Kinetic Constants for Blood—Brain Barrier Amino Acid Transport in Conscious Rats

The kinetic constants for large neutral amino acid (LNAA) transport across the blood-brain barrier (BBB) of conscious rats were determined in four brain regions: cortex, caudate-putamen, hippocampus, and thalamus-hypothalamus. Indwelling external carotid artery catheters allowed for single-bolus (200 microliters) injections directly into the arterial system of unanesthetized and lightly restrained animals. Our results showed lower brain uptake index values for conscious rats compared to previous reports for anesthetized animals which are consistent with higher rates of cerebral blood flow in the conscious animals. Km values were lower in the conscious animals and ranged from 29% to 87% of the Km values in pentobarbital-anesthetized animals whereas the KD values were about twofold higher in the conscious animals. No apparent regional differences were observed. Influx rates were determined which take into consideration flow rates and plasma amino acid concentrations. Our results showed an average amino acid influx value of 5.2 nmol/min/g, which is 53% higher than the average influx in pentobarbital-anesthetized animals. The present results in conscious animals regarding the low Km of LNAA transport across the BBB lend further support to the importance of fluctuations in plasma amino acid concentrations and LNAA transport competitive effects on brain amino acid availability.     

大鼠、小鼠胰蛋白酶的分离纯化及动力学性质

采用STI-Sepharose 4B亲和层析的方法,从鼠新鲜胰脏中分离得到纯的胰蛋白酶。大鼠胰蛋白酶的比活为24 615BAEEU/mg蛋白,总活性回收率47％,小鼠胰蛋白酶的比活为32 768BAEEU/mg蛋白,总活性回收率55％。经SDS-聚丙烯酰胺凝胶电泳鉴定,大鼠、小鼠胰蛋白酶均呈现单一蛋白带,两者的分子量都是24kD。用等电聚焦电泳测定,二者的等电点均为p19.5以上。对它们的动力学性质作了研究,大鼠胰蛋白酶的Km值为2.33×10~(-4)mol/L,K,值为0.92×10~(-5)mol/L,小鼠胰蛋白酶的Km值为5.60×10~(-4)mol/L,K:值为1.27×10~(-5)mol/L。     

Formation of kynurenic and xanthurenic acids from kynurenine and 3-hydroxykynurenine in the dinoflagellate Lingulodinium polyedrum: role of a novel,oxidative pathway

The dinoflagellate Lingulodinium polyedrum (syn. Gonyaulax polyedra) was used as a model organism for studying the effects of high and low physiological oxidative stress on the formation of kynurenic and xanthurenic acids from kynurenine and 3-hydroxykynurenine. Cell were incubated with the precursors and exposed to light (high physiological stress due to photosynthetically formed oxidants) or kept in darkness (low stress). In cultures of less than 0.5 ml cell volume/l of medium, cells took up approximately one half of 0.1 mM extracellular kynurenine within 18 h. The amino acid was partially converted to kynurenic acid, most of which was released to the medium; however, intracellular concentrations of the product were by approximately 10-fold higher than extracellular levels. Rates of kynurenic acid release exceeded by far those explained by kynurenine and tryptophan aminotransferase activities, the latter representing an additional source of kynurenic acid formation via indole-3-pyruvic acid. Light enhanced the release of kynurenic acid by approximately 4-fold; these rates were further increased by exposure to continuous light. Diurnal rhythmicity of kynurenic acid release was clearly exogenous and did not match with the circadian pattern of kynurenine or tryptophan aminotransferase activities; no rhythm was detected in constant darkness. Similar findings were obtained on turnover of 3-hydroxykynurenine to xanthurenic acid and release of the product to the medium. However, light/dark differences were relatively smaller, and additional products were formed, according to HPLC data obtained with electrochemical detection. Results are most easily explained on the basis of a recently discovered pathway of kynurenic acid formation from kynurenine, involving either non-enzymatic oxidation by H(2)O(2) or, at higher rates, enzymatic catalysis by hemoperoxidase. A corresponding mechanism may exist for the hydroxylated analogue.     

大肠杆菌亮氨酰－tRNA合成酶LeuRS67R的纯化及其动力学研究

本实验室已得到的亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因,与文献相比,６７位氨基酸残基由Ｈｉｓ变为Ａｒｇ,此酶被定名为ＬｅｕＲＳ６７Ｒ。我们从该基因与ｐＵＣ１９重组质粒的大肠杆菌ＴＧ１转化子ＴＧ１－９１中得到ＬｅｕＲＳ的高表达,粗抽液中ＬｅｕＲＳ的表达量在转化子中比在宿主菌ＴＧ１中高２０倍以上。用三步拉层析得到电泳一条带的酶,其比活为１７８９单位／毫克。测定其动力学常数,氨酰化活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０２７ｍｍｏｌ／Ｌ、０．４７ｍｍｏｌ／Ｌ,Ｋｃａｔ值分别为３．５～５．１ｓ－１。ＡＴＰ－ＰＰｉ交换活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０３ｍｍｏｌ／Ｌ、１．０ｍｍｏｌ／Ｌ,Ｌｃａｔ值分别为１４０～１５５ｓ－１。此结果与从野生型大肠杆菌Ｋ－１２中提纯的ＬｅｕＲＳ的动力学常数差别很小,６７位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫(Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定,并构建了融合表达质粒pET-ESTA2。转化大肠杆菌BL21后,在异丙基硫代牛乳糖苷(IPTG)的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达。表达产物经亲和层析纯化获得了1条带的重组蛋白。与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高。表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高。羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

Activation of bile salt dependent lipase by (lyso)phosphatidic acid and platelet activating factor

The activity of breast milk BSDL was assayed with or without phospholipids as extra-intestinal effector candidates. Phosphatidic acid, lysophosphatidic acid and platelet activating factor but not phosphatidylcholine and lysophosphatidylcholine stimulated BSDL activity at least as efficiently as taurocholate. The apparent dissociation constants of PA and LPA at saturating concentrations of three different substrates were between 0.1 and 13.4 μM and that of PAF was below or equal to 200 pM. Kinetic data suggested the existence of at least one binding site for each of these effectors. PA, LPA and PAF are likely extra-intestinal modulators of BSDL activity.     

Rapid screening of monoamine oxidase B inhibitors in natural extracts by capillary electrophoresis after enzymatic reaction at capillary inlet

A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N′-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH = 7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K_i, K_m and IC₅₀ based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD = 1.8%) and peak area (RSD = 3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.     

The tryptophan oxidation pathway in mosquitoes with emphasis on xanthurenic acid biosynthesis

Oxidation of tryptophan to kynurenine and 3-hydroxykynurenine (3-HK) is the major catabolic pathway in mosquitoes. However, 3-HK is oxidized easily under physiological conditions, resulting in the production of reactive radical species. To overcome this problem, mosquitoes have developed an efficient mechanism to prevent 3-HK from accumulating by converting this chemically reactive compound to the chemically stable xanthurenic acid. Interestingly, 3-HK is a precursor for the production of compound eye pigments during the pupal and early adult stages; consequently, mosquitoes need to preserve and transport 3-HK for compound eye pigmentation in pupae and adults. This review summarizes the tryptophan oxidation pathway, compares and contrasts the mosquito tryptophan oxidation pathway with other model species, and discusses possible driving forces leading to the functional adaptation and evolution of enzymes involved in the mosquito tryptophan oxidation pathway.     

Relations between biochemical thermodynamics and biochemical kinetics

The parameters in steady-state or rapid-equilibrium rate equations for enzyme-catalyzed reactions depend on the temperature, pH, and ionic strength, and may depend on the concentrations of specific species in the buffer. When the complete rate equation (i.e. the equation with parameters for the reverse reaction as well as the forward reaction) is determined, there are one or more Haldane relations between some of the kinetic parameters and the apparent equilibrium constant for the reaction that is catalyzed. When the apparent equilibrium constant can be calculated from the kinetic parameters, the equilibrium composition can be calculated. This is remarkable because the kinetic parameters all depend on the properties of the enzymatic site, but the apparent equilibrium constant and the equilibrium composition do not. The effects of ionic strength and pH on the unoccupied enzymatic site and the occupied enzymatic site have to cancel in the Haldane relation or in the calculation of the apparent equilibrium constant using the rate constants for the steps in the mechanism. Several simple enzymatic mechanisms and their complete rate equations are discussed.     

Anaerobic digestion of glycerol derived from biodiesel manufacturing

The anaerobic digestion of glycerol derived from biodiesel manufacturing, in which COD was found to be 1010 g/kg, was studied in batch laboratory-scale reactors at mesophilic temperature using granular and non-granular sludge. Due to the high KOH concentration of this by-product, H₃PO₄ was added to recover this alkaline catalyst as agricultural fertilizer (potassium phosphates). Although it would not be economically viable, a volume of glycerol was distilled and utilised as reference substrate. The anaerobic revalorisation of glycerol using granular sludge achieved a biodegradability of around 100%, while the methane yield coefficient was 0.306 m³ CH₄/kg acidified glycerol. Anaerobic digestion could be a good option for revalorising this available, impure and low priced by-product derived from the surplus of biodiesel companies. The organic loading rate studied was 0.21–0.38 g COD/g VSS d, although an inhibition phenomenon was observed at the highest load.     

Application of a chemoenzymatic glycosylation method to α-chymotrypsin and Candida rugosa lipase surface modifications

A recently developed chemoenzymatic glycosylation procedure has been successfully applied on two hydrolytic enzymes, α-chymotrypsin and Candida rugosa lipase. First, a number of sucrose molecules have been bound to the surface lysine residues and then, lengthening of the glycosidic chains has been carried out by the action of a levansucrase from Bacillus subtilis. For both steps, reaction conditions have been studied in order to obtain a range of glycosylation degrees. The influence of glycoside binding on biocatalyst surface characteristics has been assessed and a progressive increase in global enzyme hydrophilic character with glycosylation has been observed. Besides, the study of hydrolytic activity and kinetic constants showed that the performed modifications brought about a certain decrease in enzyme hydrolytic activity and very slight variations in enzyme-substrate affinity.     

高等植物Na+吸收、转运及细胞内Na+稳态平衡研究进展

盐胁迫是影响农业生产的重要环境因素之一。本文对植物Na 吸收的机制和途径、Na 在植物体内的长距离转运以及细胞内Na 稳态平衡的研究进展进行了概述。参与植物Na 吸收与转运的蛋白和通道可能包括HKT、LCT1、AKT和NSCC等。其中,HKT是植物体内普遍存在的一类转运蛋白,能够介导Na 的吸收,其结构中的带电氨基酸残基对于其离子选择性有着非常明显的影响。LCT1是从小麦中发现的一类能够介导低亲和性阳离子吸收的蛋白,然而在典型的土壤Ca2 浓度下LCT1并不能发挥吸收Na 的功能。AKT家族的成员在高盐环境下可能也参与了Na 的吸收。目前虽然还没有克隆到编码NSCC蛋白的基因,但是NSCC作为植物吸收Na 的主要途径的观点已被广泛接受。SOS1和HKT参与了Na 在根部与植株地上部的长距离转运过程,它们在木质部和韧皮部的Na 装载和卸载中发挥重要作用,从而影响植物的抗盐性。另外,由质膜Na /H 逆向转运蛋白SOS1、蛋白激酶SOS2以及Ca2 结合蛋白SOS3组成的SOS复合体对细胞的Na 稳态具有重要的调节作用,单子叶和双子叶植物之间的这种调节机制在结构和功能上具有保守性。SOS复合体与其它位于质膜或液泡膜上的Na /H 逆向转运蛋白以及H 泵一起调节着细胞的Na 稳态。     

Cooperative effect of the camphor imine ligand in alkynols activation by trans-[MCl2(YNC10H14O)2] (M = Pd or Pt)

The cooperative effect of the camphor imine ligand (YNC₁₀H₁₄O) in the cyclization of 5-hexyn-1-ol and 4-pentyn-1-ol promoted by complexes trans-[MCl₂(YNC₁₀H₁₄O)₂] (M = Pd, Y = NH₂, NHMe, NMe₂, OH, Ph; Pt, Y = NH₂, NHMe, NMe₂) is established from a direct relation between the constants calculated for conversion of 5-hexyn-1-ol (A) into 2-methyl-2-pent-4-ynyloxy-tetrahydropyran (B) and 4-pentyn-1-ol (C) into 2-methyl-2-pent-4-ynyloxy-tetrahydrofuran (D) and the basic character of the camphor imine substituent (Y). In the catalytic process acid-base interactions between the alkynol and the coordinated camphor imine are supported by the structural characterization of [PdCl₄][Me₂NHNC₁₀H₁₄O]₂.     

Determination of kinetic parameters of enzyme-catalyzed reactions with a minimum number of velocity measurements

Duggleby [Duggleby, R.G., 1979. Experimental designs for estimating kinetic parameters for enzyme-catalyzed reactions. J. Theor. Biol. 81, 672-684] discussed the “design of several replicate measurements of the velocity at as many experimental conditions as there are parameters to be estimated.” He discussed the application of this method to A→products, without and with competitive inhibition, and commented briefly on A+B→products. The availability of computer applications that can solve large sets of simultaneous equations makes it possible to use this method to calculate kinetic parameters for more complicated enzyme mechanisms. This article is concerned with rapid-equilibrium rate equations, but this method can also be used with steady-state rate equations. Computer programs are provided for the calculation of the three kinetic parameters for ordered A+B→products from three velocity measurements and for the calculation of the four kinetic parameters for random A+B→products from four velocity measurements. Computer programs are also provided for competitive inhibition, uncompetitive inhibition, and mixed inhibition of ordered A+B→products.     

Interaction of cryptogein with its binding sites in tobacco plasma membrane studied using the piezoelectric biosensor

Elicitins are low-molecular-weight proteins representing the elicitor family secreted by many species of the oomycete Phytophthora. Elicitins induce a hypersensitive reaction in tobacco, a process that is triggered by binding of elicitin to the high-affinity site on the plasma membrane. Specific interaction of cryptogein with the binding sites on tobacco plasma membranes was studied using the piezoelectric biosensor in real time in a flow-through mode. Cryptogeins (wild-type and mutant forms) were covalently immobilized on the sensing surface, and membrane vesicles containing receptors were in solution. Kinetic characterization of the interaction provided values of kinetic rate association (k_a) = 5.74 · 10⁶ M¹ s⁻¹ and kinetic rate dissociation (k_d) = 6.87 10⁻⁴ s⁻¹ constants, respectively. The kinetic equilibrium dissociation constant was calculated as K_D = 12.0 nM. The piezoelectric biosensor appeared to be a convenient tool for studying interactions of receptors embedded in membrane vesicles.     

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.     

Determination of kinetic constants for a two-stage anaerobic upflow packed-bed reactor for dairy wastewater

A two-stage upflow packed-bed (reactors in series) system was used for the treatment of dairy wastewater. Nylon pads were used as supporting media for the biomass. This investigation aimed at the determination of various kinetic constants for substrate, biomass and biogas based on various models. The maximum loadings that could be applied to reactor I and reactor II were 14·29 and 5·0 kg of chemical oxygen demand (COD) per m³ per day, respectively. The maximum COD removal efficiencies at various loading rates were in the ranges of 93·8–98·5% and 72·5–84% for the two reactor systems, respectively. The combined biogas yield was between 0·196 and 0·386 m³ gas/kg COD_a.