Rate Constants For Incorporation Research Articles

Simulating actin networks in synaptic spine heads using dynamical graph grammars.

There is a morphodynamic component to synaptic learning by which dendritic (postsynaptic) spine head size changes can strengthen or weaken the synaptic connection between two neurons, in response to signals from the axon of a presynaptic neuron. These morphological factors are in turn sculpted by the graph-like dynamics of the actin cytoskeleton. In this project, we seek to use Dynamical Graph Grammars (DGGs) [1,2] implemented within a computer algebra system to model how networks of actin filaments can dynamically grow or shrink and reshape the spine head. We designed and implemented several DGG sub-grammar mathematical models including actin network growth, isotropic/anisotropic filament forces, filament-membrane mechanical interaction, and Hessian Boltzmann sampling of random molecular displacements, to regulate the generation and deletion of graph objects. From first principles expressed in about a dozen DGG rules we simulate emergent biomechanics of a simplified network of actin polymers and its interaction with membrane, using very rough parameter estimates, all in two dimensions. We refined this model by incorporating rate constants used in previous models (e.g. [3,4,5]) in each sector, and we are currently recapitulating the previously observed sub-grammar behaviors.

Transient-state kinetic analysis of multi-nucleotide addition catalyzed by RNA polymerase I

RNA polymerases execute the first step in gene expression: transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multisubunit RNA polymerases (Pol I, Pol II, and Pol III). RNA polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell’s most energy consuming tasks. While a kinetic mechanism for nucleotide addition catalyzed by Pol I has been reported, it remains unclear to what degree different nucleotide sequences impact the incorporation rate constants. Furthermore, it is currently unknown if the previous investigation of a single-nucleotide incorporation was sensitive to the translocation step. Here, we show that Pol I exhibits considerable variability in both kmax and K1/2values using an in vitro multi-NTP incorporation assay measuring AMP and GMP incorporations. We found the first two observed nucleotide incorporations exhibited faster kmax-values (∼200 s−1) compared with the remaining seven positions (∼60 s−1). Additionally, the average K1/2 for ATP incorporation was found to be approximately threefold higher compared with GTP, suggesting Pol I has a tighter affinity for GTP compared with ATP. Our results demonstrate that Pol I exhibits significant variability in the observed rate constant describing each nucleotide incorporation. Understanding of the differences between the Pol enzymes will provide insight on the evolutionary pressures that led to their specialized roles. Therefore, the findings resulting from this work are critically important for comparisons with other polymerases across all domains of life.

HIV Reverse Transcriptase Pre-Steady-State Kinetic Analysis of Chain Terminators and Translocation Inhibitors Reveals Interactions between Magnesium and Nucleotide 3'-OH.

Deoxythymidine triphosphate analogues with various 3′ substituents in the sugar ring (−OH (dTTP)), −H, −N3, −NH2, −F, −O–CH3, no group (2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3′-OH but with 4′ additions (4′-C-methyl, 4′-C-ethyl) or sugar ring modifications (d-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg2+ with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants (kobs) compared to dTTP ranging from about 2-fold (3′-H, −N3, and d4TTP with high Mg2+) to >10-fold (3′-NH2 and 3′-F with low Mg2+), while 3′-O-CH3 dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg2+ and the 3′-OH, kobs using 5 μM dTTP and 0.5 mM Mg2+ was only modestly slower (1.6-fold) than with 6 mM Mg2+, while analogues with 3′ alterations incorporated 2.8–5.1-fold slower in 0.5 mM Mg2+. In contrast, 4′-C-methyl and d-carba dTTP, which retain the 3′-OH, were not significantly affected by Mg2+. Consistent with these results, analogues with 3′ modifications were better inhibitors in 6 versus 0.5 mM Mg2+. Equilibrium dissociation constant (KD) and maximum incorporation rate (kpol) determinations for dTTP and analogues lacking a 3′-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogues but did not significantly affect KD, results consistent with a role for 3′-OH/Mg2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg2+ and the 3′-OH of the incoming nucleotide and suggest that inhibitors with 3′-OH groups may have advantages in low free Mg2+ in physiological settings.

A method of incorporating rate constants as kinetic constraints in molecular dynamics simulations

From the point of view of statistical mechanics, a full characterization of a molecular system requires an accurate determination of its possible states, their populations, and the respective interconversion rates. Toward this goal, well-established methods increase the accuracy of molecular dynamics simulations by incorporating experimental information about states using structural restraints and about populations using thermodynamics restraints. However, it is still unclear how to include experimental knowledge about interconversion rates. Here, we introduce a method of imposing known rate constants as constraints in molecular dynamics simulations, which is based on a combination of the maximum-entropy and maximum-caliber principles. Starting from an existing ensemble of trajectories, obtained from either molecular dynamics or enhanced trajectory sampling, this method provides a minimally perturbed path distribution consistent with the kinetic constraints, as well as modified free energy and committor landscapes. We illustrate the application of the method to a series of model systems, including all-atom molecular simulations of protein folding. Our results show that by combining experimental rate constants and molecular dynamics simulations, this approach enables the determination of transition states, reaction mechanisms, and free energies. We anticipate that this method will extend the applicability of molecular simulations to kinetic studies in structural biology and that it will assist the development of force fields to reproduce kinetic and thermodynamic observables. Furthermore, this approach is generally applicable to a wide range of systems in biology, physics, chemistry, and material science.

(Invited) In-Situ Co-Doping in CVD Si1-xGex Epitaxial Growth Based on the Langmuir-Type Mechanism

High performance Si-based devices require atomically ordered interface of heterostructures and doping profiles as well as strain engineering due to the introduction of Ge and C into Si. Our interest is the generalization of atomic-order surface reaction processes for group IV semiconductors based on thermodynamical treatments [1-6]. In this work, in-site co-doping of dopant (P or B) and C in CVD Si, and Si1-xGex (100) epitaxial growth using SiH4-GeH4-PH3 or B2H6-SiH3CH3-H2-He gas mixtures [1- 7] is reviewed based on the Langmuir-type surface adsorption and reaction scheme.The adsorption rate constants of SiH4 and GeH4 and reaction rate constants of SiH4 at each site of Si-Si, Si-Ge and Ge-Ge on (100) surface in ref. 2 are used.For heavy doping of P, the adsorption and desorption rate constants of PH3, the incorporation rate constants of P, the segregation coefficient of P between the surface coverage of PH3 molecules and the concentration of P incorporated at each pair site, and growth rate constant of GeH4 at the site where PH3 molecules have been adsorbed are obtained from the experimental data, assuming that GeH4 molecules decomposes at P-occupied sites and P atoms of 3, 2, 1 atomic layer is formed self-limitedly on the Si-Si, Si-Ge, Ge-Ge sites by PH3, respectively [8].For heavy doping of B, the adsorption and desorption rate constants of B2H6, the incorporation rate constants of B at each pair site, and growth rate constants of SiH4 and GeH4 and B precipitation rate constant of B2H6 at the site where B2H6 molecules have been adsorbed are obtained from the experimental data, assuming that SiH4, GeH4 and B2H6 adsorb/react partially at the B-occupied sites [8].For heavy doping of C, the adsorption and desorption constants of SiH3CH3 and incorporation rate of C at each pair site are obtained from the experimental data [9], assuming that the sites where SiH3CH3 molecules have been adsorbed become inactive for both the SiH4 and GeH4 adsorption/reactions on the surface.For in-situ co-doping of P and C, the calculated growth rate, P concentration, Ge fraction and C concentration using the above fitting parameters are in good agreement with the experimental data [10] for the C concentration below 1021 cm-3. It is proposed that the decrease of growth rate and the increase of P concentration by SiH3CH3 addition for higher GeH4 and lower PH3 partial pressure is caused mainly by the increase of inactive site density for both the SiH4 and GeH4 due to the adsorption of SiH3CH3 at Ge-Ge pair sites.For in-situ co-doping of B and C, the calculated values of B concentration correspond to the experimental data [11] except for the samples grown at P GeH4=1.2 Pa and P SiH3CH3=0.09 Pa. For those samples (Ge fraction>0.44 and C concentration>8 x 1020 cm-3), it was suggested that the B and C atoms exist at interstitial site [8]. Therefore, it is considered that the incorporation rate constants for interstitial B are different from that for substitutional B. It should be noted that the calculated C concentration, which is almost independent of B concentration, results from C precipitation by reaction of SiH3CH3 gas at B-occupied sites.These results open the way for generalization of atomically controlled surface reaction process in group IV semiconductors by CVD.[1] J. Murota: The Electrochemical Society Extended s, Fall Meeting, Detroit, Oct.17-21, 1982, Abs.No.226, p.363.[2] H. Ishii, Y. Takahashi and J. Murota: Appl. Phys. Lett., 47, 863 (1985).[3] J. Murota and S. Ono: Jpn. J. Appl. Phys., 33, 2290 (1994).[4] B. Tillack, B. Heinemann, D. Knoll: Thin Solid Films, 369, 189 (2000).[5] J. Murota, M. Sakuraba and B. Tillack: Jpn. J. Appl. Phys., 45, 6767 (2006).[6] B. Tillack, Y. Yamamoto, D. Bolze, B. Heinemann, H. Rücker, D. Knoll, J. Murota and W. Mehr: Thin Solid Films, 508, 279 (2006).[7] J. Murota, Y. Yamamoto, I. Costina, B. Tillack, V. Le Thanh, R. Loo and M. Caymax: ECS J. Solid State Sci. Technol., 7(6), P305 (2018).[8] J. Murota: ECS Trans., 90(1), 43 (2019).[9] A. Ichikawa, Y. Hirose, T. Ikeda, T. Noda, M. Fujiu, T. Takatsuka, A. Moriya, M. Sakuraba, T. Matsuura and J. Murota: Thin Solid Films, 369(1-2), 167 (2000).[10] D. Lee, T. Noda, H. Shim, M. Sakuraba, T. Matsuura and J. Murota: Jpn. J. Appl. Phys., 40, 2697(2001).[11] T. Noda, D. Lee, H. Shim, M. Sakuraba, T. Matsuura and J. Murota: Thin Solid Films. 380, 57 (2000).

Dietary lysine deficiency increases the incorporation rate of nitrogen-free compounds in muscle of pacu (Piaractus mesopotamicus)

Isotopic experiments are conducted to evaluate turnover rates in fish tissue, especially as a function of nutrition. In 2007, Cerling et al. proposed an approach they named the reaction progress variable, which among other functions, allows to correct for growth during the period of data collection and to treat the isotopic incorporation system as a linear function, irrespective of the growth rate model. The objective of present study was to apply “the reaction progress variable” approach to describe stable isotope turnover in muscle tissue of pacu fed a lysine-deficient and a lysine-supplemented diet in order to determine the incorporation rates of nitrogen-free compounds, considering the correction for increases in mass during turnover. Since in muscle tissue carbon compounds are proteins, nucleic acids, lipids and carbohydrates and nitrogen compounds include only proteins and nucleic acids, the incorporation rates of nitrogen-free compounds were calculated by the difference between the incorporation rates of carbon and nitrogen compounds. The δ13C and δ15N values were adjusted for the increase in mass of fish to determine incorporation rate constants through linear fit. High turnover rates were observed when the increase in mass was considered. There was no effect of the diets on the incorporation rates of carbon compounds into muscle tissue of juvenile pacu. Fish fed the lysine-deficient diet exhibited a higher metabolic δ15N turnover rate, probably because of the greater degradation of nitrogen compounds; consequently, the incorporation rate of nitrogen-free compounds was 1.23% day−1. A higher incorporation rate of nitrogen compounds was observed in fish fed the lysine-supplemented diet and the incorporation rate of nitrogen-free compounds was therefore lower in this group (0.05% day−1). The incorporation rate of nitrogen-free compounds (e.g., lipids and glycogen) into muscle tissue was higher in juvenile pacu fed the lysine-deficient diet compared to animals receiving the lysine-supplemented diet. We therefore propose to adopt the reaction progress variable approach as a more useful tool for describing isotopic incorporation datasets.

A Population Pharmacokinetic Analysis of Dextroamphetamine in the Plasma and Hair of Healthy Adults.

Hair is an attractive matrix for amphetamine drug testing; however, little is known about the rate at which amphetamines are deposited into hair. Therefore, the purpose of this study was to determine the pharmacokinetics of oral dextroamphetamine in plasma and quantify the rate of deposition into hair in healthy adults using a linked population pharmacokinetic model. Healthy adults >18 years of age received dextroamphetamine 10 mg orally for 7 days. Plasma samples were collected over 48 h following the final dose, and hair was collected 5 weeks following the first dose. NONMEM 7.2 was used to estimate dextroamphetamine oral absorption rate constant, apparent clearance and volume of distribution of the plasma compartment, the plasma to hair incorporation rate constant, and the apparent volume of distribution in the hair compartment. Dextroamphetamine pharmacokinetics were well-described by a one-compartment model with combined additive and proportional error for the plasma compartment, which was linked to a single compartment for the hair. Apparent clearance and volume of distribution in the plasma compartment were scaled by current body weight (centered on the mean). Melanin hair concentration was included as a significant covariate on the hair compartment. Absorption rate constant, clearance, and volume of distribution for the plasma compartment were estimated as 0.527 h(-1) (95% CI 0.467-0.586), 28.7 L/h (95% CI 27.1-30.3), and 377 L (95% CI 326-428), respectively. The incorporation rate constant from plasma to hair was 1.60e(-6) h(-1) (95% CI 1.06e(-6)-2.14e(-6)) and apparent volume of distribution in hair was 17.7 mg (95% CI 12.5-22.8). A one-compartment plasma model linked to a single compartment for hair successfully described the pharmacokinetics of dextroamphetamine in healthy adults. The volume of distribution and clearance of dextroamphetamine increased with weight, and the volume of distribution of the hair compartment increased with greater melanin concentrations.

Significant contribution of the 3'→5' exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ.

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.

Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation.

The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis. The flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

Patterns of extracellular enzyme activities and microbial metabolism in an Arctic fjord of Svalbard and in the northern Gulf of Mexico: contrasts in carbon processing by pelagic microbial communities

The microbial community composition of polar and temperate ocean waters differs substantially, but the potential functional consequences of these differences are largely unexplored. We measured bacterial production, glucose metabolism, and the abilities of microbial communities to hydrolyze a range of polysaccharides in an Arctic fjord of Svalbard (Smeerenburg Fjord), and thus to initiate remineralization of high-molecular weight organic matter. We compared these data with similar measurements previously carried out in the northern Gulf of Mexico in order to investigate whether differences in the spectrum of enzyme activities measurable in Arctic and temperate environments are reflected in “downstream” aspects of microbial metabolism (metabolism of monomers and biomass production). Only four of six polysaccharide substrates were hydrolyzed in Smeerenburg Fjord; all were hydrolyzed in the upper water column of the Gulf. These patterns are consistent on an interannual basis. Bacterial protein production was comparable at both locations, but the pathways of glucose utilization differed. Glucose incorporation rate constants were comparatively higher in Svalbard, but glucose respiration rate constants were higher in surface waters of the Gulf. As a result, at the time of sampling ca. 75% of the glucose was incorporated into biomass in Svalbard, but in the northern Gulf of Mexico most of the glucose was respired to CO2. A limited range of enzyme activities is therefore not a sign of a dormant community or one unable to further process substrates resulting from extracellular enzymatic hydrolysis. The ultimate fate of carbohydrates in marine waters, however, is strongly dependent upon the specific capabilities of heterotrophic microbial communities in these disparate environments.

Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.

Fluorescence-Based Assay to Measure the Real-time Kinetics of Nucleotide Incorporation during Transcription Elongation

Understanding the mechanism and fidelity of transcription by the RNA polymerase (RNAP) requires measurement of the dissociation constant (Kd) of correct and incorrect NTPs and their incorporation rate constants (kpol). Currently, such parameters are obtained from radiometric-based assays that are both tedious and discontinuous. Here, we report a fluorescence-based assay for measuring the real-time kinetics of single-nucleotide incorporation during transcription elongation. The fluorescent adenine analogue 2-aminopurine was incorporated at various single positions in the template or the nontemplate strand of the promoter-free elongation substrate. On addition of the correct NTP to the T7 RNAP-DNA, 2-aminopurine fluorescence increased rapidly and exponentially with a rate constant similar to the RNA extension rate obtained from the radiometric assay. The fluorescence stopped-flow assay, therefore, provides a high-throughput way to measure the kinetic parameters of RNA synthesis. Using this assay, we report the kpol and Kd of all four correct NTP additions by T7 RNAP, which showed a range of values of 145–190 s−1 and 28–124 μM, respectively. The fluorescent elongation substrates were used to determine the misincorporation kinetics as well, which showed that T7 RNAP discriminates against incorrect NTP both at the nucleotide binding and incorporation steps. The fluorescence-based assay should be generally applicable to all DNA-dependent RNAPs, as they use similar elongation substrates. It can be used to elucidate the mechanism, fidelity, and sequence dependency of transcription and is a rapid means to screen for inhibitors of RNAPs for therapeutic purposes.

Selective Area Metal–Organic Vapor Phase Epitaxy of Nitride Semiconductors for Multicolor Emission

Selective area metal-organic vapor phase epitaxy (SA-MOVPE) allows in-plane control of emission wavelength by tailored width of masks. For InGaN/GaN multiple quantum wells (MQWs), modulation of luminescence wavelength was achieved based on a balance between vapor-phase diffusion of group-III precursors and their surface incorporation. For the basic understanding of the SA-MOVPE of nitride semiconductors, thickness profiles of GaN, InN, AlN, and InGaN layers around relatively wide (>10 mum) masks were investigated. The effective lateral diffusion length D/k <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">s</sub> , which is the ratio of the vapor-phase mass diffusivity of a precursor to its surface incorporation rate constant, was extracted for GaN and InN. The value was much larger for InN due to smaller surface incorporation rate. In the SA-MOVPE of InGaN bulk layer at around 800degC, indium incorporation rate seems to be limited by the surface flux of a gallium precursor, resulting in no variation in the indium content. Varied width of the InGaN wells by the existence of masks seems to govern the shift in the luminescence wavelength from InGaN/GaN MQWs. Therefore, design of the thickness distribution of GaN based on the quantitative model is essential to the controlled in-plane color modulation of solid-state lighting devices using SA-MOVPE.

PLD-deposited (BaxSr1-x)(CoyFe1-y)O3-δ Thin-Film Microelectrodes: Structure Aspects and Oxygen Incorporation Kinetics

Among the mixed conducting perovskites, (BaxSr1-x)(CoyFe1-y)O3-d (BSCF) attracts special attention due to its exceptionally high effective oxygen incorporation rate constant. The structure of the dense BSCF thin films prepared by pulsed laser deposition (PLD) was investigated. Long-term stability of the cubic perovskite phase for different cation compositions at intermediate temperatures was evaluated. The surface resistance Rs associated with the oxygen incorporation reaction for different cation compositions was determined from impedance spectroscopy measurements on dense thin-film microelectrodes without interference from electrode morphology. The correlation between some bulk properties and Rs was studied.

Kinetic analysis of surface adsorption layer for InGaAsP-related binary materials using in situ RAS

Kinetic analysis of the surface adsorption layer on GaAs, InAs, InP, and GaP surfaces during metalorganic vapor phase epitaxy (MOVPE) was performed by in situ monitoring of the temporal behavior of surface reconstructions using reflectance anisotropy spectroscopy (RAS). When the flow of group-III sources was switched on/off, the transient RA signal exhibited two time constants t 0 and t 1 in response to the generation/extinction of the adsorption layer. It was considered that t 0, when the supply of group-III sources was started, corresponds to the adsorption of group-III atoms from the gas phase to the adsorption layer, and that t 1, when the supply of group-III sources was terminated, is associated with the incorporation of adsorbed group-III atoms to the crystal. The rate constant of adsorption, 1/ t 0, was approximately 2 s −1 for all surfaces except for GaP. The rate constant of incorporation, 1/ t 1, ranged from 0.1 to 10 s −1. It was found that 1/ t 1 for the As-containing materials was faster than that for P-related materials, and 1/ t 1 for In-containing materials was faster than that for the Ga-containing materials. For the As-containing materials, 1/ t 1 agreed with the incorporation rate constants of adsorbed group-III precursors obtained using the Langmuir–Hinshelwood model by the analysis of selective-area growth (SAG).

Kinetic Effect of a Downstream Strand and Its 5′-Terminal Moieties on Single Nucleotide Gap-filling Synthesis Catalyzed by Human DNA Polymerase λ

During short-patch base excision repair, the excision of a 5'-terminal 2-deoxyribose-5-phosphate moiety of the downstream strand by the 5'-2-deoxyribose-5-phosphate lyase activity of either DNA polymerase beta or lambda is believed to occur after each respective enzyme catalyzes gap-filling DNA synthesis. Yet the effects of this 5'-terminal 2-deoxyribose-5-phosphate moiety on the polymerase activities of these two enzymes have never been quantitatively determined. Moreover, x-ray crystal structures of truncated polymerase lambda have revealed that the downstream strand and its 5'-phosphate group of gapped DNA interact intensely with the dRPase domain, but the kinetic effect of these interactions is unclear. Here, we utilized pre-steady state kinetic methods to systematically investigate the effect of a downstream strand and its 5'-moieties on the polymerase activity of the full-length human polymerase lambda. The downstream strand and its 5'-phosphate were both found to increase nucleotide incorporation efficiency (kp/Kd) by 15 and 11-fold, respectively, with the increase procured by the effect on the nucleotide incorporation rate constant kp rather than the ground state nucleotide binding affinity Kd. With 4 single nucleotide-gapped DNA substrates containing a 1,2-dideoxyribose-5-phosphate moiety, a 2-deoxyribose-5-phosphate mimic, we measured the incorporation efficiencies of 16 possible nucleotides. Our results demonstrate that although this 5'-terminal 2-deoxyribose-5-phosphate mimic does not affect the fidelity of polymerase lambda, it moderately decreased the polymerase efficiency by 3.4-fold. Moreover, this decrease in polymerase efficiency is due to a drop of similar magnitude in kp rather than Kd. The implication of the downstream strand and its 5'-moieties on the kinetics of gap-filling synthesis is discussed.

Up-regulation of the Fidelity of Human DNA Polymerase λ by Its Non-enzymatic Proline-rich Domain

DNA repair pathways are essential for maintaining genome stability. DNA polymerase beta plays a critical role in base-excision repair in vivo. DNA polymerase lambda, a recently identified X-family homolog of DNA polymerase beta, is hypothesized to be a second polymerase involved in base-excision repair. The full-length DNA polymerase lambda is comprised of three domains: a C-terminal DNA polymerase beta-like domain, an N-terminal BRCA1 C-terminal domain, and a previously uncharacterized proline-rich domain. Strikingly, pre-steady-state kinetic analyses reveal that, although human DNA polymerase lambda has almost identical fidelity to human DNA polymerase beta, the C-terminal DNA polymerase beta-like domain alone displays a dramatic, up to 100-fold loss in fidelity. We further demonstrate that the non-enzymatic proline-rich domain confers the increase in fidelity of DNA polymerase lambda by significantly lowering incorporation rate constants of incorrect nucleotides. Our studies illustrate a novel mechanism, in which the DNA polymerase fidelity is controlled not by an accessory protein or a proofreading exonuclease domain but by an internal regulatory domain.

Pre-steady-state Kinetic Characterization of the DinB Homologue DNA Polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Dispersion, temperature and torque models for an internal mixer

AbstractModels based on kinetic, thermodynamic and rheological equations have been developed to compute dispersion extent, batch temperature and relative batch viscosity at discrete intervals during a mix cycle in an internal mixer. The calculated relative bulk viscosity can be linked empirically to a measured variable such as rotor torque or power consumption. Solution of the models over successive time intervals allows the computation of complete dispersion, temperature, and torque/power profiles for a mix cycle. The mix models exhibit good fits to experimental torque and temperature curves for mixing natural rubber with carbon black over a wide range of carbon black loadings (0 to 50 phr) for rotor speeds ranging from 40 to 70 RPM in two lab‐scale internal mixers. The models are shown to be suitable for moderately reinforcing (ASTM N660) as well as highly reinforcing (ASTM N121) grades of carbon black. Rate constants for filler dispersion, incorporation and erosion can be extracted from the models and can be interpolated to generate dispersion, temperature and torque curves at new conditions of carbon black loading and rotor speed. The mix models could thus be used for process‐control purposes and for specifying drop targets based on batch properties in addition to the time‐temperature‐energy criteria typically used today. Polym. Eng. Sci. 44:1247–1257, 2004. © 2004 Society of Plastics Engineers.

Kinetics of the reactions of divalent copper, zinc, cobalt, and nickel with a deformed water soluble centrally monoprotic porphyrin

The kinetics of incorporation of Cu 2+, Zn 2+, Co 2+, and Ni 2+ into the free base H 2-BrP(4) 4+ and centrally monoprotic H-BrP(4) 3+ forms of the deformed and water soluble beta-octabromo-tetrakis( N-methyl-4-pyridyl)porphyrin were studied at 25 °C, I=0.10 (NaNO 3/HNO 3). Below pH 7, the reactions were first-order in porphyrin and metal, and the rate constants increased with an increase in pH. The monocation H 3-BrP(4) 5+ did not incorporate metal ions, and H-BrP(4) 3+ reacted several hundred times faster than H 2-BrP(4) 4+. The incorporation rate constants for the pre-deformed H 2-BrP(4) 4+ were ∼10 3 times larger as compared to the more planar tetrakis( N-methyl-4-pyridyl)porphyrin. For both porphyrins, the reactivity order was Cu 2+>Zn 2+>Co 2+>Ni 2+. While Cu 2+ reacted ∼10 4 times more rapidly than did Ni 2+, the ratios of the metalation rate constants of H-BrP(4) 3+ to the water exchange rate constants of the metal ions varied by no more than a factor of 4 across the series.