首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.

Background

Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.

Results

The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.

Conclusions

Fluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.  相似文献   

2.
Enzymatic O2‐dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O2 diffusion restriction. Internal O2 supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space‐averaged intraparticle O2 concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O2 sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005–12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O2 concentration differences between bulk liquid and the intraparticle environment. We show that the O2 gradient at apparent steady state increased with increasing enzyme loading, so that O2 eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O2 concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O2 diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross‐linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O2 concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O2 dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support. Biotechnol. Bioeng. 2013; 110: 2086–2095. © 2013 Wiley Periodicals, Inc.  相似文献   

3.
Cross‐linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross‐linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7‐aminocephalosporanic acid. The CLEAs had a Kcat/KM of 0.9 M?1/S?1 for 7‐ACA (7‐aminocephalosporanic acid) and 0.1 M?1/S?1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis–Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36–42, 2016  相似文献   

4.
Summary Foliar fertilization has received considerable attention in recent years. Because of the importance of amides and urea as N sources, this work was carried out to study the enzymes that catalyze the hydrolysis of these compounds in plant leaves. The methods developed for assay of these enzymes in plants involve determination by steam distillation of the NH4 +–N produced by amidase or urease activity when plant materials are incubated at 37°C with buffered (0.1M THAM pH 8.0) amide solution or buffered (0.1M THAM pH 7.5) urea solution, respectively. Amidase and urease were detected in 21 diverse plants in the families of Gramineae and Leguminosae. Results showed that amidase and urease have optimum activities at buffer pH values of 8.0 and 7.5, respectively. Both amidase and urease activities were decreased significantly upon freezing or air-drying of plant samples before enzyme assay. These differences were proportional to the original activities of fresh plant materials. Studies on the effect of temperature on amidase and urease activities showed that these enzymes are inactivated at temperatures above 60 and 70°C, respectively. The energy of activation of the reaction catalyzed by amidase and urease in plants, expressed in kJ·mole–1, ranged from 44.0 to 51.2 (avg.=47.1) and from 43.1 to 56.5 (avg.=51.2) when formamide and urea were used as substrates, respectively. The apparent Km constants of these enzymes varied among the plant samples studied. By using the Lineweaver-Burk plot, the Km values for amidase when formamide was used as a substrate ranged from 2.0 to 9.4 (avg.=5.8 mM) and for urease ranged from 0.4 to 1.6 (avg.=0.8 mM). The Vmax values of 7 plant samples, expressed in g of NH4 +–N produced/0.1 g of plant materials/2h, ranged from 137 to 514 for amidase and from 29 to 123 for urease. The importance of these enzymes in application of amides and urea to plant leaves is discussed.  相似文献   

5.
In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI Mw = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (Km = 14.3 mM) was better than that of HA–CA–glut (Km = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015  相似文献   

6.
It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [kcat 1144 ± 23.8 s?1, KM 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 106 M?1 s?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with kon = (1.6 ± 0.1) × 105 M?1 s?1 and koff = 1.4 ± 2.9 s?1 (KD ~ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O2‐producing proteins in (nitrogen‐fixing) cyanobacteria.  相似文献   

7.
Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single‐chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5‐Å‐resolution structure of this new AGU model enzyme and built an enzyme–substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on KM and kcat of the amidase. It appears that a “molecular clamp” capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.  相似文献   

8.
The rdpA gene of strains Delftia acidovorans MC1, Rhodoferax sp. P230, and Sphingobium herbicidovorans MH proved to be identical. However, when RdpA [(R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate dioxygenase] was investigated after purification from the various strains, significant differences in the kinetics and some chemical properties of the enzymes were observed. The preference for substrates ranged in the order (R)‐2‐(2,4‐dichlorophenoxy)propionate (2,4‐DP) > (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate (MCPP) >> 2,4‐dichlorophenoxyacetate (2,4‐D) ~ 4‐chloro‐2‐methylphenoxyacetate (MCPA), but detailed kinetic investigations revealed significant strain‐dependent differences in the kcat and KM values. While the KM values of RdpA from the various strains were low and their range rather narrow with 2,4‐DP (19–60 μM) and MCPP (35–64 μM), larger differences were observed with phenoxyacetates which were distinctly higher and spanned a wider range with 2,4‐D (237–935 μM) and MCPA (164–510 μM). The lowest KM values with 2,4‐D and MCPA were found for RdpA originating from strain P230. Investigation of the enzymes from the various sources by 2D gel electrophoresis revealed up to three monomeric enzyme forms which differed in the pI value. The 2D‐patterns were similar with RdpA from strains MC1 and MH, and after heterologous expression of the enzyme in Escherichia coli, but differed significantly from that of strain P230. The presence of enzyme forms and their different composition coincided apparently with the differences observed in the kinetic properties of RdpA in the various strains. The effects are discussed in terms of posttranslational modification of RdpA which appears to be different in extent and kind in the various strains.  相似文献   

9.
Lactate dehydrogenase (LDH) was adsorbed on low-(γ, η) and high -(θ, α) temperature forms of alumina. θ-Al2O3 exhibited the greatest adsorption ability. The maximum adsorption value was 30 mg LDH/g of a carrier. The conditions for irreversible adsorption have been determined. An adsorption isotherm on θ-Al2O3 for pH 6.0 has been obtained; the LDHads surface area and the carrier surface portion accessible to the enzyme molecules have been calculated. The reaction kinetic parameter were determined by taking into account the reaction proceeding in the intradiffusional region. The specific catalytic activity (Aspec) of LDHads at small surface coverage of θ-Al2O3 is five times less than Aspec of the native enzyme and KMimm with respect to NADH exceeds KMnat by two orders or magnitude. The is evidence for a strong LDH–Al2O3 interaction and a considerable deformation of the enzyme globule. Aspec and KM decrease as the amount of the enzyme attached to the carrier increases. Due to adsorption. LDH becomes thermostable and durable. The LDHads samples conserve 20–40% of their activity at room temperature during a year.  相似文献   

10.
Kinetic, thermodynamic, and structural properties of the aminoglycoside N3‐acetyltransferase‐VIa (AAC‐VIa) are determined. Among the aminoglycoside N3‐acetyltransferases, AAC‐VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (~40‐fold) resulted in ~30‐fold variation in kcat/KM. Binding of aminoglycosides to AAC‐VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3‐acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H2O and D2O for the binary enzyme–sisomicin complex but remained the same in both solvents for the ternary enzyme–CoASH–sisomicin complex. Unlike, most other aminoglycoside‐modifying enzymes, the values of ΔCp were within the expected range of protein‐carbohydrate interactions. Solution behavior of AAC‐VIa was also different from the more promiscuous aminoglycoside N3‐acetyltransferases and showed a monomer‐dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand‐protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3‐acetyltransferase. Proteins 2017; 85:1258–1265. © 2017 Wiley Periodicals, Inc.  相似文献   

11.
The transport characteristics of the plasma membrane H+‐ATPase (PMHA) and Na+‐ATPase (PMNA) from marine unicellular green alga Tetraselmis viridis Rouch. were studied using sealed plasma membrane vesicles isolated from this species. The activities of the ATPases were investigated by monitoring the ATP‐dependent pH changes in the vesicle lumen. PMHA operation led to acidification of the vesicle lumen, whereas Na+ translocation into plasma membrane vesicles catalysed by PMNA was accompanied by H+ efflux, namely the alkalization of the vesicle lumen (Balnokin et al., FEBS Lett 462: 402–406, 1999). The intravesicular acidification and alkalization were detected with the ΔpH probe acridine orange and the pH probe pyranine, respectively. PMHA and PMNA were found to operate in distinct pH regions, maximal activity of PMHA being observed at pH 6.5 and that of PMNA at pH 7.8. Kinetic studies revealed that the ATPases have similar affinities to their primary substrate, MgATP complex (an apparent Km = 34 ± 6.2 µM for PMHA and 73 ± 8.7 µM for PMNA). At the same time, the ATPases were differently affected by free Mg2+ and ATP. Free Mg2+ appeared to be a mixed‐type inhibitor for PMNA (Ki′ = 210 µM) but it did not suppress PMHA. Conversely, free ATP markedly suppressed PMHA being a mixed‐type inhibitor (Ki′ = 330 µM), but PMNA was affected by free ATP only slightly. Furthermore, the ATPases substantially differed in their sensitivities to the inhibitors of membrane ATPases, such as orthovanadate, N‐ethylmaleimide and N,N′‐dicyclohexylcarbodiimide. The differences found in the properties of the PMHA and PMNA are discussed in terms of regulation of their activities and their capacity to be involved in cytosolic ion homeostasis in T. viridis cells.  相似文献   

12.
Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a Vmax of 0.142 U/mg purified rXI, and a KM for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol KI values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low KM of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca2+ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher Vmax at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016  相似文献   

13.
Besides its amide hydrolase activity, the amidase from Brevibacterium sp. R312 also exhibits an acyl-transferase activity.

The mechanism of the transfer reaction of the acyl from acetamide to hydroxylamine was studied. This is a “Bi Bi Ping Pong” type reaction. The kinetic parameters of the reaction were determined:
– Apparent Vm = 135 μmol · min –1 · mg–1

– Acetamide Km = 18.2 mM

– Hydroxylamine Km = 131 mM  相似文献   

14.
Purification and in vitro protein‐folding schemes were developed to produce monodisperse samples of the mature wild‐type HIV‐2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2E37K (elevated Kd ~20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a Tm of 60.5 for PR2 as compared with 65.7°C for HIV‐1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the Tm of PR2 increases by 14.8°C in the presence of DRV as compared with 22.4°C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2T26A) or a deletion of the C‐terminal residues 96–99 (PR21–95), drastically increase the Kd (>105‐fold). PR2T26A and PR21–95 consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size‐exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild‐type PR2 and its active‐site mutant PR2D25N are folded dimers. Addition of twofold excess active‐site inhibitor promotes dimerization of PR2T26A but not of PR21–95, indicating that subunit interactions involving the C‐terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.  相似文献   

15.
Kinetics of net phosphate (Pi) uptake was measured on intact ectomycorrhizal and non‐mycorrhizal Pinus sylvestris seedlings using a semihydroponic cultivation method. The depletion of Pi in a nutrient solution was assessed over a 160–0.2 μM Pi gradient. Growth of the pine seedlings was P limited and measurements were performed 7 and 9 weeks after inoculation. Three ectomycorrhizal fungi were studied: Paxillus involutus, Suillus bovinus and Thelephoraterrestris. Pi uptake was extremely fast in plants colonised by P. involutus. The Pi concentration dropped below 0.2 μM within 4–5 h. In plants colonised with S. bovinus this occurred in 5–6 h and in plants associated with T. terrestris 8 h were needed to run through the whole concentration range. Non‐mycorrhizal plants of similar size and nutrient status decreased Pi to a concentration between 1 and 2 μM in 18 h. Data were curve fitted to a two‐phase Michaelis‐Menten equation. The apparent kinetic constants, Km and Vmax, for the high affinity Pi uptake system of the pine roots could be estimated accurately. Vmax of this system was up to 7 times higher in pines associated with P. involutus than in non‐mycorrhizal seedlings. The intact extraradical mycelium greatly increased the absorption surface area of the roots (Vmax). Non‐mycorrhizal plants had a Km between 7.8 and 16.4 μM Pi. Plants mycorrhizal with P. involutus had Km values between 2.4 and 7.2, plants colonised with S. bovinus had a Km between 5.1 and 12.3, and seedlings associated with T. terrestris had a Km from 4.6 to 10.1 μM Pi. All 3 ectomycorrhizal fungi had a strong impact on the Pi absorption capacity of the pine seedlings. The results also demonstrated that there is substantial heterogeneity in kinetic parameters among the different mycorrhizal root systems.  相似文献   

16.
Using whole cell invertase of Saccharomyces pastorianus, entrapped in spherical agar pellets, sucrose hydrolysis was carried out in a continuously fed fluidized bed reactor. The effective rate of reaction determined experimentally for the catalytic pellet was correlated with particle radius (R), intraparticle concentration of enzyme (Ep) and external concentration of substrate (S R). The results were elucidated by theoretical analysis incorporating internal mass transfer resistance. At high degrees of diffusional resistance, the effectiveness factor was successfully estimted from Bischoff's equation. A dimensionless number, mA ? R(k2Ep/KmD)0.5(Km/(Km + S R)), was used conveniently to predict the effectiveness factor in those cases wher the intraparticle diffusional effect was less significant. This number was employed to determine critical pellet size for an optimal reaction. The relationship between the properties of the pellet (size and intraparticle enzyme activity) and its apparent kinetic constants (k2 and Km), estimated according to Lineweaver-Burk, are discussed.  相似文献   

17.
β-Adrenergic Modulation of Glial Inwardly Rectifying Potassium Channels   总被引:1,自引:0,他引:1  
Abstract: Cultured spinal cord astrocytes (2–13 days in vitro) express several different potassium current types, including delayed rectifier, transient A-type, and inward rectifier (Kir) K+ currents. Of these, Kir is believed to be of critical importance in the modulation of extracellular [K+] in the CNS. Using the whole-cell patch-clamp technique, we analyzed modulation of Kir currents by β-adrenergic receptor activation. The selective β-adrenergic agonist isoproterenol (1–100 µM) and epinephrine (1–100 µM) each reduced peak Kir current amplitudes to 52.7 ± 12.5 and 63.6 ± 7.0%, respectively, at 100 µM. Forskolin (KD of ~25 µM), an activator of adenylate cyclase (AC), and dibutyryl-cyclic AMP (1 mM), a membrane-permeable analogue of cyclic AMP (cAMP), were each used to increase [cAMP]i, the product of AC, and resulted in similar reductions of Kir currents. By contrast, 1,9-dideoxyforskolin (1–50 µM), a forskolin analogue that does not activate AC, did not affect Kir currents, indicating that AC activity is a required element for Kir modulation. Three inhibitors of PKA—Rp-adenosine 3′,5′-cyclic monophosphothioate, H-7, and adenosine 3′,5′-cyclic monophosphate-dependent protein kinase inhibitor—failed to inhibit Kir current reduction by β-adrenergic agonists. These results indicate that β-adrenergic receptor ligands can modulate Kir currents and suggest that this modulation involves activation of AC but not protein kinase A. Such modulation may provide a mechanism by which neurons can modulate glial Kir currents and thereby may affect glial K+“spatial buffering” in the CNS.  相似文献   

18.
Abstract: Using dissociated rat carotid body (CB) cultures, we compared levels of extracellular dopamine (DA) around oxygen-sensitive glomus cells grown for ~12 days in normoxia (Nox; 20% O2), chronic hypoxia (CHox; 6% O2), or chronic nicotine (CNic; 10 µM nicotine, 20% O2), with or without acetylcholine (ACh) receptor (AChR) agonists/antagonists and blockers of DA uptake. In Nox cultures, extracellular DA, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was augmented by acute (~15-min) exposure to hypoxia (5% O2; ~6× basal), high extracellular K+ (30 mM; ~10× basal), nomifensine (1 µM; a selective DA uptake inhibitor; ~3× basal), and nicotine (100 µM; ~5× basal), but not methylcholine (300 µM; a specific muscarinic agonist). In contrast, in CHox cultures where basal DA release is markedly elevated (~9× control), the stimulatory effect of high K+ (3–4× basal) and acute hypoxia (~2× basal) on DA release persisted, but nicotine and nomifensine were no longer effective and methylcholine had a partial inhibitory effect. In CNic cultures, basal DA levels were also elevated (~9× control), similar to that in CHox cultures; however, although acute hypoxia had a stimulatory effect on DA release (~2× basal), nicotine, nomifensine, and high K+ were ineffective. The elevated basal DA in both CHox and CNic cultures was attenuated by acute or chronic treatment with mecamylamine (100 µM), a nicotinic AChR (nAChR) antagonist. In addition, long-term (16-h), but not acute (15-min), treatment with the muscarinic antagonist atropine (1 µM) produced an additional enhancement of basal DA levels in CHox cultures. Thus, after chronic hypoxia or nicotine in vitro, extracellular DA levels around CB chemoreceptor cell clusters appear to be set by a variety of factors including released ACh, positive and negative feedback regulation via nAChRs and muscarinic AChRs, respectively, and modulation of DA transporters. These results provide insight into roles of endogenous transmitters in the adaptation of CB chemoreceptors to chronic hypoxia and suggest pathways by which neuroactive drugs, e.g., nicotine, can interfere with the protective chemoreflex response against hypoxia.  相似文献   

19.
Arthrobacter sp. Q36 produces a novel enzyme, maltooligosyl trehalose synthase, which catalyzes the conversion of maltooligosaccharide into the non-reducing saccharide, maltooligosyl trehalose (α-maltooligosyl α-D-glucoside) by intramolecular transglycosylation. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, DEAE-Sephadex A-50, Ultrogel AcA44, and Butyl-Toyopearl 650M. The enzyme was specific for maltooligosaccharides except maltose, and catalyzed the conversion to form maltooligosyl trehalose. The Km of the enzyme for maltotetraose, maltopentaose, maltohexaose, and maltoheptaose were 22.9mM, 8.7mM, 1.4mM, and 0.9mM, respectively. The enzyme had a molecular mass of 81,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.1 by gel isoelectrofocusing. The N-terminal and C-terminal amino acids of the enzyme were methionine and serine, respectively. The enzyme showed the highest activity at pH 7.0 and 40°C, and was stable from pH 6.0 to 9.5 and up to 40°C. The enzyme activity was inhibited by Hg2+ and Cu2+.  相似文献   

20.
Ferrocene‐incorporated selenoureas 1‐(4‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P4Me), 1‐(3‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P3Me), and 1‐(2‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P2Me) were synthesized and characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, atomic absorption spectroscopy, CHNS, and single‐crystal X‐ray diffraction. DNA interaction of the compounds was investigated with cyclic voltammetry, UV–visible spectroscopy, and viscometry, which is a prerequisite for anticancer agents. Drug‐DNA binding constant was found to vary in the sequence: KP4Me (4.9000 × 104 M?1) > KP2Me (2.318 × 104 M?1) > KP3Me (1.296 × 104 M?1). Antioxidant (1,1‐diphenyl‐2‐picrylhydrazyl), antifungal (against Faussarium solani and Helmentosporium sativum), and antibacterial (against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) activities have also been reported in addition.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号