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1.
Abstract

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpqruvyl group from phospho(enol)pqruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may hate potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction. GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P. suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction. GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might he formed between the enzyme. EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.  相似文献   

2.
The binding of trimethoprim and [1,3,2-amino-15N3]-trimethoprim to Lactobacillus casei dihydrofolate reductase has been studied by 15N and 1H NMR spectroscopy. 15N NMR spectra of the bound drug were obtained by using polarisation transfer pulse sequences. The 15N chemical shifts and 1H-15N spin-coupling constants show unambiguously that the drug is protonated on N1 when bound to the enzyme.The N1-proton resonance in the complex has been assigned using the 15N-enriched molecule. The temperature-dependence of the linewidth of this resonance has been used to estimate the rate of exchange of this proton with the solvent: 160±10s-1 at 313 K, with an activation energy of 75 (±9) kJ·mole-1. This is considerably faster than the dissociation rate of the drug from this complex, demonstrating that there are local fluctuations in the structure of the complex.  相似文献   

3.
The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpyruvyl group from phospho(enol)pyruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may have potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction, GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P, suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction, GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might be formed between the enzyme, EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.  相似文献   

4.
13C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1–2 ppm, although T2 measurements indicate that 0.1–0.2 ppm could be obtained. We have prepared a DMPC – 13C4-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90° (or of the magic angle) with B0. We have measured T2s, CSAs, and linewidths for the choline 13C--methyl, the cholesterol-C4 carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of B0 field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and 13C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of ±0.30° ), 13C linewidth of 0.2–0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90°, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 ± 2 Hz between the choline methyl carbons was determined.  相似文献   

5.
This study is designed to examine the participation of the major red cell membrane protein, band 3 protein, in the chain which transmits information from the cardiac glycoside site on the external face of the cell (Na+ + K+)-ATPase to the megadalton glycolytic enzyme complex within the cell. The experiments show that the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, affects the resonance of 2,3-diphosphoglycerate, as does the cardiac glycoside cation transport inhibitor, ouabain. Resonance shifts induced by the cardiac glycoside alone are modulated by addition of the anion transport inhibitor which indicates that there is coupling in the red cell between the (Na+ + K+)-ATPase and band 3 protein. Band 3 protein was separated from the membrane and partially purified following the technique of Yu and Steck ((1975) J. Biol. Chem. 250, 9170–9175). When glyceraldehyde-3-phosphate dehydrogenase was added to the separated band 3 protein preparation, addition of cardiac glycosides caused shifts in the 31P resonance of glyceraldehyde 3-phosphate. These experiments indicate that there is coupling between the (Na+ + K+)-ATPase and band 3 protein in the separated preparation and suggest that the anion and cation transport systems may be closely related spatially and functionally in the intact red cell.  相似文献   

6.
Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme–DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is PvuII endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here 31P NMR spectroscopy is applied to demonstrate the unique spectral response of DNA to sequence-specific protein interactions. The 31P NMR spectrum of a noncognate DNA exhibits only spectral broadening upon the addition of enzyme. However, when enzyme is added to target DNA, a number of 31P resonances shift dramatically. The magnitudes of the chemical shifts (2–3 ppm) are among the largest observed. Site-specific substitution with phosphoramidates and phosphorothioates are used analyze these effects. While such spectral features have been interpreted as indicative of DNA backbone distortions, FRET analysis indicates that this does not occur in PvuII-cognate DNA complexes in solution. The distinct 31P spectral signature observed for cognate DNA mirrors that observed for the enzyme, underscoring the unique features of cognate complex formation.  相似文献   

7.
Erythrocyte membrane potential can be estimated by measuring the transmembrane concentration (activity) distribution of a membrane-permeable ion. We present here the study of difluorophosphate (DFP) as a 19F NMR probe of membrane potential. This bicarbonate and phosphate analogue has a pKa of 3.7±0.2 (SD, n = 4) and therefore exists almost entirely as a monovalent anion at physiological pH. When it is incorporated into red cell suspensions, it gives two well resolved resonances that arise from the intra- and extracellular populations; the intracellular resonance is shifted 130 Hz to higher frequency from that of the extracellular resonance. Hence the transmembrane distribution of DFP is readily assessed from a single 19F NMR spectrum and the membrane potential can be calculated using the Nernst equation. The membrane potential was independent of, DFP concentration in the range 4 to 59 mM, and haematocrit of the cell suspensions of 31.0 to 61.4%. The membrane potential determined by using DFP was 0.94±0.26 of that estimated from the transmembrane pH difference. The distribution ratios of intracellular/extracellular DFP were similar to those of the membrane potential probes, hypophosphite and trifluoroacetate. DFP was found to be transported across the membranes predominantly via the electrically-silent pathway mediated by capnophorin. Using magnetization transfer techniques, the membrane influx permeability-coefficient of cells suspended in physiological medium was determined to be 7.2±2.5 × 10–6 cm s–1 (SD, n=4). Offprint requests to: P. W Kuchel  相似文献   

8.
Du W  Liu WS  Payne DJ  Doyle ML 《Biochemistry》2000,39(33):10140-10146
The inhibitor binding synergy mechanism of the bi-substrate enzyme Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been investigated with a linkage thermodynamics strategy, involving direct binding experiments of one ligand conducted over a range of concentration of the other. The results demonstrate that binding of the inhibitor glyphosate (GLP) is highly synergistic with both a natural substrate shikimate-3-phosphate (S3P) and activating monovalent cations. The synergy between GLP and S3P binding was determined to be 1600-fold and is in qualitative agreement with previous work on Escherichia coli EPSPS. The binding molar ratios of S3P and GLP were measured as 1.0 and 0.7 per EPSPS, respectively. Monovalent cations that have been shown previously to stimulate S. pneumoniae EPSPS catalytic activity and its inhibition by GLP were found here to exhibit a similar rank-order with respect to their measured GLP binding synergies (ranging from 0 to > or =3000-fold increase in GLP affinity). The cation specificity and the sub-millimolar concentrations where these effects occur strongly suggest the presence of a specific cation binding site. Analytical ultracentrifugation data ruled out GLP-binding synergy mechanisms that derive from, or are influenced by, changes in oligomerization of S. pneumoniae EPSPS. Rather, the data are most consistent with an allosteric mechanism involving changes in tertiary structure. The results provide a quantitative framework for understanding the inhibitor binding synergies in S. pneumoniae EPSPS and implicate the presence of a specific cation binding regulatory site. The findings will help to guide rational design of novel antibiotics targeting bacterial EPSPS enzymes.  相似文献   

9.
O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of l-cysteine, the replacement of the β-acetoxy group of O-acetyl-l-serine (OAS) by a thiol. The 5′-phosphate of the PLP cofactor is very tightly bound to the enzyme; it accepts 8 hydrogen bonds from enzyme side chains and a pair of water molecules, and is in close proximity to a helix dipole. Histidine-152 (H152) is one of the residues that, via a water molecule, is responsible for positioning the 5′-phosphate. Mutation of H152 to alanine was predicted to increase the freedom of the 5′-phosphate, and as a result the cofactor, giving a decrease in the overall rate of the reaction. The H152A mutant enzyme was thus prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and 31P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. UV-visible absorbance and visible CD spectra are consistent with a shift in the ketoeneamine to enolimine tautomeric equilibrium toward the neutral enolimine in the internal Schiff base of the free enzyme (ISB), the amino acid external Schiff base (ESB), and the α-aminoacrylate intermediate (AA). 31P NMR spectra clearly indicate the presence of two conformers (presumably open and closed forms of the enzyme) that interconvert slowly on the NMR time scale in the ISB and ESB. Kinetic data suggest the decreased rate of the enzyme likely reflects a decrease in the amount of active enzyme as a result of an increased flexibility of the cofactor which results in substantial nonproductive binding of OAS in its external Schiff base, and a stabilization of the external Schiff bases of OAS and S-carboxynitrophenyl-l-cysteine. The nonproductive binding and stabilization of the external Schiff bases are thus linked to the shift in the tautomeric equilibrium and increase in the rate of interconversion of the open and closed forms of the enzyme. The location of the 5′-phosphate in the cofactor-binding site determines additional interactions between the cofactor and enzyme in the closed (ESB) form of the enzyme, consistent with an increased rate of interconversion of the open and closed forms of the enzyme upon increasing the rate of flexibility of the cofactor.  相似文献   

10.
We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to  ∼ 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-13C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-13C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional 13C-13C spectrum.  相似文献   

11.
It has been reported recently that the phosphorylated form of the immunomodulator FTY720 activates sphingosine 1-phosphate G protein-coupled receptors [1] and [2]. Therefore, understanding the biology of this new class of receptors will be important in clarifying the immunological function of bioactive lysosphingolipid ligands. The S1P4 receptor has generated interest due to its lymphoid tissue distribution. While the S1P4 receptor binds the prototypical ligand, S1P, a survey of other lysosphingolipids demonstrated that 4d-hydroxysphinganine 1-phosphate, more commonly known as phytosphingosine 1-phosphate (PhS1P), binds to S1P4 with higher affinity. Using radiolabeled S1P (S133P), the affinity of PhS1P for the S1P4 receptor is 1.6 nM, while that of S1P is nearly 50-fold lower (119±20 nM). Radiolabeled PhS1P proved to be superior to S133P in routine binding assays due to improved signal-to-noise ratio. The present study demonstrates the utility of a novel radiolabeled probe, PhS133P, for in vitro studies of the S1P4 receptor pharmacology.  相似文献   

12.
Summary According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patchclamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K+ channels and a sustained activation of anion channels. The K+ channels are inwardly rectifying with a single-channel slope conductance of 56 ± 3 pS at –50 mV (cell negative) and of 29 ± 2 pS at 0 mV PD across the patch 150 mmol/liter K+ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 ± 4 pS at –50 mV (cell negative) and of 25 ± 3 pS at 0 mV PD across the patch (150 mmol/liter Cl in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1–10 mol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.The authors gratefully acknowledge the valuable discussion with Drs. P. Deetjen, E. Wöll and F. Friedrich, the skilled technical assistance of G. Siber and S. David, and the excellent mechanic and electronic support by K.-H. Streicher, Ing. M. Hirsch and M. Plank. This study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung, Grant No. P5813 and P6792M.  相似文献   

13.
Potentiometric, conductometric and 31P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP6 ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP6 aggregates. 31P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T1 relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP6 molecule results in an unstable heterogeneous system.  相似文献   

14.
Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys66 as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. 1H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys66 for the epimerization reaction with participation of neighboring Arg43, Asp126, and Glu180 residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.  相似文献   

15.
C, N CP MAS and high resolution multinuclear NMR study of methyl

Four new derivatives of methyl

were studied by 1H, 13C, 15N NMR in CDCl3 solutions and by 13C, 15N NMR in the solid state. The replacement of one aryl substituent by another has no influence on the proton and carbon chemical shifts within the sugar moiety, in solution. The differences in 13C chemical shifts Δ = δliquid - δsolid are significant for C-3 (deshielding of -3.4 to -3.8 ppm), C-5 and OMe but not observed for C-2, where the ureido substituent is linked, thus indicating that this fragment of the structure is rigid. The values of Δ in 15N chemical shifts of N-3′ are -2.3 to -2.8 ppm (increase of shielding in the solids); the effect of replacement of substituent at aromatic ring is larger than the contribution of intermolecular H-bond interaction. The values of 15.5–16.1 Hz for 1JC-1′-N and 21.2–21.5 Hz for 1JCO-N indicate that the two C---N-3′ bonds are of significant double bond character.  相似文献   

16.
The effect of lipid peroxidation on the affinity of specific active sites of Na+, K+-ATPase for ATP (substrate), K+ and Na+ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl2 at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na+, K+-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K+ and Na+ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na+, K+-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K+ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na+, K+-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.  相似文献   

17.
Although hypercholesterolemia is a well-known risk factor for atherosclerosis, little is known about the effect of hypercholesterolemia on cardiac contractile function. The objective of this study was to examine the effect of hypercholesterolemia on myocardial contractility. Fifteen New Zealand white rabbits were fed standard chow (control group) and another 15 were fed a cholesterolenriched diet (HC group) for 12 weeks. The contractile response of ventricular muscle strips was measured in various extracellular calcium concentrations and at different pacing rates. The whole-cell calcium current recording, and mRNA and protein levels of cellular calcium-handling proteins were also analyzed. With 2 mM Ca2+ and stimulation at 3 Hz, the contractile force of HC strips was less than that of the controls (3.63±0.20 vs. 4.61±0.50 mN, p<0.05). The time to peak tension was longer for HC strips (93.3±2.16 vs. 82.2±2.81 ms, p < 0.05). The peak L-type calcium inward current density was slightly higher in HC myocytes but did not reach statistical significance (–14.90±0.94 vs. –12.44±0.84 pA/pF, p=0.15). The mRNA level of sarcoplasmic reticulum Ca2+-ATPase (SERCA), normalized to GAPDH, was significantly lower in the HC than that in the control group (2.85±0.14 vs. 7.67±0.67, p<0.05), as was the ryanodine receptor (RyR; 0.42±0.06 vs. 0.71±0.13, p<0.05). The mRNA of the Na+/Ca2+ exchanger (NCX) was statistically higher in the HC group (0.90±0.12 vs. 0.48±0.05, p<0.05). Western blot experiments revealed that protein expression of SERCA in the HC strips decreased, but that of the NCX increased. The protein expression of the dihydropyridine receptor was similar between these two groups. We concluded that hypercholesterolemia results in suppression of the maximal contractile function and in a longer systolic contractile time course. These changes may partially be mediated through a decrease in SERCA and RyR but an increase in NCX expression.  相似文献   

18.
Complexation of d-gluconate (Gluc) with Ca2+ has been investigated via 1H, 13C and 43Ca NMR spectroscopy in aqueous solutions in the presence of high concentration background electrolytes (1 M ? I ? 4 M (NaCl) ionic strength). From the ionic strength dependence of its formation constant, the stability constant at 6 ? pH ? 11 and at I → 0 M has been derived (). The protonation constant of Gluc at I = 1 M (NaCl) ionic strength was also determined and was found to be log Ka = 3.24 ± 0.01 (13C NMR) and log Ka = 3.23 ± 0.01 (1H NMR). It was found that 1H and 13C NMR chemical shifts upon complexation (both with H+ and with Ca2+) do not vary in an unchanging way with the distance from the Ca2+/H+ binding site. From 2D 1H-43Ca NMR spectra, simultaneous binding of Ca2+ to the alcoholic OH on C2 and C3 was deduced. Molecular modelling results modulated this picture by revealing structures in which the Gluc behaves as a multidentate ligand. The five-membered chelated initial structure was found to be thermodynamically more stable than that derived from a six-membered chelated initial structure.  相似文献   

19.
The roles of the Na+/H+ exchange system in the development and cessation of reperfusion induced ventricular arrhythmias were studied in the isolated perfused rat heart. The hearts were perfused in the working heart mode with modified Krebs Henseleit bicarbonate (KHB) buffer and whole heart ischemia was induced by a one-way ball valve with 330 beat/min pacing. Ischemia was continued for 15 min followed by 20 min of aerobic reperfusion (control). Amiloride (1.0mM), an inhibitor of the Na+/H+ exchange system, was added to the KHB buffer only during reperfusion (group B) or only during ischemic periods (group C). Electrocardiographic and hemodynamic parameters were monitored throughout the perfusion. Coronary effluent was collected through pulmonary artery cannulation and PO2, PCO2, HCO 3 and pH were measured by blood-gas analyzer.The incidence of reperfusion induced ventricular arrhythmias was 100%, 100% and 0% in control, group B and group C, respectively. The mean onset time of termination of reperfusion arrhythmias was significantly shorter in group B than in control. PCO2 increased from 39.0±0.9 to 89.3±6.0 mmHg at the end of ischemia in control and from 40.6±0.4 to 60.5±5.8 in group C, the difference between groups was statistically significant. HCO 3 level decreased from 21.8±0.1 to 18.3±0.5 mmol/l in control, however, this decrease was significantly inhibited in group C (from 22.0±0.5 to 20.3±0.2). The increase in PCO2 and the decrease in HCO 3 in group B were similar over time to those observed in control. The decrease in pH produced by ischemia was marked in control (from 7.35±0.01 to 6.92±0.04) and group B (from 7.34±0.01 to 6.94±0.02), whereas a decrease in pH was significantly prevented in group C (from 7.34±0.01 to 7.15±0.04). There were no significant differences in PCO2, HCO 3 or pH among the three groups during reperfusion.These experiments provide evidence that amiloride significantly prevented the incidence of reperfusion arrhythmias when added only during ischemia and significantly terminated reperfusion arrhythmias when added only during reperfusion. Amiloride may prevent a decrease in pH, due to alterations in PCO2 and/or HCO 3 . These changes in PCO2 and HCO 3 might be indirectly influenced by inhibition of the Na+/H+ exchange system via Cl/HCO 3 exchange. The mechanism by which amiloride terminates reperfusion arrhythmias seems to involve electrophysiological effects which were not directly addressed in this experiment.  相似文献   

20.
Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 μM [γ-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7–15 μM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.  相似文献   

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