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Menten Constant (menten + constant)
Selected AbstractsElectrochemical Cholesterol Sensor Based on Tin Oxide-Chitosan Nanobiocomposite FilmELECTROANALYSIS, Issue 8 2009Anees Abstract A chitosan (CS)-tin oxide (SnO2) nanobiocomposite film has been deposited onto an indium-tin-oxide glass plate to immobilize cholesterol oxidase (ChOx) for cholesterol detection. The value of the Michaelis,Menten constant (Km) obtained as 3.8,mM for ChOx/CS-SnO2/ITO is lower (8,mM) than that of a ChOx/CS/ITO bioelectrode revealing enhancement in affinity and/or activity of ChOx towards cholesterol and also revealing strong binding of ChOx onto CS-SnO2/ITO electrode. This ChOx/CS-SnO2/ITO cholesterol sensor retains 95% of enzyme activity after 4,6 weeks at 4,°C with response time of 5,s, sensitivity of 34.7,,A/mg dL,1 cm2 and detection limit of 5,mg/dL. [source] Electrostatic Assembly of a Redox Catalysis System for Detection of GlutamateELECTROANALYSIS, Issue 24 2006Alice Abstract Interfacial assemblies capable of determining glutamate by redox catalysis are prepared by electrostatic assembly of alternating layers of ferrocene poly(allylamine) polymer and glutamate oxidase on a gold electrode. Deposition of the polymer was confirmed in cyclic voltammetry measurements by the presence of a surface wave corresponding to the oxidation of the ferrocene group. In the presence of glutamate in the adjacent electrolyte solution, the current increases and approaches a pseudosteady state, consistent with redox catalysis. Electrodes modified with glutamate oxidase had a linear response to glutamate up to 0.0045,M with sensitivity of 20,,A/cm2 and a limit of detection of 31.4,,M glutamate. An apparent Michaelis,Menten constant of 0.40(±0.13),mM for the confined glutamate oxidase was determined for this assembly. When used in flow-injection experiments, glucose oxidase modified electrodes responded to transient zones of glucose; however, the detection limits of the assemblies to the flowing stream were substantially higher than found for measurements on static solutions. [source] Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme,zymogen complexFEBS JOURNAL, Issue 1 2005Matilde Esther Fuentes A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme,zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis,Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time,concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin. [source] Monoamine oxidase activity in kidney and heart of Piaractus mesopotamicus (Holmberg)JOURNAL OF FISH BIOLOGY, Issue 6 2007C. M. C. Salles The values of Michaelis,Menten constant (KM) and maximum velocity (VMAX) for kidney and heart monoamine oxidase (MAO) from pacu Piaractus mesopotamicus were determined. The mean ±s.e. KM values were 17·28 ± 2·27 ,M for kidney and 15·38 ± 1·86 ,M for heart. MAO activities were 111·60 ± 3·25 and 15·12 ± 0·30 nmols min,1 g,1 of wet tissue for kidney and heart, respectively. In addition, MAO inhibitory studies in these two tissues indicate that this enzyme may be a different isoform of MAO. [source] Transport characteristics of N -acetyl- l -aspartate in rat astrocytes: involvement of sodium-coupled high-affinity carboxylate transporter NaC3/NaDC3-mediated transport systemJOURNAL OF NEUROCHEMISTRY, Issue 3 2005Takuya Fujita Abstract We investigated in the present study the transport characteristics of N -acetyl- l -aspartate in primary cultures of astrocytes from rat cerebral cortex and the involvement of NA+ -coupled high-affinity carboxylate transporter NaC3 (formerly known as NaDC3) responsible for N -acetyl- l -aspartate transport. N -acetyl- l -aspartate transport was NA+ -dependent and saturable with a Michaelis,Menten constant (Km) of ,110 µm. NA+ -activation kinetics revealed that the NA+ to- N -acetyl- l -aspartate stoichiometry was 3 : 1 and concentration of Na+ necessary for half-maximal transport (KNAm) was 70 mm. NA+ -dependent N -acetyl- l -aspartate transport was competitively inhibited by succinate with an inhibitory constant (Ki) of 14.7 µm, which was comparable to the Km value of NA+ -dependent succinate transport (29.4 µm). l -Aspartate also inhibited NA+ -dependent [14C]N -acetyl- l -aspartate transport with relatively low affinity (Ki = 2.2 mm), whereas N -acetyl- l -aspartate was not able to inhibit NA+ -dependent aspartate transport in astrocytes. In addition, Li+ was found to have a significant inhibitory effect on the NA+ -dependent N -acetyl- l -aspartate transport in a concentration-dependent manner. Furthermore, RT,PCR and western blot analyses revealed that NaC3 is expressed in primary cultures of astrocytes. Taken collectively, these results indicate that NaC3 expressed in rat cerebrocortical astrocytes is responsible for NA+ -dependent N -acetyl- l -aspartate transport. This transporter is likely to be an essential prerequisite for the metabolic role of N -acetyl- l -aspartate in the process of myelination. [source] On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar,von Caemmerer,Berry leaf photosynthesis modelPLANT CELL & ENVIRONMENT, Issue 2 2004G. J. ETHIER ABSTRACT Virtually all current estimates of the maximum carboxylation rate (Vcmax) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the maximum electron transport rate (Jmax) for C3 species implicitly assume an infinite CO2 transfer conductance (gi). And yet, most measurements in perennial plant species or in ageing or stressed leaves show that gi imposes a significant limitation on photosynthesis. Herein, we demonstrate that many current parameterizations of the photosynthesis model of Farquhar, von Caemmerer & Berry (Planta 149, 78,90, 1980) based on the leaf intercellular CO2 concentration (Ci) are incorrect for leaves where gi limits photosynthesis. We show how conventional A,Ci curve (net CO2 assimilation rate of a leaf ,An, as a function of Ci) fitting methods which rely on a rectangular hyperbola model under the assumption of infinite gi can significantly underestimate Vcmax for such leaves. Alternative parameterizations of the conventional method based on a single, apparent Michaelis,Menten constant for CO2 evaluated at Ci[Km(CO2)i] used for all C3 plants are also not acceptable since the relationship between Vcmax and gi is not conserved among species. We present an alternative A,Ci curve fitting method that accounts for gi through a non-rectangular hyperbola version of the model of Farquhar et al. (1980). Simulated and real examples are used to demonstrate how this new approach eliminates the errors of the conventional A,Ci curve fitting method and provides Vcmax estimates that are virtually insensitive to gi. Finally, we show how the new A,Ci curve fitting method can be used to estimate the value of the kinetic constants of Rubisco in vivo is presented [source] Real-Time Monitoring of Mass-Transport-Related Enzymatic Reaction Kinetics in a Nanochannel-Array ReactorCHEMISTRY - A EUROPEAN JOURNAL, Issue 33 2010Su-Juan Li Abstract To understand the fundamentals of enzymatic reactions confined in micro-/nanosystems, the construction of a small enzyme reactor coupled with an integrated real-time detection system for monitoring the kinetic information is a significant challenge. Nano-enzyme array reactors were fabricated by covalently linking enzymes to the inner channels of a porous anodic alumina (PAA) membrane. The mechanical stability of this nanodevice enables us to integrate an electrochemical detector for the real-time monitoring of the formation of the enzyme reaction product by sputtering a thin Pt film on one side of the PAA membrane. Because the enzymatic reaction is confined in a limited nanospace, the mass transport of the substrate would influence the reaction kinetics considerably. Therefore, the oxidation of glucose by dissolved oxygen catalyzed by immobilized glucose oxidase was used as a model to investigate the mass-transport-related enzymatic reaction kinetics in confined nanospaces. The activity and stability of the enzyme immobilized in the nanochannels was enhanced. In this nano-enzyme reactor, the enzymatic reaction was controlled by mass transport if the flux was low. With an increase in the flux (e.g., >50,,L,min,1), the enzymatic reaction kinetics became the rate-determining step. This change resulted in the decrease in the conversion efficiency of the nano-enzyme reactor and the apparent Michaelis,Menten constant with an increase in substrate flux. This nanodevice integrated with an electrochemical detector could help to understand the fundamentals of enzymatic reactions confined in nanospaces and provide a platform for the design of highly efficient enzyme reactors. In addition, we believe that such nanodevices will find widespread applications in biosensing, drug screening, and biochemical synthesis. [source] Methane oxidation kinetics differ in European beech and Norway spruce soilsEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 4 2009D. M. Degelmann Summary Coniferous forest soils often consume less of the greenhouse gas methane (CH4) than deciduous forest soils. The reasons for this phenomenon have not been resolved. It might be caused by differences in the diffusive flux of CH4 through the organic layer, pH or different concentrations of potentially inhibitory compounds. Soil samples were investigated from three adjacent European beech (Fagus sylvatica) and Norway spruce (Picea abies) stands in Germany. Maximal CH4 oxidation velocities (Vmax(app)) and Michaelis Menten constants (KM(app)), retrieved from intact soil cores at constant CH4 concentrations, temperature and matric potential, were twice as great in beech as in spruce soils. Also atmospheric CH4 oxidation rates measured in homogenized soil samples displayed the same trend. Greatest atmospheric CH4 oxidation rates were detected in the Oa horizon or in the upper 5 cm of the mineral soil. In contrast to the beech soils, the Oa horizon of the spruce soils consumed no CH4. A differential effect due to divergent diffusive flux through the litter layer was not found. pH and ammonium concentration were similar in samples from both forest soil types. Ethylene accumulation in all soils was negligible under oxic conditions. These collective results suggest that the different atmospheric CH4 uptake by beech and spruce soils is caused by different CH4 oxidizing capacities of methanotrophic communities in the Oa horizon and top mineral soil. [source] The effect of temperature on C4 -type leaf photosynthesis parametersPLANT CELL & ENVIRONMENT, Issue 9 2007RAIA-SILVIA MASSAD ABSTRACT C4 -type photosynthesis is known to vary with growth and measurement temperatures. In an attempt to quantify its variability with measurement temperature, the photosynthetic parameters , the maximum catalytic rate of the enzyme ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) (Vcmax), the maximum catalytic rate of the enzyme phosphoenolpyruvate carboxylase (PEPC) (Vpmax) and the maximum electron transport rate (Jmax) , were examined. Maize plants were grown in climatic-controlled phytotrons, and the curves of net photosynthesis (An) versus intercellular air space CO2 concentrations (Ci), and An versus photosynthetic photon flux density (PPFD) were determined over a temperature range of 15,40 °C. Values of Vcmax, Vpmax and Jmax were computed by inversion of the von Caemmerer & Furbank photosynthesis model. Values of Vpmax and Jmax obtained at 25 °C conform to values found in the literature. Parameters for an Arrhenius equation that best fits the calculated values of Vcmax, Vpmax and Jmax are then proposed. These parameters should be further tested with C4 plants for validation. Other model key parameters such as the mesophyll cell conductance to CO2 (gi), the bundle sheath cells conductance to CO2 (gbs) and Michaelis,Menten constants for CO2 and O2 (Kc, Kp and Ko) also vary with temperature and should be better parameterized. [source] The 1.35,Å resolution structure of the phosphatase domain of the suppressor of T-cell receptor signaling protein in complex with sulfateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Jean Jakoncic The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis,Menten constants at acidic pH. [source] Cloning of deoxynucleoside monophosphate kinase genes and biosynthesis of deoxynucleoside diphosphatesBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Jie Bao Abstract The genes encoding four deoxynucleoside monophosphate kinase (dNMP kinase) enzymes, including ADK1 for deoxyadenylate monophosphate kinase (AK), GUK1 for deoxyguanylate monophosphate kinase (GK), URA6 for deoxycytidylate monophosphate kinase (CK), and CDC8 for deoxythymidylate monophosphate kinase (TK), were isolated from the genome of Saccharomyces cerevisiae ATCC 2610 strain and cloned into E. coli strain BL21(DE3). Four recombinant plasmids, pET17b-JB1 containing ADK1, pET17b-JB2 containing GUK1, pET17b-JB3 containing URA6, and pET17b-JB4 containing CDC8, were constructed and transformed into E. coli strain for over-expression of AK, GK, CK, and TK. The amino acid sequences of these enzymes were analyzed and a putative conserved peptide sequence for the ATP active site was proposed. The four deoxynucleoside diphosphates (dNDP) including deoxyadenosine diphosphate (dADP), deoxyguanosine diphosphate (dGDP), deoxycytidine diphosphate (dCDP), and deoxythymidine diphosphate (dTDP), were synthesized from the corresponding deoxynucleoside monophosphates (dNMP) using the purified AK, GK, CK, and TK, respectively. The effects of pH and magnesium ion concentration on the dNDP biosynthesis were found to be important. A kinetic model for the synthetic reactions of dNDP was developed based on the Bi,Bi random rapid equilibrium mechanism. The kinetic parameters including the maximum reaction velocity and Michaelis,Menten constants were experimentally determined. The study on dNDP biosynthesis reported in this article are important to the proposed bioprocess for production of deoxynucleoside triphosphates (dNTP) that are used as precursors for in vitro DNA synthesis. There is a significant advantage of using enzymatic biosyntheses of dNDP as compared to the chemical method that has been in commercial use. © 2005 Wiley Periodicals, inc. [source] | ||||||||||||||||