general relationships, which will be true of all mechanisms giving rise to a particular type of behaviour (here Michaelis-Menten kinetics), provide a test for internal consistency in a set of experimental data. Other relationships, which are specific, can be derived from individual reaction schemes, with the use of traditional procedures in kinetic analysis. The specific relationships include constants for infinite trans entry and exit in addition to constants involved in the general relationships. In conjunction, the general and specific relationships provide a stringent test of mechanism. A set of results that fails to satisfy the general relationships must be rejected; here systematic error or unexpected changes in the transport system in different experiments may have distorted the calculated constants, or the system may not actually obey Michaelis-Menten kinetics. Results in accord with the general relationships, on the other hand, can be applied in specific tests of mechanism. The usefulness of the theorem is illustrated in the cases of the glucose-transport and choline-transport systems of erythrocytes. Experimental results taken from several studies in the literature, which were in accord with hyperbolic substrate kinetics, had previously been shown to disagree with relationships derived for the carrier model, and the model was rejected. The new analysis shows that the data violated the general relationships and therefore cannot decide the issue. More recent results on the glucose-transport system satisfy the general relations and agree with the carrier model.
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PAOLI, Paolo, Paolo CIRRI, Lucia CAMICI, Giampaolo MANAO, Gianni CAPPUGI, Gloriano MONETI, Giuseppe PIERACCINI, Guido CAMICI, and Giampietro RAMPONI. "Common-type acylphosphatase: steady-state kinetics and leaving-group dependence." Biochemical Journal 327, no. 1 (October 1, 1997): 177–84. http://dx.doi.org/10.1042/bj3270177.
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A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 °C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme–substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O–inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. All these findings, as well as the strong dependence of kcat upon the leaving group pKa, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.
11
Cornish-Bowden, A., and A. C. Storer. "Mechanistic origin of the sigmoidal rate behaviour of rat liver hexokinase D (‘glucokinase’)." Biochemical Journal 240, no. 1 (November 15, 1986): 293–96. http://dx.doi.org/10.1042/bj2400293.
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Two recent proposals to account for the kinetic co-operativity of hexokinase D (‘glucokinase’) from rat liver are examined. A model in which the deviations from Michaelis-Menten kinetics result from a random order of binding of the substrates [Pettersson (1986) Biochem. J. 233, 347-350] accounts satisfactorily for the behaviour as a function of glucose concentrations, but it also predicts observable substrate inhibition by MgATP, which is in fact not observed. An alternative proposal in which the deviations arise from recycling of an enzyme-MgADP complex [Pettersson (1986) Eur. J. Biochem. 154, 167-170] also accounts satisfactorily for some of the data, but the required enzyme-MgADP complex could not be detected in isotope-exchange measurements. Thus the mnemonical mechanism proposed originally [Storer & Cornish-Bowden (1977) Biochem. J. 165, 61-69], which explains the deviations in terms of a relatively slow interconversion between two forms of free enzyme, remains the most parsimonious explanation of the behavior of hexokinase D.
12
Goldson-Barnaby, Andrea, and Christine H. Scaman. "Purification and Characterization of Phenylalanine Ammonia Lyase from Trichosporon cutaneum." Enzyme Research 2013 (September 12, 2013): 1–6. http://dx.doi.org/10.1155/2013/670702.
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Trichosporon cutaneum phenylalanine ammonia lyase was selected as a model to investigate the dual substrate activity of this family of enzymes. Sequencing of the PAL gene identified an extensive intron region at the N-terminus. Five amino acid residues differing from a prior report were identified. Highest Phe : Tyr activities (1.6 ±0.3 : 0.4±0.1 μmol/h g wet weight) were induced by Tyr. The enzyme has a temperature optimum of 32°C and a pH optimum of 8–8.5 and shows no metal cofactor dependence. Michaelis-Menten kinetics (Phe, Km 5.0 ± 1.1 mM) and positive allostery (Tyr, K′ 2.4 ± 0.6 mM, Hill coefficient 1.9±0.5) were observed. Anion exchange chromatography gave a purification fold of 50 with 20% yield. The His-Gln motif (substrate selectivity switch region) indicates the enzyme’s ability to act on both substrates.
13
Wolfe, R. R., F. Jahoor, and H. Miyoshi. "Evaluation of the isotopic equilibration between lactate and pyruvate." American Journal of Physiology-Endocrinology and Metabolism 254, no. 4 (April 1, 1988): E532—E535. http://dx.doi.org/10.1152/ajpendo.1988.254.4.e532.
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When an isotopic tracer is infused for the purpose of determining the rate of turnover or oxidation of a substrate, it is assumed that the resulting isotopic enrichment by the tracer will reflect the kinetics of only the pool of interest. However, this may not be the case when carbon-labeled lactate is infused, since rapid isotopic exchange with the intracellular pyruvate and alanine pools could potentially occur. Therefore we have determined the extent of isotopic exchange occurring during the infusion of [3-13C]lactate into six anesthetized dogs. In the steady state, pyruvate enrichment was 91 +/- 2.2% (means +/- SE) of the lactate enrichment, and alanine enrichment was 81 +/- 3.3% of the pyruvate enrichment and 72 +/- 2.6% of the lactate enrichment. In contrast, when [3-13C]alanine was infused (n = 2), pyruvate (and lactate) enrichment was 9.9% of the alanine enrichment. We therefore conclude that there is rapid isotopic equilibration between lactate and pyruvate but that interaction with alanine reflects the true metabolic flux rates, rather than isotopic exchange. Consequently, lactate kinetics, as traditionally determined, more accurately reflect whole body pyruvate kinetics.
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Furumai, H., and B. E. Rittmann. "Interpretation of bacterial activities in nitrification filters by a biofilm model considering the kinetics of soluble microbial products." Water Science and Technology 30, no. 11 (December 1, 1994): 147–56. http://dx.doi.org/10.2166/wst.1994.0555.
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Activities of heterotrophic bacteria in nonsteady-state biofilms were evaluated using a simplified biofilm model in which formation and exchange of soluble microbial products (SMP) by nitrifiers and heterotrophs were considered. The model was applied to experimental results for a trace-level substrate removal. The model predictions indicated that SMP from nitrifiers contributed to supporting heterotrophic growth and their substrate removal potential. The biological interactions were more significant in cases of low influent substrate COD concentrations and increased with higher influent ammonium concentration. The introduction of SMP kinetics into the model captured the key aspects of removal and formation of COD components in biofilms receiving low influent substrate concentrations, such as nitrification filters for drinking water treatment and wastewater reuse.
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Galezowski, Wlodzimierz, and Arnold Jarczewski. "Kinetics, isotope effects of the reaction of 1-(4-nitrophenyl)-1-nitroalkanes with DBU in tetrahydrofuran and chlorobenzene solvents." Canadian Journal of Chemistry 68, no. 12 (December 1, 1990): 2242–48. http://dx.doi.org/10.1139/v90-345.
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The kinetics of the reaction of[Formula: see text](R = Me, Et, i-Pr; NPNE, NPNP, MNPNP respectively; L is H or D) with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) base in tetrahydrofuran (THF) and chlorobenzene (CB) solvents are reported. The products of these proton transfer reactions are ion pairs absorbing at λmax = 460–480 nm. The equilibrium constants in THF were [Formula: see text]and in CB [Formula: see text]for NPNE, NPNP, MNPNP respectively. The thermodynamic parameters of the reactions are also quoted. The substrate reacts with DBU in both THF and CB solvents in a normal second-order proton transfer reaction. In the case of deuteron transfer, isotopic D/H exchange is much faster than internal return. The reactions show low values of enthalpy of activation ΔH* = 14.3, 18.1, 24.2 and 13.0, 15.1, 18.6 kJmol−1 for NPNE, NPNP, and MNPNP in THF and CB respectively, and large negative entropies of activation −ΔS* = 141, 139, 146; 140, 146, 160 J mol−1 deg−1 for the same sequence of substrates and solvents. The kinetic isotope effects are large, (kH/kD)20°c = 12.2, 13.0, 10.1; 12.9, 12.0, 10.2 for the above sequence of substrates and solvents, and show no difference with changes in either steric hindrance of the C-acids or polarity of the solvents. Keywords: proton transfer, kinetic isotope effect.
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Yi, Jian Hua, Mei Li Li, and Zhen Bao Zhu. "Kinetics of LIS-100 Cation Exchange Resin to Remove Reaction Substrate of Non-Enzymatic Browning in Clarified Apple Juice." Advanced Materials Research 699 (May 2013): 770–74. http://dx.doi.org/10.4028/www.scientific.net/amr.699.770.
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In order to inhibit non-enzymatic browning in clarified Fuji apple juice during storage, four kinds of cation exchange resins were compared for their abilities to exchange and remove amino acids, the reaction substrates of non-enzymatic browning in apple juice. The favorite resin, LSI-100, was screened to carry out static and dynamic adsorption experiments. The results showed that LSI-100 cation exchange resin had the best capacity for adsorption and removal amino acids. The equilibrium time of LSI-100 resin for amino acids was 3.5h.And the isotherm of LSI-100 resin could be described by Freundlich at 20°C. Additionally, the flow rates, temperature and concentrations of amino acids in apple juice affected the dynamic kinetic curves of LSI-100 cation exchange resin and the better dynamic exchange and adsorption parameters were as follows: flow rate 4BV/h, temperature 50°C,and amino acid concentration 30mg/100g apple juice.
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Frumence, Etienne, Sandrine Genetet, Pierre Ripoche, Achille Iolascon, Immacolata Andolfo, Caroline Le Van Kim, Yves Colin, Isabelle Mouro-Chanteloup, and Claude Lopez. "Rapid Cl−/HCO3−exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells." American Journal of Physiology-Cell Physiology 305, no. 6 (September 15, 2013): C654—C662. http://dx.doi.org/10.1152/ajpcell.00142.2013.
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Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.
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Saeidian, Shahriar. "Kinetics Properties of Polyphenol Oxidase in Hawthorn (Crataegus spp)." International Letters of Natural Sciences 25 (September 2014): 29–38. http://dx.doi.org/10.18052/www.scipress.com/ilns.25.29.
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Polyphenol oxidase (PPO) from hawthorn was extracted and partially purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. The activity of polyphenol oxidase was investigated in Crataegus spp. Spectrophotometric method was used to assay the enzyme activity and the kinetic constants - maximum enzyme velocity (Vmax) and Michealis - Menten constant (Km). Of the substrates tested, catechol was the best substrate for PPO with a Km value of 2.2 mM. The optimum pH for PPO activity was found to be 7. The enzyme showed high activity over a broad pH range of 4 - 8. The optimal pH and temperature for enzyme activity were found to be 7 and 40-45 °C, respectively. km value for hawthorn PPO is calculated 22 mM for catechol and 6.7 mM for pyrogallol and 9.7 mM for L-dopa. As can be seen, affinity of PPOs for various substrates varies widely. The enzyme showed a broad activity over a broad pH and temperature range. The thermal inactivation studies showed that the enzyme is heat resistant. The enzyme showed the highest activity toward pyrogallol and no activity toward tyrosine. Of the inhibitors tested, the most potent inhibitors were kojic acid, cysteine and glycine , respectively
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Tiihonen, K., and M. Nikinmaa. "MEMBRANE PERMEABILITY AND UTILIZATION OF l-LACTATE AND PYRUVATE IN CARP RED BLOOD CELLS." Journal of Experimental Biology 178, no. 1 (May 1, 1993): 161–72. http://dx.doi.org/10.1242/jeb.178.1.161.
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l-Lactate and pyruvate permeability and utilization in carp (Cyprinus carpio) red blood cells was studied in vitro with tracer methods. Transport of lactate and pyruvate across the carp red blood cell membrane is rapid. At low plasma concentrations, lactate and pyruvate are transported into carp red blood cells mainly via a specific monocarboxylate carrier. This is shown by a study of the saturation kinetics and by inhibition using alpha-cyano-4-hydroxycinnamic acid and, more powerfully, p-chloromercuriphenylsulphonic acid. At higher plasma concentrations both simple diffusion and, apparently, the band 3 anion exchange system become increasingly important transport pathways. Carbon dioxide production rates from lactate and pyruvate as a function of their extracellular concentrations showed saturation kinetics. The transport rates of lactate and pyruvate are considerably higher than those required for their maximal rate of oxidation. The rapid transport of lactate and pyruvate into carp red blood cells thus guarantees that substrate availability is not the rate-limiting factor for the oxidation of these substrates.
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Tanner, Adam, and David J. Hopper. "Conversion of 4-Hydroxyacetophenone into 4-Phenyl Acetate by a Flavin Adenine Dinucleotide-Containing Baeyer-Villiger-Type Monooxygenase." Journal of Bacteriology 182, no. 23 (December 1, 2000): 6565–69. http://dx.doi.org/10.1128/jb.182.23.6565-6569.2000.
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ABSTRACT An arylketone monooxygenase was purified from Pseudomonas putida JD1 by ion exchange and affinity chromatography. It had the characteristics of a Baeyer-Villiger-type monooxygenase and converted its substrate, 4-hydroxyacetophenone, into 4-hydroxyphenyl acetate with the consumption of one molecule of oxygen and oxidation of one molecule of NADPH per molecule of substrate. The enzyme was a monomer with an M r of about 70,000 and contained one molecule of flavin adenine dinucleotide (FAD). The enzyme was specific for NADPH as the electron donor, and spectral studies showed rapid reduction of the FAD by NADPH but not by NADH. Other arylketones were substrates, including acetophenone and 4-hydroxypropiophenone, which were converted into phenyl acetate and 4-hydroxyphenyl propionate, respectively. The enzyme displayed Michaelis-Menten kinetics with apparent Km values of 47 μM for 4-hydroxyacetophenone, 384 μM for acetophenone, and 23 μM for 4-hydroxypropiophenone. The apparentKm value for NADPH with 4-hydroxyacetophenone as substrate was 17.5 μM. The N-terminal sequence did not show any similarity to other proteins, but an internal sequence was very similar to part of the proposed NADPH binding site in the Baeyer-Villiger monooxygenase cyclohexanone monooxygenase from anAcinetobacter sp.
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Pokorny, Diana, Lothar Brecker, Mateja Pogorevc, Walter Steiner, Herfried Griengl, Thomas Kappe, and Douglas W. Ribbons. "Proton-Nuclear Magnetic Resonance Analyses of the Substrate Specificity of a β-Ketolase from Pseudomonas putida, Acetopyruvate Hydrolase." Journal of Bacteriology 181, no. 16 (August 15, 1999): 5051–59. http://dx.doi.org/10.1128/jb.181.16.5051-5059.1999.
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ABSTRACT A revised purification of acetopyruvate hydrolase from orcinol-grown Pseudomonas putida ORC is described. This carbon-carbon bond hydrolase, which is the last inducible enzyme of the orcinol catabolic pathway, is monomeric with a molecular size of ∼38 kDa; it hydrolyzes acetopyruvate to equimolar quantities of acetate and pyruvate. We have previously described the aqueous-solution structures of acetopyruvate at pH 7.5 and several synthesized analogues by1H-nuclear magnetic resonance (NMR)-Fourier transform (FT) experiments. Three 1H signals (2.2 to 2.4 ppm) of the methyl group are assigned unambiguously to the carboxylate anions of 2,4-diketo, 2-enol-4-keto, and 2-hydrate-4-keto forms (40:50:10). A1H-NMR assay for acetopyruvate hydrolase was used to study the kinetics and stoichiometries of reactions within a single reaction mixture (0.7 ml) by monitoring the three methyl-group signals of acetopyruvate and of the products acetate and pyruvate. Examination of 4-tert-butyl-2,4-diketobutanoate hydrolysis by the same method allowed the conclusion that it is the carboxylate 2-enol form(s) or carbanion(s) that is the actual substrate(s) of hydrolysis. Substrate analogues of 2,4-diketobutanoate with 4-phenyl or 4-benzyl groups are very poor substrates for the enzyme, whereas the 4-cyclohexyl analogue is readily hydrolyzed. In aqueous solution, the arene analogues do not form a stable 2-enol structure but exist principally as a delocalized π-electron system in conjugation with the aromatic ring. The effects of several divalent metal ions on solution structures were studied, and a tentative conclusion that the enol forms are coordinated to Mg2+ bound to the enzyme was made. 1H–2H exchange reactions showed the complete, fast equilibration of 2H into the C-3 of acetopyruvate chemically; this accounts for the appearance of2H in the product pyruvate. The C-3 of the product pyruvate was similarly labelled, but this exchange was only enzyme catalyzed; the methyl group of acetate did not undergo an exchange reaction. The unexpected preference for bulky 4-alkyl-group analogues is discussed in an evolutionary context for carbon-carbon bond hydrolases. Routine one-dimensional 1H-NMR in normal1H2O is a new method for rapid, noninvasive assays of enzymic activities to obtain the kinetics and stoichiometries of reactions in single reaction mixtures. Assessments of the solution structures of both substrates and products are also shown.
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Post, M. A., and D. C. Dawson. "Basolateral Na(+)-H+ antiporter. Mechanisms of electroneutral and conductive ion transport." Journal of General Physiology 103, no. 5 (May 1, 1994): 895–916. http://dx.doi.org/10.1085/jgp.103.5.895.
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The basolateral Na-H antiporter of the turtle colon exhibits both conductive and electroneutral Na+ transport (Post and Dawson. 1992. American Journal of Physiology. 262:C1089-C1094). To explore the mechanism of antiporter-mediated current flow, we compared the conditions necessary to evoke conduction and exchange, and determined the kinetics of activation for both processes. Outward (cell to extracellular fluid) but not inward (extracellular fluid to cell) Na+ or Li+ gradients promoted antiporter-mediated Na+ or Li+ currents, whereas an outwardly directed proton gradient drove inward Na+ or Li+ currents. Proton gradient-driven, "counterflow" current is strong evidence for an exchange stoichiometry of > 1 Na+ or Li+ per proton. Consistent with this notion, outward Na+ and Li+ currents generated by outward Na+ or Li+ gradients displayed sigmoidal activation kinetics. Antiporter-mediated proton currents were never observed, suggesting that only a single proton was transported per turnover of the antiporter. In contrast to Na+ conduction, Na+ exchange was driven by either outwardly or inwardly directed Na+, Li+, or H+ gradients, and the activation of Na+/Na+ exchange was consistent with Michaelis-Menten kinetics (K1/2 = 5 mM). Raising the extracellular fluid Na+ or Li+ concentration, but not extracellular fluid proton concentration, inhibited antiporter-mediated conduction and activated Na+ exchange. These results are consistent with a model for the Na-H antiporter in which the binding of Na+ or Li+ to a high-affinity site gives rise to one-for-one cation exchange, but the binding of Na+ or Li+ ions to other, lower-affinity sites can give rise to a nonunity, cation exchange stoichiometry and, hence, the net translocation of charge. The relative proportion of conductive and nonconductive events is determined by the magnitude and orientation of the substrate gradient and by the serosal concentration of Na+ or Li+.
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Zhang, Guosheng, Na Liu, Yuan Luo, Haibo Zhang, Long Su, Kokyo Oh, and Hongyan Cheng. "Efficient Removal of Cu(II), Zn(II), and Cd(II) from Aqueous Solutions by a Mineral-Rich Biochar Derived from a Spent Mushroom (Agaricus bisporus) Substrate." Materials 14, no. 1 (December 23, 2020): 35. http://dx.doi.org/10.3390/ma14010035.
Full textAbstract:
This study evaluated the novel application of a mineral-rich biochar derived from a spent Agaricus bisporus substrate (SAS). Biochars with various pyrolysis temperatures (350–750 °C) were used to remove Cu(II), Zn(II), and Cd(II) from aqueous solutions. The adsorption characteristics and removal mechanisms of the biochars were investigated. The adsorption kinetics and isotherm data were fitted well by pseudo-second-order and Freundlich models. The Langmuir maximum removal capacity (Qmax) values of Cu(II), Zn(II), and Cd(II) were ordered as SAS750 > SAS350 > SAS550, and the Qmax values of SAS750 were 68.1, 55.2, and 64.8 mg·g−1, respectively. Overall, the removal mechanisms of biochar at a low production temperature (350 °C) to Cu(II), Zn(II), and Cd(II) were mainly via ion exchange (54.0, 56.0, and 43.0%), and at a moderate production temperature (550 °C), removal mechanisms were mainly via coordination with π electrons (38.3, 45.9, and 55.0%), while mineral precipitation (65.2, 44.4, and 76.3%, respectively) was the dominant mechanism at a high produced temperature (750 °C). The variation of the mutual effect of minerals and heavy metals was the predominant factor in the sorption mechanism of mineral precipitation and ion exchange. The results demonstrated that spent Agaricus bisporus substrate biochar is a potential candidate for the efficient removal of heavy metals, which provides a utilization route for spent mushroom substrates.
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Saeidian, Shahriar. "Kinetics Properties of Polyphenol Oxidase in Hawthorn (<i>Crataegus spp</i>)." International Letters of Natural Sciences 25 (September 2, 2014): 29–38. http://dx.doi.org/10.56431/p-64t842.
Full textAbstract:
Polyphenol oxidase (PPO) from hawthorn was extracted and partially purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. The activity of polyphenol oxidase was investigated in Crataegus spp. Spectrophotometric method was used to assay the enzyme activity and the kinetic constants - maximum enzyme velocity (Vmax) and Michealis - Menten constant (Km). Of the substrates tested, catechol was the best substrate for PPO with a Km value of 2.2 mM. The optimum pH for PPO activity was found to be 7. The enzyme showed high activity over a broad pH range of 4 - 8. The optimal pH and temperature for enzyme activity were found to be 7 and 40-45 °C, respectively. km value for hawthorn PPO is calculated 22 mM for catechol and 6.7 mM for pyrogallol and 9.7 mM for L-dopa. As can be seen, affinity of PPOs for various substrates varies widely. The enzyme showed a broad activity over a broad pH and temperature range. The thermal inactivation studies showed that the enzyme is heat resistant. The enzyme showed the highest activity toward pyrogallol and no activity toward tyrosine. Of the inhibitors tested, the most potent inhibitors were kojic acid, cysteine and glycine , respectively
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Restrepo, D., B. L. Cronise, R. B. Snyder, L. J. Spinelli, and P. A. Knauf. "Kinetics of DIDS inhibition of HL-60 cell anion exchange rules out ping-pong model with slippage." American Journal of Physiology-Cell Physiology 260, no. 3 (March 1, 1991): C535—C544. http://dx.doi.org/10.1152/ajpcell.1991.260.3.c535.
Full textAbstract:
According to the ping-pong model of band 3-mediated anion exchange, the transport protein has a single transport site, which can exist in either an inward-facing or an outward-facing conformation. Anions bind to these unloaded forms of the carrier, and translocation takes place only when a suitable anion is bound to the transport site. In a previous paper [Am. J. Physiol. 257 (Cell Physiol. 26): C520-C527, 1989], we had shown that the substrate kinetics of Cl-Cl exchange in the promyelocytic HL-60 cell cannot be explained by this simple ping-pong model of anion exchange but is consistent with a simultaneous model according to which both extracellular and intracellular anions must bind before simultaneous translocation can take place. In the present paper we show that external 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibits anion exchange in HL-60 cells by competing with Cl- for binding to the outward-facing transport site. Furthermore, there is a linear dependence of the slope of the Dixon plot for inhibition by DIDS on the reciprocal of the intracellular Cl- concentration. This result clearly rules out a simple ping-pong scheme. In addition, the data also rule out a ping-pong model in which some translocation of the unloaded carrier is allowed (ping-pong model with slippage). The observed inhibition kinetics can be modeled by a simultaneous model of Cl-Cl exchange with competitive inhibition by DIDS.
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Meikle, P. J., A. M. Whittle, and J. J. Hopwood. "Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation." Biochemical Journal 308, no. 1 (May 15, 1995): 327–33. http://dx.doi.org/10.1042/bj3080327.
Full textAbstract:
Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.
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Benveniste, I., B. Gabriac, and F. Durst. "Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction." Biochemical Journal 235, no. 2 (April 15, 1986): 365–73. http://dx.doi.org/10.1042/bj2350365.
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NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2′,5′-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.
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BRITTON, Hubert G. "Isomerization of the free enzyme versus induced fit: effects of steps involving induced fit that bypass enzyme isomerization on flux ratios and countertransport." Biochemical Journal 321, no. 1 (January 1, 1997): 187–99. http://dx.doi.org/10.1042/bj3210187.
Full textAbstract:
In a single-substrate–single-product enzyme reaction, ‘countertransport’, which indicates that the ratio of the forward to the reverse fluxes is less than that expected from the Independence Relationship, is regarded as strong evidence for the free enzyme existing in two states, one of which combines with the substrate and the other with the product, with a slow isomerization between the two conditions. To account for positive and negative co-operativity, found with some enzymes, additional induced-fit reactions bypassing at least part of the isomerization have been proposed. The effects of such additional steps have been examined, using two models: in one, (a), the enzyme passes through an intermediate state during its isomerization, and both substrate and product may react with this state to give rise to the binary complexes; in the other, (b), the substrate may react with the enzyme as soon as the product is released and similarly with the reverse reaction, the isomerization thereby being bypassed completely. In the presence of such additional steps, the following can be concluded. (i) The data should be analysed in terms of the flux ratios, rather than observation of the amount of countertransport. (ii) The additional bypassing steps markedly change the pattern of dependence of the flux ratio on substrate and product concentrations. At high substrate and product concentrations, the ratio remains very dependent on how far the reaction is from equilibrium, and the kinetics are asymmetric. (iii) The mechanism causing the flux ratio to be less than that given by the Independence Relationship differs from that previously described, in that, at least in part, it arises from a 1:1 exchange between substrate and product. (iv) Despite this novel mechanism, there must be two states of the enzyme, combining respectively with substrate and product, and these must not be in rapid exchange. Thus countertransport remains very strong evidence for the existence of two such states. It is no longer a requirement that the enzyme states should be linked by an isomerization step. (v) Under no conditions can the flux ratio exceed that given by the Independence Relationship. (vi) Under unusual conditions the isomerization of the enzyme in model (b) may be undetectable by steady-state kinetics. (vii) Measurements of the coefficients in the flux ratio equations enable limits to be set to certain ratios of the rate constants. In addition to these conclusions, methods are described for (viii) analysing flux ratio data for the presence of induced fit steps and (ix) determining flux ratios from induced transport curves. The derivation of steady state–velocity equations show that: (x) both models may give rise to positive and negative ‘co-operativity’ and sigmoid substrate–velocity curves, but that, under conditions giving rise to sigmoid curves, the deviation of the flux ratio from that required by the Independence Relationship may be difficult to demonstrate because of the asymmetry of the system. Under all conditions the fluxes at equilibrium should obey hyperbolic kinetics.
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Hilgemann, Donald W., and Chin-Chih Lu. "Gat1 (Gaba:Na+:Cl−) Cotransport Function." Journal of General Physiology 114, no. 3 (September 1, 1999): 459–76. http://dx.doi.org/10.1085/jgp.114.3.459.
Full textAbstract:
We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na+:Cl−) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, Ein and Eout. In the Ein state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the Eout state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one γ-aminobutyric acid (GABA) molecule can bind from the extracellular side. When Ein sites are empty, a transition to the Eout state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the Eout state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *Ein state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the Ein state. GABA–GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the Ein state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current–voltage relations. Experimental results that are simulated accurately include (a) all current–voltage relations, (b) all substrate dependencies described to date, (c) cis–cis and cis–trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre–steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA–GABA exchange, and (i) the existence of inward transport current and GABA–GABA exchange in the nominal absence of extracellular chloride.
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Dangprapai, Yodying, and Stephen H. Wright. "Interaction of H+ with the extracellular and intracellular aspects of hMATE1." American Journal of Physiology-Renal Physiology 301, no. 3 (September 2011): F520—F528. http://dx.doi.org/10.1152/ajprenal.00075.2011.
Full textAbstract:
Human multidrug and toxin extrusion 1 (hMATE1, SLC47A1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules. Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake, i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction, i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, we measured the time course of hMATE1-mediated efflux of the prototypic MATE1 substrate, [3H]1-methyl-4-phenylpyridinium, under a variety of intra- and extracellular pH conditions, from Chinese hamster ovary cells that stably expressed the transporter. In this study, we showed that an IC50/ Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] was not significantly different ( P = 0.6). Furthermore, kinetics of interaction between intracellular H+ and inward-facing hMATE1 determined using the efflux protocol revealed an IC50 for H+ of 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1.
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James, S. R., R. A. Demel, and C. P. Downes. "Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C." Biochemical Journal 298, no. 2 (March 1, 1994): 499–506. http://dx.doi.org/10.1042/bj2980499.
Full textAbstract:
The activity of a beta-isoform of phospholipase C (PLC) partially purified from turkey erythrocyte cytosol was assayed using phospholipid monolayers formed at an air-water interface. PLC was rapidly purified at least 8000-fold by a sequence of ion-exchange, hydrophobic and heparin chromatographies. 33P-labelled substrates were prepared using partially purified PtdIns kinase and PtdIns4P 5-kinases, respectively, and purified by h.p.l.c. using an amino-cyano analytical column. Using such 33P-labelled phosphoinositides of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphates and their subsequent dissolution and quenching in the subphase. Under these conditions, PtdIns4P hydrolysis obeyed approximately first-order kinetics whereas PtdIns(4,5)P2 hydrolysis was zero-order at least until 80% of the substrate had been degraded. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure and with the most permissive pressures in the middle of the range investigated. The optimum surface pressure for hydrolysis of PtdIns4P was approx. 25 mN/m, but for PtdIns(4,5)P2 the maximum activity occurred at the markedly higher surface pressure of 30 mN/m. These data are discussed in terms of the substrate specificity and likely regulation of PLC beta isoforms engaged in degrading their substrate in biological membranes.
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Jarczewski, Arnold, Grzegorz Schroeder, Wlodzimierz Galezowski, Kenneth T. Leffek, and Urszula Maciejewska. "The kinetics, isotope effects, and mechanism of the reaction of 2,2-di(4-nitrophenyl)-1,1,1-trifluoroethane with alkoxide bases in alcohol solvents." Canadian Journal of Chemistry 63, no. 3 (March 1, 1985): 576–80. http://dx.doi.org/10.1139/v85-094.
Full textAbstract:
The reaction between 2,2-di(4-nitrophenyl)-1,1,1-trifluoroethane and the alkoxide bases ŌCH3, ŌC2H5, ŌnC4H9, ŌCH(CH3)2, and ŌC(CH3)3 in their corresponding alcohol solvents is a multistep reaction with several intermediates: 2,2-di(4-nitrophenyl)-1,1-difluoro-1-alkoxyethane (A), 2,2-di(4-nitrophenyl)-1-fluoro-1-alkoxyethene (B), 2,2-di(4-nitrophenyl)-1,1-dialkoxyethene (C), 2,2-di(4-nitrophenyl)-1,1-difluoroethene (D), and 4,4′-dinitrobenzophene (E). Rate constants and activation parameters have been measured for the appearance of the two stable products B and C. The kinetic deuterium isotope effects for the appearance of B fell in the range of kH/kD = 1 to 2 at 25 °C for the primary and secondary alkoxides, whereas kH/kD = 5.4 at 30 °C for the appearance of D with tert-butoxide. Exchange experiments showed that H/D exchange took place between the substrate and solvent to the extent of 100% with methoxide, 50% with ethoxide and isopropoxide, and 0% with tert-butoxide. It is concluded the HF elimination from the substrate follows an (ElcB)R mechanism with methoxide/methanol, changing to (ElcB)I or E2 with tert-butoxide/tert-butanol.
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Allen, Karen N., Arnon Lavie, Gregory K. Farber, Arthur Glasfeld, Gregory A. Petsko, and Dagmar Ringe. "Isotopic Exchange plus Substrate and Inhibition Kinetics of D-Xylose Isomerase Do Not Support a Proton-Transfer Mechanism." Biochemistry 33, no. 6 (February 15, 1994): 1481–87. http://dx.doi.org/10.1021/bi00172a026.
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Morris, A. J., K. J. Murray, P. J. England, C. P. Downes, and R. H. Michell. "Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase." Biochemical Journal 251, no. 1 (April 1, 1988): 157–63. http://dx.doi.org/10.1042/bj2510157.
Full textAbstract:
An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 × 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5′-[gamma−thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.
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Schneider, H. "Placental transport function." Reproduction, Fertility and Development 3, no. 4 (1991): 345. http://dx.doi.org/10.1071/rd9910345.
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Placental transport provides a means of supplying nutrients to and removing metabolites from the fetus. Transport is based on substrate exchange and net flux from mother to fetus or vice versa and can be a result of a concentration difference or of unidirectional carrier-mediated transport. Blood flow regulates delivery to and removal from the area of placental exchange, and rapidly crossing compounds are dependent on blood flow for their rate of passage. There are substantial species differences in terms of flow rates normalized for fetal weight and also in terms of vascular arrangement. The barrier can be overcome via paracellular water-filled channels or via a transcellular route. Hydrophilic molecules that are not actively transported diffuse through paracellular channels, and the placentae of rodents and primates are much more permeable than the placenta of the sheep. Many different substrates such as glucose, amino acids, electrolytes and vitamins are transported by carrier systems. Transport proteins are located in the microvillous and basal membranes of the trophoblast. Asymmetry in the kinetics of binding results in differences in influx and efflux at the interface with maternal and fetal blood, allowing directional net flux across the placenta. Immunoglobulins are believed to cross by receptor-mediated endocytosis.
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Rahimizadeh, Mohammad, Karen Kam, Stephen I. Jenkins, Robert S. McDonald, and Paul HM Harrison. "Kinetics of glycoluril template-directed Claisen condensations and mechanistic implications." Canadian Journal of Chemistry 80, no. 5 (May 1, 2002): 517–27. http://dx.doi.org/10.1139/v02-071.
Full textAbstract:
Eight N-acetyl-N-aroyl-glycolurils were prepared and found to undergo efficient tert-butoxide-promoted Claisen-like condensation between the two acyl moieties. The kinetics for formation of each of the N-(aroylacetyl)gly coluril products were monitored by UV spectroscopy. The reaction exhibited pseudo-first-order kinetics in substrate in the presence of excess base. For the parent benzoyl compound the observed first-order rate constant (kobs) was linearly dependent on the concentration of the base, tert-butoxide. A Hammett plot of the resulting apparent second-order rate constants (kapp) vs. σ for each of the eight aroyl derivatives was linear and had a positive ρ value 1.04 ± 0.04), demonstrating that the substituent on the aromatic ring exerts a significant effect upon the condensation reaction. The corresponding plot for three [D3]acetyl analogues was also linear, but the slope was reduced by 20% relative to the protonated compounds. The isotope effect (kHapp/kDapp) thus increased from 1.4 (benzoyl) to 2.6 (p-nitrobenzoyl). The results are consistent with a three-step mechanism in which both deprotonation of the acetyl entity and the ensuing nucleophilic attack of the resulting enolate on the benzoyl group are partially rate-determining steps. The tetrahedral intermediate thus produced rapidly collapses to the product. For the [D3]acetyl benzoyl derivative, exchange of substrate deuterium with solvent hydrogen due to reprotonation of the enolate intermediate occurs at a rate that is similar to that of condensation, but the enolate partitions towards the product when electron withdrawing groups are present in the aroyl ring. Thus, despite the presence of a large excess of co-solvent tert-butanol, the efficiency with which the enolate undergoes condensation remains high. The clean kinetics observed allows further exploration of the details of this intramolecular Claisen-like condensation process.Key words: Claisen condensation, glycoluril, kinetics, Hammett, mechanism.
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Weber, J. M. "Effect of endurance swimming on the lactate kinetics of rainbow trout." Journal of Experimental Biology 158, no. 1 (July 1, 1991): 463–76. http://dx.doi.org/10.1242/jeb.158.1.463.
Full textAbstract:
The lactate turnover rate of rainbow trout (Oncorhynchus mykiss) was measured by bolus injection of [U-14C]lactate at rest and during prolonged swimming at 85% Ucrit to determine the importance of this metabolic fuel for endurance locomotion in fish, to assess whether lactate exchange between white and red muscle could be a possible mechanism for supplying oxidizable fuel to their lateral red muscle, and to compare the contribution of lactate to total energy provision between teleost and mammalian species. Turnover rate only increased from 4.41 +/− 0.33 to 9.71 +/− 1.69 mumol kg-1 min-1 between rest and prolonged swimming, and the contribution of lactate oxidation to total metabolism declined during exercise. Lactate exchange between white and red muscle is, therefore, not a significant mechanism to fuel the active lateral red musculature during prolonged swimming. The lactate turnover rate of teleosts is one or two orders of magnitude lower than in mammals of equivalent size, but lactate has the same importance as a fuel in both vertebrate groups. However, lactate turnover rate and oxidation rate do not scale with body mass in the same fashion as does metabolic rate. The slope of the mammalian relationship for whole-body lactate turnover and oxidation is much lower (0.58) than the slope of the classic relationship for metabolic rate (0.75), indicating that lactate is a much more important oxidative substrate for small than for large animals.
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Horton, Tracy J., Emily K. Miller, Deborah Glueck, and Kathleen Tench. "No effect of menstrual cycle phase on glucose kinetics and fuel oxidation during moderate-intensity exercise." American Journal of Physiology-Endocrinology and Metabolism 282, no. 4 (April 1, 2002): E752—E762. http://dx.doi.org/10.1152/ajpendo.00238.2001.
Full textAbstract:
Resting and exercise fuel metabolism was assessed in three different phases of the menstrual cycle, characterized by different levels of estrogen relative to progesterone: early follicular (EF, low estrogen and progesterone), midfollicular (MF, elevated estrogen, low progesterone), and midluteal (ML, elevated estrogen and progesterone). It was hypothesized that exercise glucose utilization and whole body carbohydrate oxidation would decrease sequentially from the EF to the MF to the ML phase. Normal-weight healthy females, experiencing a regular menstrual cycle, were recruited. Subjects were moderately active but not highly trained. Testing occurred after 3 days of diet control and after an overnight fast (12–13 h). Resting (2 h) and exercise (50% maximal O2 uptake, 90 min) measurements of whole body substrate oxidation, tracer-determined glucose flux, and substrate and hormone concentrations were made. No significant difference was observed in whole body fuel oxidation during exercise in the three phases (nonprotein respiratory exchange ratio: EF 0.84 ± 0.01, MF 0.85 ± 0.01, ML 0.85 ± 0.01) or in rates of glucose appearance or disappearance. There were, however, significantly higher glucose ( P < 0.05) and insulin ( P < 0.001) concentrations during the first 45 min of exercise in the ML phase vs. EF and MF phases. In conclusion, whole body substrate oxidation and glucose utilization did not vary significantly across the menstrual cycle in moderately active women, either at rest or during 90 min of moderate-intensity exercise. During the ML phase, however, this similar pattern of substrate utilization was associated with greater glucose and insulin concentrations. Both estrogen and progesterone are elevated during the ML phase of the menstrual cycle, suggesting that one or both of these sex steroids may play a role in this response.
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Sebastián, David, Giovanni Lemes, José M. Luque-Centeno, María V. Martínez-Huerta, Juan I. Pardo, and María J. Lázaro. "Optimization of the Catalytic Layer for Alkaline Fuel Cells Based on Fumatech Membranes and Ionomer." Catalysts 10, no. 11 (November 20, 2020): 1353. http://dx.doi.org/10.3390/catal10111353.
Full textAbstract:
Polymer electrolyte fuel cells with alkaline anion exchange membranes (AAEMs) have gained increasing attention because of the faster reaction kinetics associated with the alkaline environment compared to acidic media. While the development of anion exchange polymer membranes is increasing, the catalytic layer structure and composition of electrodes is of paramount importance to maximize fuel cell performance. In this work, we examine the preparation procedures for electrodes by catalyst-coated substrate to be used with a well-known commercial AAEM, Fumasep® FAA-3, and a commercial ionomer of the same nature (Fumion), both from Fumatech GmbH. The anion exchange procedure, the ionomer concentration in the catalytic layer and also the effect of membrane thickness, are investigated as they are very relevant parameters conditioning the cell behavior. The best power density was achieved upon ion exchange of the ionomer by submerging the electrodes in KCl (isopropyl alcohol/water solution) for at least one hour, two exchange steps, followed by treatment in KOH for 30 min. The optimum ionomer (Fumion) concentration was found to be close to 50 wt%, with a relatively narrow interval of functioning ionomer percentages. These results provide a practical guide for electrode preparation in AAEM-based fuel cell research.
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Zander, Catherine B., Thomas Albers, and Christof Grewer. "Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2." Journal of General Physiology 141, no. 6 (May 13, 2013): 659–72. http://dx.doi.org/10.1085/jgp.201210948.
Full textAbstract:
Neutral amino acid exchange by the alanine serine cysteine transporter (ASCT)2 was reported to be electroneutral and coupled to the cotransport of one Na+ ion. The cotransported sodium ion carries positive charge. Therefore, it is possible that amino acid exchange is voltage dependent. However, little information is available on the electrical properties of the ASCT2 amino acid transport process. Here, we have used a combination of experimental and computational approaches to determine the details of the amino acid exchange mechanism of ASCT2. The [Na+] dependence of ASCT2-associated currents indicates that the Na+/amino acid stoichiometry is at least 2:1, with at least one sodium ion binding to the amino acid–free apo form of the transporter. When the substrate and two Na+ ions are bound, the valence of the transport domain is +0.81. Consistently, voltage steps applied to ASCT2 in the fully loaded configuration elicit transient currents that decay on a millisecond time scale. Alanine concentration jumps at the extracellular side of the membrane are followed by inwardly directed transient currents, indicative of translocation of net positive charge during exchange. Molecular dynamics simulations are consistent with these results and point to a sequential binding process in which one or two modulatory Na+ ions bind with high affinity to the empty transporter, followed by binding of the amino acid substrate and the subsequent binding of a final Na+ ion. Overall, our results are consistent with voltage-dependent amino acid exchange occurring on a millisecond time scale, the kinetics of which we predict with simulations. Despite some differences, transport mechanism and interaction with Na+ appear to be highly conserved between ASCT2 and the other members of the solute carrier 1 family, which transport acidic amino acids.
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Kien, C. L. "Isotopic dilution of CO2 as an estimate of CO2 production during substrate oxidation studies." American Journal of Physiology-Endocrinology and Metabolism 257, no. 2 (August 1, 1989): E296—E298. http://dx.doi.org/10.1152/ajpendo.1989.257.2.e296.
Full textAbstract:
The rate of oxidation of a substrate is often divided by the numerical estimate of a correction factor for the proportion of labeled CO2 that is excreted during the course of the experiment. This factor, derived from tracer studies of bicarbonate kinetics, equals the product of the net rate of respiratory CO2 excretion and the ratio of the plateau isotopic enrichment of CO2 to the rate of infusion of labeled bicarbonate. The inverse of this ratio may be equated to the rate of appearance of unlabeled CO2. When one substitutes the expression for the correction factor into a typical equation for the rate of substrate oxidation, the rate of appearance of CO2 substitutes for both net CO2 excretion and the correction factor in the original oxidation equation. Thus the rate of appearance of CO2 is an index of CO2 production that takes into account both net fixation of CO2 and isotopic exchange of labeled CO2; it also may have application as an index of net CO2 production.
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Wang, Jiali, Yang Dong, and Christof Grewer. "Functional and Kinetic Comparison of Alanine Cysteine Serine Transporters ASCT1 and ASCT2." Biomolecules 12, no. 1 (January 11, 2022): 113. http://dx.doi.org/10.3390/biom12010113.
Full textAbstract:
Neutral amino acid transporters ASCT1 and ASCT2 are two SLC1 (solute carrier 1) family subtypes, which are specific for neutral amino acids. The other members of the SLC1 family are acidic amino acid transporters (EAATs 1–5). While the functional similarities and differences between the EAATs have been well studied, less is known about how the subtypes ASCT1 and 2 differ in kinetics and function. Here, by performing comprehensive electrophysiological analysis, we identified similarities and differences between these subtypes, as well as novel functional properties, such as apparent substrate affinities of the inward-facing conformation (in the range of 70 μM for L-serine as the substrate). Key findings were: ASCT1 has a higher apparent affinity for Na+, as well as a larger [Na+] dependence of substrate affinity compared to ASCT2. However, the general sequential Na+/substrate binding mechanism with at least one Na+ binding first, followed by amino acid substrate, followed by at least one more Na+ ion, appears to be conserved between the two subtypes. In addition, the first Na+ binding step, presumably to the Na3 site, occurs with high apparent affinity (<1 mM) in both transporters. In addition, ASCT1 and 2 show different substrate selectivities, where ASCT1 does not respond to extracellular glutamine. Finally, in both transporters, we measured rapid, capacitive charge movements upon application and removal of amino acid, due to rearrangement of the translocation equilibrium. This charge movement decays rapidly, with a time constant of 4–5 ms and recovers with a time constant in the 15 ms range after substrate removal. This places a lower limit on the turnover rate of amino acid exchange by these two transporters of 60–80 s−1.
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Kim, Yong Keun, Jin Sup Jung, and Sang Ho Lee. "Dicarboxylate transport in renal basolateral and brush-border membrane vesicles." Canadian Journal of Physiology and Pharmacology 70, no. 1 (January 1, 1992): 106–12. http://dx.doi.org/10.1139/y92-015.
Full textAbstract:
Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na+–H+ exchange. The Na+-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na+-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na+-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis–Menten kinetics, with an apparent Km of 22.20 ± 4.08 and 71.52 ± 0.14 μM and a Vmax of 39.0 ± 3.72 and 70.20 ± 0.96 nmol/(mg·min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na+-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.Key words: dicarboxylate transport, brush border membrane, basolateral membrane, inhibitors, rabbit kidney.
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Prato, José Gregorio, Fernando Carlos Millán, Luisa Carolina González, Anita Cecilia Ríos, Esteban López, Iván Ríos, Siboney Navas, Andrés Márquez, Julio César Carrero, and Juan Isidro Díaz. "Adsorption of Phosphate and Nitrate Ions on Oxidic Substrates Prepared with a Variable-Charge Lithological Material." Water 14, no. 16 (August 9, 2022): 2454. http://dx.doi.org/10.3390/w14162454.
Full textAbstract:
This work evaluates phosphate and nitrate ion adsorption from aqueous solutions on calcined adsorbent substrates of variable charge, prepared from three granulometric fractions of an oxidic lithological material. The adsorbent material was chemically characterized, and N2 gas adsorption (BET), X-ray diffraction, and DTA techniques were applied. The experimental conditions included the protonation of the beds with HCl and H2SO4 and the study of adsorption isotherms and kinetics. The lithological material was moderately acidic (pH 5) with very little solubility (electrical conductivity 0.013 dS m−1) and a low cation exchange capacity (53.67 cmol (+) kg−1). The protonation reaction was more efficient with HCl averaging 0.745 mmol versus 0.306 mmol with H2SO4. Likewise, the HCl-treated bed showed a better adsorption of PO4−3 ions (3.296 mg/100 g bed) compared to the H2SO4-treated bed (2.579 mg/100 g bed). The isotherms showed great affinity of the PO4−3 ions with the oxide surface, and the data fit satisfactorily to the Freundlich model, suggesting a specific type of adsorption, confirmed by the pseudo-second-order kinetic model. In contrast, the nitrate ions showed no affinity for the substrate (89.7 µg/100 g for the HCl-treated bed and 29.3 µg/100 g bed for the H2SO4-treated bed). Amphoteric iron and aluminum oxides of variable charges present in the lithological material studied allow for their use as adsorbent beds as an alternative technique to eliminate phosphates and other ions dissolved in natural water.
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Čičmanec, Pavel, Jiří Kotera, Jan Vaculík, and Roman Bulánek. "Influence of Substrate Concentration on Kinetic Parameters of Ethanol Dehydration in MFI and CHA Zeolites and Relation of These Kinetic Parameters to Acid–Base Properties." Catalysts 12, no. 1 (January 3, 2022): 51. http://dx.doi.org/10.3390/catal12010051.
Full textAbstract:
The catalytic activity of zeolites is often related to their acid–base properties. In this work, the relationship between the value of apparent activation energy of ethanol dehydration, measured in a fixed bed reactor and by means of a temperature-programmed surface reaction (TPSR) depending on the amount of ethanol in the zeolite lattice and the value of activation energy of H/D exchange as a measure of acid–base properties of MFI and CHA zeolites, was studied. Tests in a fixed bed reactor were unable to provide reliable reaction kinetics data due to internal diffusion limitations and rapid catalyst deactivation. Only the TPSR method was able to provide activation energy values comparable to the activation energy values obtained from the H/D exchange rate measurements. In addition, for CHA zeolite, it has been shown that the values of ethanol dehydration activation energies depend on the amount of ethanol in the CHA framework, and this effect can be attributed to the substrate clustering effects supporting the deprotonation of zeolite Brønsted centers.
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Banik, Swarnali, Shrutidhara Biswas, and Srabani Karmakar. "Extraction, purification, and activity of protease from the leaves of Moringa oleifera." F1000Research 7 (July 30, 2018): 1151. http://dx.doi.org/10.12688/f1000research.15642.1.
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Background: Proteases cleave proteins, thereby providing essential amino acids for protein synthesis, and degrade misfolded and damaged proteins to maintain homeostasis. Proteases also serve as signaling molecules, therapeutic agents and find wide applications in biotechnology and pharmaceutical industry. Plant-derived proteases are suitable for many biomedical applications due to their easy availability and activity over a wide range of pH, temperature, and substrates. Moringa oleifera Lam (Moringaceae) is a very common food plant with medicinal property and geographically distributed in tropical countries. Here, we isolate proteases from the leaves of Moringa oleifera and characterize its enzymatic activity. Methods: Proteases were isolated from the aqueous leaf extract of Moringa oleifera by ammonium sulfate precipitation and purified by ion exchange chromatography. Subsequently, the enzyme kinetics was determined using casein as a substrate and calibrated over different pH and temperature range for maximal activity. Results: We obtained purified fraction of the protease having a molecular weight of 51 kDa. We observed that for the maximal caseinolytic activity of the protease, a pH of 8 and temperature of 37ºC was found to be most effective. Conclusion: The plant-derived proteolytic enzymes are finding increasing clinical and industrial applications. We could extract, purify and characterize the enzymatic activity of proteases from the leaves of Moringa oleifera. Further molecular characterization, substrate specificity and activity of the extracted protease are required for determining its suitability as a proteolytic enzyme for various applications.
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Lönn, Björn, Rosemary Brown, Robin Pfeiffer, Henrik Frederiksen, and Björn Wickman. "Platinum-Based Nanoparticle Fuel Cell Catalysts Synthesized By Sputtering Onto Liquid Substrates." ECS Meeting Abstracts MA2022-01, no. 35 (July 7, 2022): 1450. http://dx.doi.org/10.1149/ma2022-01351450mtgabs.
Full textAbstract:
The quest of finding cheaper and better fuel cell catalyst materials has been long ongoing and remains a crucial step in making fuel cells a commercially viable technology. Platinum (Pt) is still the conventional catalyst used in proton exchange membrane (PEM) fuel cells, however, with slow cathode kinetics and high material cost. Platinum-rare earth (RE) and lanthanide alloys have been found to greatly enhance the catalytic activity towards the oxygen reduction reaction (ORR).1 Increases in ORR activity of up to one order of magnitude compared to pure platinum has been observed for these alloys, but high oxygen affinity of the RE alloying agents considerably complicates their synthesis. Sputter deposition into low vapor pressure liquid substrates, such as ionic liquids and certain polymers enables fabrication of clean nanoparticles, without the presence of air and other contaminates, making it a potential technique for Pt-RE nanoparticle synthesis. Here, we will present our recent work on magnetron sputtering of Pt 2 and Pt-alloys into different liquid substrates, including polyethylene glycol and three imidazolium-based ionic liquids. We have investigated the influence of substrate temperature on Pt nanoparticle growth during sputtering, and in post-sputtering heat treatment.2 We show that whilst substrate temperature influences the Pt nanoparticle size, the effect is not as great as for other materials. Better understanding the growth processes of Pt nanoparticles sputtered into liquids is an important step towards tunable sizes and catalytic activities of Pt-RE nanocatalysts. Escudero-Escribano, María, et al. "Tuning the activity of Pt alloy electrocatalysts by means of the lanthanide contraction." Science 352.6281 (2016): 73-76. Brown, Rosemary, et al. "Plasma-Induced Heating Effects on Platinum Nanoparticle Size During Sputter Deposition Synthesis in Polymer and Ionic Liquid Substrates." Langmuir 37.29 (2021): 8821-8828.
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Chinkes, D. L., X. J. Zhang, J. A. Romijn, Y. Sakurai, and R. R. Wolfe. "Measurement of pyruvate and lactate kinetics across the hindlimb and gut of anesthetized dogs." American Journal of Physiology-Endocrinology and Metabolism 267, no. 1 (July 1, 1994): E174—E182. http://dx.doi.org/10.1152/ajpendo.1994.267.1.e174.
Full textAbstract:
We have developed a new model to quantify regional pyruvate and lactate transmembrane transport, shunting, exchange, production, and oxidation in vivo. The method is based on the systemic continuous infusion of pyruvate or lactate stable isotopic carbon tracers and the measurement of pyruvate and lactate enrichment and concentration in the artery and vein of that region (e.g., leg or gut), the pyruvate and lactate enrichment of intracellular free water in the tissue as measured by biopsy, and the rate of blood flow through the tissue. The purpose of the experiment was to measure the pyruvate and lactate kinetics in leg muscle and gut in anesthetized dogs (n = 6). The transmembrane transport and degree of shunting of pyruvate and lactate were comparable in muscle and gut. When modified for substrate inflow, interconversion between pyruvate and lactate took place at a rate twice as fast in muscle as in the gut, and production and oxidation of pyruvate was approximately 50% greater in muscle than in the gut. Thus our new model enables quantitation of many aspects of lactate and pyruvate kinetics. We conclude that in anesthetized animals the muscle is the tissue most responsible for whole body peripheral pyruvate and lactate kinetics.
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Laeverenz Schlogelhofer, Hannah, François J. Peaudecerf, Freddy Bunbury, Martin J. Whitehouse, Rachel A. Foster, Alison G. Smith, and Ottavio A. Croze. "Combining SIMS and mechanistic modelling to reveal nutrient kinetics in an algal-bacterial mutualism." PLOS ONE 16, no. 5 (May 20, 2021): e0251643. http://dx.doi.org/10.1371/journal.pone.0251643.
Full textAbstract:
Microbial communities are of considerable significance for biogeochemical processes, for the health of both animals and plants, and for biotechnological purposes. A key feature of microbial interactions is the exchange of nutrients between cells. Isotope labelling followed by analysis with secondary ion mass spectrometry (SIMS) can identify nutrient fluxes and heterogeneity of substrate utilisation on a single cell level. Here we present a novel approach that combines SIMS experiments with mechanistic modelling to reveal otherwise inaccessible nutrient kinetics. The method is applied to study the onset of a synthetic mutualistic partnership between a vitamin B12-dependent mutant of the alga Chlamydomonas reinhardtii and the B12-producing, heterotrophic bacterium Mesorhizobium japonicum, which is supported by algal photosynthesis. Results suggest that an initial pool of fixed carbon delays the onset of mutualistic cross-feeding; significantly, our approach allows the first quantification of this expected delay. Our method is widely applicable to other microbial systems, and will contribute to furthering a mechanistic understanding of microbial interactions.
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Portman, Michael A., Kun Qian, Julia Krueger, and Xue-Han Ning. "Direct action of T3on phosphorylation potential in the sheep heart in vivo." American Journal of Physiology-Heart and Circulatory Physiology 288, no. 5 (May 2005): H2484—H2490. http://dx.doi.org/10.1152/ajpheart.00848.2004.
Full textAbstract:
Thyroid acting through ligand binding to nuclear receptors modifies myocardial respiratory kinetics and oxidative phosphorylation in the heart. Direct nongenomic action of thyroid hormone on high-energy phosphate concentrations and respiratory kinetics has never been proven in vivo but might be responsible for observed changes in oxygen utilization efficiency immediately after triiodothyronine (T3) administration. We tested the hypothesis that T3directly and rapidly modifies myocardial high-energy phosphate concentrations and phosphorylation potential in vivo. Anesthetized sheep (age 28–40 days) thyroidectomized shortly after birth (Thy) and euthyroid age-matched controls (Con) underwent median sternotomy and received T3infusion (0.8 μg/kg), followed by epinephrine infusion to increase myocardial oxygen consumption (MV̇o2).31P magnetic resonance spectra were monitored via a surface coil over the left ventricle. T3increased phosphocreatine (PCr)/ATP and decreased ADP in Thy animals without causing a change in MV̇o2. T3produced no changes in high-energy phosphates in Con animals. T3did not modify the PCr/ATP or ADP response to epinephrine and elevation in MV̇o2in either group. Cardiac mitochondria isolated from Thy and Con animals showed no change in respiratory rate or ADP/ATP exchange efficiency after T3incubation. T3infusion in a hypothyroid state decreases ADP concentration, thereby altering the equilibrium between phosphorylation potential and myocardial respiratory rate. These T3-induced effects are not due to changes in ADP/ATP exchange efficiency through action at the adenine nucleotide translocator but may be due to T3mediation of substrate utilization, confirmed in other models.