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1.
Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity
Christopher J. Winters W. Brian Reeves Thomas E. Andreoli 《The Journal of membrane biology》1990,118(3):269-278
Summary We evaluated the effects of vawrying aqueous Cl– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl– concentrations and/or PGO on the relation between holding voltageV
H
-mV) and open-time probability (P
o).Reducingcis Cl– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P
v) with a half-maximal reduction inP
o atcis Cl–170mM. Channel activity was sustained by equimolar replacement ofcis Cl– with F–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl– concentration andP
0 at 10mm Cl–. Reducingcis Cl– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl– channels. 相似文献
2.
Cultured mouse MTAL cells contain more mRNA encoding the Cl– channel mcClC-Ka, which mediates CTAL Cl– absorption, than mRNA encoding the Cl– channel mmClC-Ka, which mediates MTAL Cl– absorption. mmClC-Ka and mcClC-Ka have three functional differences: 1) mmClC-Ka open time probability, P
o, increases with increasing cytosolic Cl–, but variations in cytosolic Cl– do not affect P
o in mcClC-Ka; 2) mmClC-Ka is gated by (ATP + PKA), while (ATP + PKA) have no effect on P
o in mcClC-Ka; and 3) mmClC-Ka channels have single-ion occupancy, while mcClC-Ka channels have multi-ion occupancy. Using basolateral vesicles from MTAL cells fused into bilayers, we evaluated the effects of 1 mM cytosolic phenylglyoxal (PGO), which binds covalently to lysine or arginine, on Cl– channels. With PGO pretreatment, Cl– channels were uniformly not gated either with increases in cytosolic-face Cl– or with (ATP + PKA) at 2 mm cytosolic-face Cl–; and they exhibited multi-ion occupancy kinetics typical for mcClC-Ka channels. Thus, in basolateral MTAL membranes, blockade of Cl– access to arginine or lysine residues on mmClC-Ka by PGO results in Cl– channels having the functional characteristics of mcClC-Ka channels. 相似文献
3.
An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings 总被引:3,自引:3,他引:0
Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H+-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H+-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg2+-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H+-translocating PPase. 相似文献
4.
Glucose biosensor based on the room-temperature phosphorescence of TiO2/SiO2 nanocomposite 总被引:1,自引:0,他引:1
The direct immobilization of glucose oxidase (GOD) on TiO2/SiO2 nanocomposite and its application as glucose biosensor were investigated. The room-temperature phosphorescence of TiO2/SiO2 nanocomposite can be quenched by hydrogen peroxide (H2O2). The detection of glucose may be accomplished by monitoring the formation of hydrogen peroxide which generated in the oxidation process of glucose with the catalysis of GOD. To our surprise, by using a 96-hole polyporous plate accessory of fluorescence spectrophotometer, the biosensor exhibits excellent linear response to glucose concentrations ranging from 1.0 × 10−9 to 1.0 × 10−2 M with a detection limit of 1.2 × 10−10 M. The TiO2/SiO2 nanocomposite can be used as both supporting material and signal transducer. The phosphorescence intensity and color of the biosensor change obviously and even could be observed with naked eyes by continuous addition of glucose. Based on the room-temperature phosphorescence of TiO2/SiO2 nanocomposite, a new method of solid substrate-room-temperature phosphorimetry (SS-RTP) for glucose determination is proposed. A glucose biosensor was fabricated with wide determination concentration range, low detection limit, high sensitivity, and fast response time. And the biosensor has been successfully applied to the determination of glucose in human blood serum. The coacervation of GOD enzyme and its interaction with TiO2/SiO2 nanocomposite enlarge the surface area and enhance the chemical stability of GOD. The nice biocompatibility, large surface area, good chemical stability and nontoxicity of the TiO2/SiO2 nanocomposite have made this material suitable for functioning as biosensor. 相似文献
5.
Christopher J. Winters W. Brian Reeves Thomas E. Andreoli 《The Journal of membrane biology》1993,135(2):145-152
A unique property of basolateral membrane Cl– channels from the mTAL is that the Cl– concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P
0
). The K
1/2 for maximal activation of P
0
by Cl– facing intracellular domains of these channels is 10 mm Cl–. The present experiments evaluated the nature of these Cl–-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl– channel faces, could augment P
0
with a K
1/2 in the range of 10 mm isethionate without affecting conductance (g
Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P
0
usually seen when the Cl– concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl– channel activity was increased by first raising the Cl– concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl– channel faces had no effect on P
0
. We conclude that the intracellular aspects of these Cl– channels contain Cl–-interactive loci (termed [Cl]
i
) which are accessible to impermeant anions in intracellular fluids and which contain arginineand lysine-rich domains which can be inactivated, at low ambient Cl– or isethionate concentrations, by interactions with PGO or TNBS.We acknoeledge the able technical assistance of Anna Grace Stewart. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veteterans Administration Merit Review Grants to T. E.Andreoli and to W. B. Reeves. C. J. Winters is a Veterans Administration Associate Investigator. 相似文献
6.
The effects of the arginine modifying reagent phenylglyoxal (PGO) on solute transport was studied in two cellular systems: protoplasts isolated from the mesophyll of Vicia faba L. and XD cell suspension culture of Nicotiana tabacum L. cv. Xanthi. The solutes in the case of the protoplasts were the non‐metabolizable glucose analog 3‐O‐methyl‐D‐glucose (MeG), and a non‐metabolizable amino acid analog α‐aminoisobutyric acid (AIB), whereas the solutes for the cell suspension were AIB and nitrate. Solute transport in both systems was rapidly inhibited by PGO. Exposure of the protoplasts to light enhanced the initial rate of MeG uptake. PGO rapidly inhibited MeG uptake in both the light and the dark, the half‐time for inactivation being less than 3 min. Flux analysis of double‐labeled MeG showed that initial MeG uptake was mediated mainly by the plasma membrane transport system and that it was inhibited by PGO. Maximal inhibition of initial MeG uptake rate was observed at PGO concentrations of 1 m M and above. PGO treatment altered rapidly the equilibrium distribution of the ΔpH probe dimethyloxazolidine (DMO) in both cellular systems, indicating dissipation of ΔpH between cell and medium. In the protoplasts, PGO inhibited both DMO and MeG uptake at pH 5.5; however, at pH 7.0, where ΔpH is minimal, only MeG uptake was inhibited. Our results suggest that PGO has two effects on glucose uptake: an indirect effect through ΔpH dissipation and a direct effect through interaction with essential arginyl residues in the glucose transporter. 相似文献
7.
Amphora coffeaeformis (Ag.) Kütz. var. perpusilla (Grun.) Cleve took up glucose by an inducible transport system. The system was induced by d -fructose, d -mannose, as well as glucose. Some d -pentoses also induced a glucose uptake system but it may not be the same one as that induced by hexose. d -fructose, d -mannose and 2-deoxy-d -glucose inhibited 2 mM glucose uptake at equimolar concentration, but d -pentoses did not. The uptake system decayed in ca. 5 h in the absence of glucose. The half-saturation constant for uptake, K8 was ca. 0.1 mM glucose with a maximum uptake rate, Vmax= 0.4 nmol/106 cells-min?1. 相似文献
8.
In order to study the effect of side-chain length on the conformation of polypeptides, conformational changes of various ionic polypeptides with various lengths of side chain, poly-Nε-glutaryl-L -lysine (PGL), poly-Nδ-glutaryl-L -ornithine (PGO), poly-Nε-succinyl-L -lysine (PSL), and poly-Nδ-succinyl-L -ornithine (PGO), were investigated by ORD, potentiometric titration, and dilatometric measurements in aqueous solution. The results of optical rotation and potentiometric titration measurements indicate strongly that the α-helix stability increases in the sequence PSO < PSL ~ PGO < PGL, which corresponds to increased side-chain length. The volume change associated with the helix–coil transition also increased in the above sequence. This series of polymers seems to be more hydrophobic compared with poly-L -glutamic acid or poly-L -lysine, as suggested from the values of enthalpy and entropy changes for coil–helix transitions. 相似文献
9.
Electrically Contacted Bienzyme‐Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and Multifuel‐Driven Biofuel Cells
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The capping of electron relay units in mesoporous carbon nanoparticles (MPC NPs) by crosslinking of different enzymes on MPC NPs matrices leads to integrated electrically contacted bienzyme electrodes acting as dual biosensors or as functional bienzyme anodes and cathodes for biofuel cells. The capping of ferrocene methanol and methylene blue in MPC NPs by the crosslinking of glucose oxidase (GOx) and horseradish peroxidase (HRP) yields a functional sensing electrode for both glucose and H2O2, which also acts as a bienzyme cascaded system for the indirect detection of glucose. A MPC NP matrix, loaded with ferrocene methanol and capped by GOx/lactate oxidase (LOx), is implemented for the oxidation and detection of both glucose and lactate. Similarly, MPC NPs, loaded with 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid), are capped with bilirubin oxidase (BOD) and catalase (Cat), to yield a bienzyme O2 reduction cathode. A biofuel cell that uses the bienzyme GOx/LOx anode and the BOD/Cat cathode, glucose and/or lactate as fuels, and O2 and/or H2O2 as oxidizers is assembled, revealing a power efficiency of ≈90 μW cm?2 in the presence of the two fuels. The study demonstrates that multienzyme MPC NP electrodes may improve the performance of biofuel cells by oxidizing mixtures of fuels in biomass. 相似文献
10.
Alignment of 23 branching enzyme (BE) amino acid sequences from various species showed conservation of two arginine residues. Phenylglyoxal (PGO) was used to investigate the involvement of arginine residues of maize BEI and BEII in catalysis. BE was significantly inactivated by PGO in triethanolamine buffer at pH 8.5. The inactivation followed a time- and concentration-dependent manner and showed pseudo first-order kinetics. Slopes of 0.73 (BEI) and 1.05 (BEII) were obtained from double log plots of the observed rates of inactivation against the concentrations of PGO, suggesting that loss of BE activity results from as few as one arginine residue modified by PGO. BE inactivation was positively correlated with [14C]PGO incorporation into BE protein and was considerably protected by amylose and/or amylopectin, suggesting that the modified arginine residue may be involved in substrate binding or located near the substrate-binding sites of maize branching enzymes I and II.Abbreviations BE
branching enzyme
- BCA
bicinchoninic acid
- BSA
bovine serum albumin
- Glc-1-P
glucose-1-phosphate
- IPTG
isopropyl-d-thiogalactoside
- PGO
phenylglyoxal
- PMSF
phenylmethylsulfonyl fluoride
- SDS-PAGE
sodium docecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid
- TEA
triethanolamine 相似文献
11.
M.R. Dzoljic 《Prostaglandins & other lipid mediators》1978,15(2):317-324
The experiments were carried out to investigate the effects of prostaglandins (PGs) on the sleep pattern in the cat, and in normal and EFAD rats.The data indicate that the duration of slow wave sleep (SWS) was significantly longer in EFAD rats compared with the normal rats. However, no difference in the REM sleep was observed between the two groups. Intraventricular (i.vc.) administration of PGE1, PGE2 and PGF2α increased wakefulness without a significant alteration of REM sleep.PGE1 administered i.vc. did not alter the duration of SWS or REM sleep in the chronic cat, but induced ponto-geniculo-occipital (PGO) waves (spikes) which are the phasic phenomenon of REM sleep.The fact that previous administration of 5-hydroxytryptophane abolished the PGE1-induced PGO spiking, might indicate that this drug triggered the spikes mainly via the functional inhibition of the serotonergic system. 相似文献
12.
《Biosensors》1986,2(2):71-87
Glucose dehydrogenase (GDH), one of the recently discovered NAD(P)+-independent ‘quinoprotein’ class of oxidoreductase enzymes, was purified from Acinetobacter calcoaceticus LMD 79.41 and immobilised on a 1,1'-dimethylferrocene-modified graphite foil electrode.The second-order rate constant (ks) for the transfer of electrons between GDH and ferrocenemonocarboxylic acid (FMCA) in a homogeneous system, determined using direct current (DC) cyclic voltammetry, was found to be 9.4 × 106 litres mol−1 s−1. This value of ks for GDH was more than 40 times greater than that for the flavoprotein glucose oxidase (GOD) under identical conditions. Such high catalytic activities were also observed when GDH was immobilised in the presence of an insoluble ferrocene derivative; a biosensor based on GDH was found to produce more than twice the current density of similar GOD-based electrodes. The steady-state current produced by the GDH-based electrode was limited by the enzymic reaction since methods which increased the enzyme loadings elevated the upper limit of glucose detection from 5 mM to 15 mM.The temperature, pH, stability and response characteristics of the GDH-based glucose sensor illustrate its potential usefulness for a variety of practical applications. In particular, the high catalytic activity and oxygen insensitivity of this biosensor make it suitable for in vivo blood glucose monitoring in the management of diabetes. 相似文献
13.
Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc 总被引:3,自引:0,他引:3
Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat-and batch cultures were compared. Strain PP 799 transported and phosphorylated glucose via an intact PTS, while strain PP 952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP 799 and PP 952 exhibited similar Y
Glc values. Assuming equal Y
ATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.Abbreviations PTS
phosphoenolpyruvate: carbohydrate phosphotransferase system
- HPr
histidine-containing phosphocarrier protein
- GalP
galactose permease 相似文献
14.
A platinized carbon paste prepared via electrodeposition had a preferential electrocatalytic action toward H2O2. Therefore, we have developed a new amperometric glucose biosensor based on the immobilization of glucose oxidase on to the electrochemically platinized carbon paste. The proposed biosensor is free of potential interferences due to its cathodic detection of glucose at the potential of 0.0 V (vs. Ag/AgCl). It also shows acceptable analytical performance in terms of linearity (6 × 10−5 to 1.2 × 10−2 M, r = 0.998), detection limit (2 × 10−5 M), response time (20–30 s), reproducibility (RSD = 4.4%), and storage life (t
0.80 = 45 days). All these advantages of the biosensor raise potential possibilities for its medical or other biotechnical applications. 相似文献
15.
A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen + phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml−1. The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response. 相似文献
16.
We have developed a method for separating reduced, neutral oligosaccharides by high-performance liquid chromatography on columns of MicroPak AX-5 (Varian Associates) with a mobile phase consisting of acetonitrile:H2O. Individual glucose oligomers containing from 1 to 20 glucose moieties can be separated in a single 1-h analysis with a solvent program of decreasing acetonitrile concentration. We have applied this method to both the analysis and preparative isolation of glycoprotein-derived oligosaccharides obtained by enzymatic release with endoglycosidases or chemical release by hydrazinolysis. Introduction of 3H by reduction with NaB3H4 permits the detection of subnanomole quantities of oligosaccharides. This method offers previously unattainable rapidity and resolution for the analysis of oligosaccharides. 相似文献
17.
Background
Because H2O2 is generated by various oxidase-catalyzed reactions, a highly sensitive determination method of H2O2 is applicable to measurements of low levels of various oxidases and their substrates such as glucose, lactate, glutamate, urate, xanthine, choline, cholesterol and NADPH. We propose herein a new, highly sensitive method for the measurement of H2O2 and glucose using fluorescence correlation spectroscopy (FCS).Methodology/Principal Findings
FCS has the advantage of allowing us to determine the number of fluorescent molecules. FCS measures the fluctuations in fluorescence intensity caused by fluorescent probe movement in a small light cavity with a defined volume generated by confocal illumination. We thus developed a highly sensitive determination system of H2O2 by FCS, where horseradish peroxidase (HRP) catalyzes the formation of a covalent bond between fluorescent molecules and proteins in the presence of H2O2. Our developed system gave a linear calibration curve for H2O2 in the range of 28 to 300 nM with the detection limit of 8 nM. In addition, by coupling with glucose oxidase (GOD)-catalyzed reaction, the method allows to measure glucose in the range of 80 nM to 1.5 µM with detection limit of 24 nM. The method was applicable to the assay of glucose in blood plasma. The mean concentration of glucose in normal human blood plasma was determined to be 4.9 mM.Conclusions/Significance
In comparison with commercial available methods, the detection limit and the minimum value of determination for glucose are at least 2 orders of magnitude more sensitive in our system. Such a highly sensitive method leads the fact that only a very small amount of plasma (20 nL) is needed for the determination of glucose concentration in blood plasma. 相似文献18.
Johan A. Hellebust 《Journal of phycology》1971,7(1):1-4
Growth rate as well as rate of glucose uptake of C. cryptica depends on glucose concentration in the medium according to saturation kinetics. The K g for growth is 1.9 × 10?5 M, and the K t, for glucose transport is 5.8 × 10?5 M. The maximum growth rate in the dark on glucose is considerably slower than the light-saturated growth rate at the same temperature, and does not appear to be determined by the capacity of the cell for glucose uptake. The glucose transport process is highly specific, and depends on energy metabolism. The Q 10 for the process is 2.2 (15–2.5 C). Glucose taken up by the cells is almost, quantitatively phosphorylated within 10 min, either through the transport process itself or by a high affinity kinase system in the cells. 相似文献
19.
《Bioscience, biotechnology, and biochemistry》2013,77(2):426-429
The author modified a respiratory gas analyzer to analyze the respiratory 13CO2 of 12 small laboratory animals all at once. To investigate the practical use of this system, mice were orally (OR) or intravenously (IV) given glucose solutions containing three different amounts of 13C-labeled glucose. Expired 13CO2 derived from exogenous glucose was detected within 10 minutes after administration in OR mice, but about 30 minutes in IV mice. The height of the peak of 13CO2 expiration was correlated with the administered 13C-glucose mass. 相似文献
20.
Glucose transport by Hymenolepis diminuta was inhibited when Cl? in the bathing medium was replaced with acetate (C2H3O2Post?), but was unaffected when Cl? was replaced with SCN?. The relative effectiveness of the anions to inhibit influx of 7.4 mM Cl? in the presence of 1 mM glucose was SCN? > Cl? > C2H3O2Post?. Glucose stimulated the influxes of 120 mM Cl? and SCN?, but had little effect on 120 mM C2H3O2Post? influx. While the diffusion rates of the anions were C2H3O2Post? > SCN? = Cl?, the preference of the glucose transport system for the anions was SCN? > Cl? > C2H3O2Post?. Efflux of Cl? was not affected by the rate of glucose influx. Finally, microelectrode recordings of worms anesthetized with 2 mM arecoline revealed a transmembrane potential (TMP) of ?45 ± 3.6 mV (inside negative). Three to four minutes after addition of glucose (5 mM) there was a progressive hyperpolarization of the TMP to ?58 mV. A revised model of the glucose transport system that is consistent with previous observations on this organism is proposed. 相似文献