A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log λP value of the solvent and the activation free energy for denaturation ( ΔG d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log λP ≤-2.3 have protective effects, whereas solvents with log λP ≥-1.8 are deleterious for penicillin acylase.     

Kinetic Characterization of Two Phosphate Uptake Systems with Different Affinities in Suspension-Cultured Catharanthus roseus Protoplasts

We studied the kinetics of inorganic phosphate (P₁) uptake from0.1–1,000 µM P₁ by protoplasts from suspension-culturedcells of Catharanthus roseus (L.) G. Don. Concentration dependenceof [³²P]P₁ uptake revealed two kinetically different uptakesystems, a high-affinity system and a low-affinity system, withK_m values of 3.0 and 47 µM, respectively. Protoplastsfrom cells grown in P_i-rich media had a medium level of thelow-affinity activity and a very low level of the high-affinityactivity. It appeared low-affinity system is expressed constitutively,while the high-affinity system is regulated by the availabilityof P_i. When cells grown in a P_i-rich media were transferredto P_i-depleted media, the high-affinity activity increased significantlyafter 2 d, but the low-affinity activity was barely changed.Upon addition of 10 mM P_i, the high level of the high-affinityactivity fell to almost undetectable level in 1d. Both uptakesystems exhibited maximum activity between pH 5 and 6. ¹ Present address: Tokyo Research Laboratories, Kyowa HakkoKogyo Co., Ltd., 3-6-6 Asahi-cho, Machida, Tokyo, 194 Japan.     

Glycosylation of Aromatic Amines I: Characterization of Reaction Products and Kinetic Scheme

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D ¹H–¹H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of α and β glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1–6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.     

Kinetic Behavior of Native Alkaline Phosphatase from Human Placenta

The kinetic behavior of human placental alkaline phosphatase, which catalyses the hydrolysis of p-nitrophenyl and of o-carboxyphenyl phosphates, was studied by means of graphical and non-linear regression statistical fitting analysis of data of rate versus substrate concentration. Non linear Lineweaver-Burk and Eadie-Hofstee plots and rational functions of degree 2:2 (F-test assessing the goodness of fit) show non-Michaelian kinetic behavior. In the same way, the behavior of the enzyme was also non-Michaelian in the simultaneous presence of these two substrates.

Norlaudanosoline is a key intermediate in the biosynthesis of the benzylisoquinoline alkaloids providing the benzyl-isoquinoline portion of the morphinan skeleton. This study examines a coupled reaction system for the production of norlaudanosoline from dopamine. In this coupled system, dopamine is enzymatically converted by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (dopaldehyde). In the presence of dopamine, this aldehyde undergoes a spontaneous Pictet-Spengler condensation to form norlaudanosoline. Three potential sources of MAO were investigated: a fungal source (Aspergillus niger), a bacterial source (Sarcina lutea) and a commercial source isolated from bovine plasma. Kinetic studies with dopamine as the substrate gave Michaelis constants (K_m) of 1.81 × 10^-5 M, 6.94 × 10^-3, and 1.61 × 10^-3 M for A. niger, S. lutea and bovine plasma oxidase, respectively. The reaction system is complicated because of the effect of the condensation reaction, so a more rigorous model was developed to account for this effect. The model was suitable for showing the effect of dopamine concentration on norlaudanosoline production alghough there were some model inadequacies. Using the model a forward rate constant for the Pictet-Spengler condensation was determined to be 6.8 × 10^-2 M^-1 s^-1 and the reverse reaction appears to be negligible. Overall conversion was 14% which is 20 times that achieved in an in situ reaction system using whole cells of Aspergillus niger.     

Selection and Characterization of Phage Miniantibodies to Actins of Different Origin

Single-chain miniantibodies (scFv’s) to actin were obtained by the phage display method. A naive combinatorial phage display library of murine scFv’s (containing 2·10⁸ independent recombinant clones) was used to select miniantibodies. After three rounds of selection two clones producing miniantibodies to chicken smooth muscle actin with affinity constants of 1.4·10⁷ and 1.2·10⁶ M⁻¹ were chosen. The isolated miniantibodies could specifically detect various plant and animal actins.__________Translated from Biokhimiya, Vol. 70, No. 8, 2005, pp. 1070–1077.Original Russian Text Copyright © 2005 by Kostesha, Laman, Shepelyakovskaya, Zaitseva, Orlov, Dykman, Brovko, Sokolov.     

Application of Kinetic and Dynamic Data for Antihistamines to Risk Characterization in Sensitive Populations

A major goal of risk assessment is to protect the health of individuals who may be more sensitive than the general population. This study compared human phar-macokinetic and pharmacodynamic data in sensitive groups (i.e., children, the elderly, diseased states, and poor metabolizers) versus young, healthy adults for the antihistamines cetirizine, fexofenadine, loratadine, azelastine, ebastine, chlorpheniramine, and diphenhydramine. The default components (3.16 each for kinetic and dynamic aspects) of the intraspecies uncertainty factor were adjusted with compound specific data for the antihistamines. The majority (16 of 18) of the composite factors (kinetics X dynamics) for the sensitive groups were less than 10. Children had the lowest composite factors for antihistamines, ranging from 1.1 to 6.3. Application of kinetic and dynamic data for antihistamines to the Renwick/International Programme on Chemical Safety (IPCS) scheme can aid in characterizing the extent of variability in sensitive populations, thereby reducing the uncertainty associated with the risk assessment of sensitive populations.     

Kinetic Modelling and Half-Life Study on Bioremediation of Soil Co-Contaminated with Lubricating Motor Oil and Lead Using Different Bioremediation Strategies

The focus of this study was to investigate the effect of nutrient supplement (urea fertilizer) and microbial species augmentation (mixed culture of Aeromonas, Micrococcus, and Serratia sp.) on biodegradation of lubricating motor oil (LMO) and lead uptake by the autochthonous microorganism in LMO and lead-impacted soil were investigated. The potential inhibitory effects of lead on hydrocarbon utilization were investigated over a wide range of lead concentrations (25–200 mg/kg) owing to the complex co-contamination problem frequently encountered in most sites. Under aerobic conditions, total petroleum hydrocarbons (TPH) removal was 45.3% in the natural attenuation microcosm while a maximum of 72% and 68.2% TPH removal was obtained in biostimulation and bioaugmentation microcosms, respectively. Lead addition, as lead nitrate, to soil samples reduced the number of hydrocarbon degraders in all samples by a wide range (11–52%) depending on concentration and similarly, the metabolic activities were affected as observed in mineralization of LMO (3–60%) in soils amended with various lead concentrations. Moreover, the uptake of lead by the autochthonous microorganisms in the soil reduced with increase in the initial lead concentration. First-order kinetics described the biodegradation of LMO very well. The biodegradation rate constants were 0.015, 0.033, and 0.030 day^?1 for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The presence of varying initial lead concentration reduced the biodegradation rate constant of LMO degradation in the biostimulation treatment microcosm. Half-life times were 46.2, 21, and 23 days for LMO degradation in natural attenuation, biostimulation and bioaugmentation treatment microcosms, respectively. The half-life time in the biostimulation treatment microcosm was increased with a range between 10.7 and 39.2 days by the presence of different initial lead concentration. The results have promising potential for effective remediation of soils co-contaminated with hydrocarbons and heavy metals.     

Kinetic model of lysozyme renaturation with the molecular chaperone GroEL

From the renaturation kinetics of denatured/reduced lysozyme assisted by the molecular chaperone GroEL, a simplified kinetic model was established based on the competition between protein folding and aggregation. In the presence of GroEL and ATP, the aggregate formation was a second order reaction. With 2 mM ATP, a renaturation yield of 90% at a high renaturation rate was obtained when the molar ratio of GroEL to lysozyme was 1:1.     

Kinetic Extractions of Nickel and Lead from Some Contaminated Calcareous Soils

The mobility of heavy metals in contaminated soils is dependent on the kinetics release from soils. Metal extraction over time is commonly used to distinguish two or more fractions of metal based on differences of extraction or release rates. Kinetic studies using 0.01 M CaCl₂, 0.01 M malic acid, and 0.01 M EDTA extractions were performed to characterize nickel (Ni) and lead (Pb) kinetic release in 10 contaminated calcareous soils. Proportions of Ni and Pb extracted with EDTA were higher than when using malic acid and CaCl₂, respectively. The release of Ni and Pb was characterized by an initial fast rate followed by a slower rate and could best be described by a two first-order reactions model with rate constants k₁ and k₂ and two metal pools: readily labile (Q ₁) and less labile (Q ₂). In an EDTA extractant, different Q₁ /Q₂ ratios for Ni and Pb were observed, indicating binding energies to soil constituents is not comparable. The k₁ of the model for Ni (average of 10 soils: 0.2204 h^?1 and 0.2359 h^?1 for 0.01 M CaCl₂ and 0.01 EDTA, respectively) was higher than Pb (0.1044 h^?1 and 0.1631 h^?1 for 0.01 M CaCl₂ and 0.01 M EDTA, respectively), indicating a higher potential of Ni for leaching and groundwater contamination in contaminated calcareous soils. Relationships between the fraction of Ni and Pb determined with the two first-order reactions model and the soil composition and Pb fractions were established. The results indicated that the efficiency of the extractions Ni and Pb from soils depends both on the Ni and Pb content and soil composition. Overall, the results indicated that the use of a 0.01 M malic acid washing solution would be preferred in the field condition.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

Kinetics of Oxygen Release from ORC

Oxygen release compounds (ORC) are one possibility to enhance aerobic degradation in contaminated aquifers. However, some applications have been reported where oxygen concentrations did not meet expectations, this was attributed to ground water composition, e.g., high pH. Column experiments have been performed and the measurements were interpreted using a numerical model to investigate oxygen release kinetics from ORC in more detail. Because the zero-order rate law recommended by the manufacturer did not reflect the measurements, a more complex kinetic scheme was developed. The simulations show a minor influence of inorganic ground water constituents on oxygen release from ORC in the columns due to buffering by mineral precipitation, but an enhanced oxygen release if aerobic degradation takes place. If ORC is applied as socks, the impact of inorganic ground water composition increases compared to the application in column experiments. A simple quadratic equation is provided to estimate oxygen release rate from the buffer capacity of the ground water versus increasing pH—a parameter easily determinable in the laboratory. For slightly mineralized waters with high pH, this equation forecasts decreased oxygen release, but no total inhibition of oxygen release.     

Kinetics of Oxygen Release from ORC

Oxygen release compounds (ORC) are one possibility to enhance aerobic degradation in contaminated aquifers. However, some applications have been reported where oxygen concentrations did not meet expectations, this was attributed to ground water composition, e.g., high pH. Column experiments have been performed and the measurements were interpreted using a numerical model to investigate oxygen release kinetics from ORC in more detail. Because the zero-order rate law recommended by the manufacturer did not reflect the measurements, a more complex kinetic scheme was developed. The simulations show a minor influence of inorganic ground water constituents on oxygen release from ORC in the columns due to buffering by mineral precipitation, but an enhanced oxygen release if aerobic degradation takes place. If ORC is applied as socks, the impact of inorganic ground water composition increases compared to the application in column experiments. A simple quadratic equation is provided to estimate oxygen release rate from the buffer capacity of the ground water versus increasing pH—a parameter easily determinable in the laboratory. For slightly mineralized waters with high pH, this equation forecasts decreased oxygen release, but no total inhibition of oxygen release.     

Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis

Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD(+). Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD(+) levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.     

Kinetic Analysis of Glucoamylase-Catalyzed Hydrolysis of Starch Granules from Various Botanical Sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k ₀) were determined to be 23.3±4.4, 14.8±6.0, 6.2±1.8, 7.1±4.1, 4.6±3.0, and 1.6±0.6 s^?1 for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k ₀ was largely influenced by the type of starch granules. A comparison of the k ₀-values in relation to the crystalline structure of the starch granules suggested that k ₀ increases as the crystalline structure becomes dense.     

基于遗传算法的谷氨酸发酵动力学参数估计

把遗传算法应用于求解谷氨酸分批发酵动力学模型参数,取交叉概率Pc=0．8、变异概率Pm=0．06、初始种群为20、遗传世代数为200代,能进一步提高谷氨酸分批发酵过程状态变量的计算值与实验值的吻合程度。模拟值与实验值对比显示,该动力学模型能很好地反映谷氨酸分批发酵过程。     

Synthesis, biophysical properties, and oxygenation potential of variable molecular weight glutaraldehyde-polymerized bovine hemoglobins with low and high oxygen affinity

In a recent study, ultrahigh molecular weight (Mw ) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model.(1) In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolybHbs, increasing the G:Hb molar ratio reduced the O2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O2 affinity PolybHbs, increasing the G:Hb molar ratio led to increased O2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O2 transport model was used to simulate O2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O2 affinity PolybHbs oxygenated the bioreactor better than high O2 affinity PolybHbs. This result points to the suitability of low O2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications.     

APRT from erythrocytes of HGPRT deficient patients: Kinetic,regulatory and thermostability properties

Adenine phosphoribosyltransferase (APRT) has been 1200-fold purified from erythrocytes of a patient with partial hipoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, Propositus, and in those of a control^HPRT+, with 20% efficiency in both proteins and specific activity of 550 and 243 nmol/h/mgprotein. The specific activity determined in the Propositus enzyme was, in all purification steps, higher than that of the control^HPRT+. Significant changes were found in their thermal stabilities. Half inactivation times at each temperature studied are greater for the Propositus enzyme in the temperature interval 60–80°C. No significant difference has been observed in the affinity constants for adenine and PRPP substrates. Studies on inhibition by the reaction product suggest that AMP is a competitive inhibitor with respect to PRPP in both enzymes, with K_i values of 150 M in Propositus and 220 M in control^HPRT+.     

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the K_on and K_off values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.     

油菜叶片硝酸还原酶同功酶的纯化与特性

通过对硝酸还原酶（ＮＲ）亲和层析洗脱过程的部分改进,从油菜叶片中分离纯化到诱导型（ｉＮＲ）及组成型（ｃＮＲ）两种硝酸还原酶同功酶。电泳分析表明两者均达到银染单带纯。ｃＮＲ分子量（ＭＷ）为４５０ｋＤ．ｉＮＲＭＷ为２２０ｋＤ;两者亚基ＭＷ均为１１０ｋＤ,但亚基数目不一样,氨基酸组成也有差异。ｉＮＲ与ｃＮＤ的等电点ｐＨ不同,分别为４．４及６．０,免疫交叉反应显示ｃＮＲ的抗原性为ｉＮＲ的７５％。     

Kinetic and thermodynamic studies of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus

Throughout evolution, emerging organisms survived by adapting existing biochemical processes to new reaction conditions. Simple protein enzymes balanced changes in structural stability with changes that permitted optimal catalysis by adjustments in both entropic and enthalpic contributions to the free energy of activation for the reaction. Study of adaptive mechanisms by large multicomponent enzymes such as the ribosome has been largely unexplored. Here we have determined the kinetic and thermodynamic parameters of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus. Activity of thermophilic enzymes can be assayed over a wide range of temperatures, enabling one to measure accurate catalytic rates and determine enthalpic and entropic contributions to the free energy of activation of the reaction. Differences in the reaction conditions used here and in published studies on mesophilic ribosomes prevent direct comparison, but our data on Thermus ribosomes suggest that these ribosomes have adapted to changing environments using the same strategies as simple protein enzymes, balancing stability and flexibility without loss of catalytic rate. This strategy must be a very ancient process, perhaps first used by primitive ribosomes in the RNA World.