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1.
Kinetic studies of the reverse reaction catalysed by adenosine triphosphate–creatine phosphotransferase. The inhibition by magnesium ions and adenosine diphosphate
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1. Kinetic investigations of the reaction catalysed by ATP–creatine phosphotransferase have been carried out. 2. No firm conclusions could be reached about the reaction of Mg2+ at the nucleotide-binding site of the enzyme. The value of the kinetic constant for this reaction depends on the value used for the apparent stability constant of the metal ion–nucleotide complex and, to a smaller extent, on the method of plotting the results. 3. At higher concentrations Mg2+ is a non-competitive inhibitor of the enzyme with respect to both MgADP− and phosphocreatine. 4. ADP3− is a competitive inhibitor of the enzyme with respect to MgADP− and a non-competitive inhibitor with respect to phosphocreatine. 5. The concentration of phosphocreatine has little, if any, effect on the kinetic constants for the nucleotide reactants. 相似文献
2.
Amino acid effector binding to rabbit muscle pyruvate kinase 总被引:1,自引:0,他引:1
l-Phenylalanine, an allosteric inhibitor of rabbit muscle pyruvate kinase, is shown to bind to the tetrameric enzyme in a ratio of 4 moles effector per mole of tetramer. This binding is slightly cooperative in the absence of divalent cation activators, but the cooperativity is strongly increased when measured in the presence of 2.5 mm Mg2+ or Mn2+. The effector affinity is somewhat decreased under these conditions. l-Alanine was known to antagonize all measured phenylalanine effects and is shown here to also bind to 4 sites on the protein. The binding is noncooperative and little affected by the presence of the divalent activating cations. Competition experiments with phenylalanine and alanine suggest competition for the same site. Substrate kinetic measurements at P-enolpyruvate and Mg2+ concentrations under 100 μm show considerable inhibition of the enzyme at phenylalanine concentrations around 100 μm, near the serum levels of the free amino acid. The approach to the phenylalanine-inhibited velocity occurs with half-times less than 1 sec. 相似文献
3.
Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg2+ or Mn2+ indicate a biphasic response to divalent cations. Apparent Km values of 23 μm for free Mg2+ and 3.3 μm for free Mn2+ are obtained at low levels of added metal, while Km values of 0.50 mm for free Mg2+ and 0.43 mm for free Mn2+ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg2+-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent Km values for free ATP of 6.0 and 1.4 μm with Mg2+ and Mn2+, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that Km values for free ATP of 8.0 and 2.0 mm are obtained for Mg2+ and Mn2+, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg2+-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with Ki values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn2+ per 469,000 g of protein. The Mn2+ binding studies yield a KD for Mn2+ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn2+ at low Mn2+ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn2+ per single high affinity Mn2+ site. The KD for Mn2+ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn2+ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn2+ in the presence of the enzyme is also consistent with the tight binding of a single Mn2+ ion per 469,000 Mr protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn2+ at the single tight site, ?b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn2+ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg2+-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg2+-ATPase described here are discussed. 相似文献
4.
Jaime Andrés Rivas-Pardo Andrés Caniuguir Christian A.M. Wilson Jorge Babul Victoria Guixé 《Archives of biochemistry and biophysics》2011,(1):60
The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme. Only Mn2+ and Mg2+ can fulfill this role binding to the same activating site but the affinity for Mn2+ is 13-fold higher compared to that of Mg2+. The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg2+ binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn2+ and metal–Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn2+ as well as the metal–nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence. 相似文献
5.
The protein-sensitized fluorescence of Tb3+ was used as a probe for cation binding sites on synaptic vesicles. Competition studies show that the order of affinity for the sites is Cu2+ > Mn2+ > Ca2+ > Mg2+ and Zn2+ is inactive. Fluorescence quenching studies indicate that the site is superficial and the effect of pH suggests that histidine is involved in the binding. Measurements of enzyme activities in the presence of lanthanides reveal that the metal binding site identified by Tb3+ fluorescence is not the Cu2+ site associated with dopamine-β-hydroxylase. Terbium inhibits Ca2+-stimulated ATPase but not Mg2+-stimulated ATPase activities of the synaptic vesicle fraction. A kinetic analysis indicates that the site monitored by Tb3+ fluorescence may be a component of the Ca2+-stimulated ATPase. It is also suggested that Mg2+ and especially Cu2+ may bind to the sites in vivo, serving as a bridge between vesicles and other synaptic components such as the presynaptic plasma membrane. 相似文献
6.
Jared C. Cochran Morgan E. Thompson F. Jon Kull 《The Journal of biological chemistry》2013,288(39):28312-28323
G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg2+) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg2+- or Mn2+-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn2+, providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn2+ rescues this effect to near wild-type activity. 2′(3′)-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region. 相似文献
7.
31P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg2+, Mn2+, and Co+2, with the phosphate groups of DNA. Mg2+ is found to have no significant effect on any of the 31P-nmr parameters (chemical shift, line-width, T1, T2, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn2+ and Co2+, on the other hand, significantly change the 31P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn2+ and Co2+, respectively, corresponding to only 15 ± 5% of the total bound Mn2+ and Co2+ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the 31P relaxation rates of DNA in the absence and presence of Mn2+ and Co2+ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10?7 and 1.4 × 10?5 s for Mn2+ and Co2+, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg2+ ions is 3–4 times weaker than the binding of either Mn2+ or Co2+. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (~15%), the higher binding strength of Mn2+ and Co2+ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg2+ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ~100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented. 相似文献
8.
Hideyuki Kajiwara Andrew M. Hemmings Hisashi Hirano 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,687(2):726
A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg2+. Mobility was also affected in the presence of La3+, Mn2+, Ca2+ and Zn2+ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca2+-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg2+ and Mn2+ ions. 相似文献
9.
The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg2+, Co2+, Mn2+, Ni2+, and Zn2+ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg2+ and varying concentrations of Mn2+ or of Co2+. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co2+ and varying Mn2+. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn2+ is a better activator of the enzyme than is Mg2+, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni2+ or Zn2+ with Mg2+ suggests that Ni2+ and Zn2+ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg2+. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity. 相似文献
10.
S.K. Mukerji 《Archives of biochemistry and biophysics》1977,182(1):352-359
Differences in the kinetic properties of corn leaf phosphoenolpyruvate (PEP) carboxylase isoenzymes were found, depending on whether Mg2+ or Mn2+ was used as the metal cofactor of the reaction. Also, differences in kinetic constants with respect to Mg2+ and Mn2+ were noticed between the two isoenzymes which further differentiates the two proteins. The catalytic activity of the enzyme in the Mg2+-activated system was dependent on a PEP-Mg2+ complex and not on the concentration of free Mg2+ or free PEP. Kinetics in the presence of total Mg2+ and those of PEP-Mg2+ suggest a negative cooperative effect with respect to ligand binding with concurrent progressive substrate activation. Magnesium ions, thus, have a special regulatory role in the corn leaf PEP carboxylase reaction. 相似文献
11.
《Journal of molecular biology》2019,431(4):673-686
PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn2+, a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg2+, the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase. 相似文献
12.
B S Dugal 《Biochemical and biophysical research communications》1973,54(4):1603-1610
Kinetic methods have been used to determine whether Mg2+ and MgATP2? play an important role in regulating pigeon kidney pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1.). Mg2+ not only forms a complex with ATP4? (MgATP2?) but is also required for the enzyme activation (and probably for the binding of MgATP2? to this enzyme). Contrary to the results of other investigators, the MgATP2? complex was not found to activate pigeon kidney pyruvate carboxylase. We could not demonstrate homotropic cooperativity with MgATP2? complex. Excess Mg2+ induces allosteric stimulation of the enzymatic activity at different concentrations of MgATP2?. With different Mg2+ concentrations, changes also occured in the apparent Km? and Vmax-values. Without excess of Mg2+ only about 2 % of the total enzymic activity available could be demonstrated in the presence of MgATP2?. It is concluded that Mg2+ exhibits a homotropic cooperative effect and is required for the activation of this enzyme. Mg2+ may bind either to a specific effector site, at the active site, or at the binding site for MgATP2? which is capable of functioning as an effector site and in this way facilitates the carboxylation of pyruvate. 相似文献
13.
Isocitrate dehydrogenase kinase/phosphatase (AceK) is a bifunctional enzyme with both kinase and phosphatase activities that are activated by Mg2+. We have studied the interactions of Mn2+and Mg2+ with AceK using isothermal titration calorimetry (ITC) combined with molecular docking simulations and show for the first time that Mn2+ also activates the enzyme activities. However, Mn2+ and Mg2+ exert their effects by different mechanisms. Although they have similar binding constants (of 1.11?×?105 and 0.98?×?105 M?1, respectively) for AceK and induce conformational changes of the enzyme, they do not compete for the same binding site. Instead Mn2+ appears to bind to the regulatory domain of AceK, and its effect is transmitted to the active site of the enzyme by the conformational change that it induces. The information in this study should be very useful for understanding the molecular mechanism underlying the interaction between AceK and metal ions, especially Mn2+ and Mg2+. 相似文献
14.
Magnetic resonance studies of the interaction of divalent metal cations with 2,3-bisphosphoglycerate
Raj K. Gupta Jeffrey L. Benovic 《Biochemical and biophysical research communications》1978,84(1):130-137
The binding of Mg2+ to intracellular 2,3-bisphosphoglycerate in the human red blood cell is significant to the function of the cell. We have studied interactions of Mg2+ and Mn2+ with 2,3-bisphosphoglycerate by magnetic resonance spectroscopy. The results of this study reveal the presence of two independent divalent metal cation binding sites of similar affinity (KD = 3.0 ± 0.5 mM) in the 2,3-bisphosphoglycerate molecule, one on each phosphoryl group, contrary to the assumption of one metal ion binding site made in the previous literature. Over the range of their intracellular concentrations, ATP and ADP, however, possess only one metal ion site in spite of the presence of multiple phosphoryl groups. These results are consistent with the chemistry of metal-chelation which requires the formation of 5- or 6-membered rings for the stability of chelate structures. 相似文献
15.
《The International journal of biochemistry》1976,7(9-10):483-496
The kinetics of the forward reaction catalyzed by choline kinase from rat liver was studied at pH 8.0. The enzyme follows 2 alternative mechanisms according to the concentrations of reactants. Studies at low ligand concentrations are consistent with a sequential order mechanism with the reactants adding in the order: choline, MgATP−2 and Mg2+1 The apparent and true values of the corresponding kinetic parameters are reported. The possible implications of the dual role of Mg2+ on the regulation of the activity of this enzyme are discussed. 相似文献
16.
Kojiro Takahashi Henry Weiner James H.J. Hu 《Archives of biochemistry and biophysics》1980,205(2):571-578
The V of horse liver aldehyde dehydrogenase is enhanced twofold in the presence of 0.5 mm Mg2+ ions when assayed in the dehydrogenase reaction. The mechanism of this activation appears to be related to the fact the enzyme changes from functioning with half-of-the-sites reactivity to functioning with all-of-the-sites reactivity. That is, the presteady-state burst magnitude increases from 2 mol NADH formed per mole of tetrameric enzyme to 4 mol formed per mole (K. Takahashi and H. Weiner, J. Biol. Chem., 1980, 255, 8206–8209). Whether this twofold enhancement correlates, in fact, to a change from half-of-the-sites to all-of-the-sites reactivity of the enzyme by Mg2+ ions was investigated by determining the Stoichiometry of coenzyme binding by fluorescence quenching and enhancement methods in the absence and presence of the metal ions. The biphasic Scatchard plots for NAD binding to the enzyme were similar in the absence and presence of Mg2+ ions, while that of NADH binding was monophasic (-Mg2+) and biphasic (+Mg2+). In the presence of p-methoxyacetophenone, a competitive inhibitor for substrate, the stoichiometric titration of coenzyme binding to the ternary complexes (enzyme-NAD(H)-inhibitor) revealed that only 2 mol of NAD or NADH bind in the absence of Mg2+ ions but 4 bind per mole of tetrameric enzyme in the presence of added metal. The fluorescence intensity of NAD's fluorescent derivative, 1,N6-ethenoadenine dinucleotide, bound to the enzyme was also doubled by the addition of Mg2+ ions.The combined binding data show that the stoichiometry of coenzyme binding to aldehyde dehydrogenase in the ternary complex increases from 2 to 4 mol binding per mole of tetrameric enzyme with the addition of Mg2+ ions. This increase in stoichiometry corresponds to the observed changes of burst magnitude obtained from the presteady-state and V in the steady-state kinetics assays. From both results of the kinetics and stoichiometry, we show that horse liver aldehyde dehydrogenase exhibits half-of-the-sites reactivity when in the tetrameric state in the absence of Mg2+ ions, and all-of-the-sites reactivity in the dimeric state in the presence of the metal. 相似文献
17.
Dai-Eun Sok 《Journal of neurochemistry》1999,72(1):355-362
Abstract : Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositollinked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H2O2. Inclusion of Cu2+ in the incubation with 1 mM H2O2 led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H2O2/Cu2+ system was much more effective than the H2O2/Fe2+ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H2O2/Cu2+-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metalcatalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H2O2/Cu2+. A similar protection was also expressed by divalent metal ions such as Mg2+ or Mn2+. Separately, it was found that H2O2/Fe2+-induced inactivation was prevented by p-nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation. 相似文献
18.
P. V. Vrzheshch 《Biophysics》2016,61(3):373-379
A quantitative evaluation of the accuracy of the rapid-equilibrium assumption in steady-state enzyme kinetics was obtained for a multipath arbitrary enzyme mechanism with a number of equilibrium segments. Explicit expressions for estimating the contribution of any equilibrium segment to the accuracy of the rapid-equilibrium assumption were obtained. This allowed us to determine the accuracy of the rapid-equilibrium assumption (Δ) in general: 1 + Δ = (1 + Δ1)(1 + Δ2)... (1 + Δk), where Δ1, Δ2,..., Δk is the contribution of each individual equilibrium segment. The accuracy depends only on the structure and properties of equilibrium segments, which have been accounted for in the rapid-equilibrium assumption, but it is independent of the number of paths in the mechanism of the enzymatic reaction and on the structure and properties of the remaining part (steady-state) of the kinetic scheme. 相似文献
19.
Two types of Na+-independent Mg2+ efflux exist in erythrocytes: (1) Mg2+ efflux in sucrose medium and (2) Mg2+ efflux in high Cl− media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na+-independent Mg2+ efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K+,Cl−- and Na+,K+,Cl−-symport, Na+/H+-, Na+/Mg2+-, Na+/Ca2+- and K+(Na+)/H+ antiport, Ca2+-activated K+ channel and Mg2+ leak flux. We suggest that, in choline Cl medium, Na+-independent Mg2+ efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg2+ efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg2+ to the same degree. The Kd value for inhibition of [14C]choline efflux and for inhibition of Mg2+ efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg2+ efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg2+ efflux was reduced to the same degree by these inhibitors as was the [14C]choline efflux. 相似文献
20.
High-conductance, Ca2+-activated K+ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid
bilayers to examine the effect of Mg2+ on the single-channel properties. Mg2+ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular
side of the channel. In contrast to other K+ channels, Mg2+ does not cause rectification of current through colonic Ca2+-activated K+ channels. In addition, cytoplasmic Mg2+ decreases the reversal potential of the channel. The Mg2+-induced decrease in channel conductance is relieved by high K+ concentrations, indicating competitive interaction between K+ and Mg2+. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect
is unspecific. The inhibition of channel current by Mg2+ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance.
But in addition to charge, other properties appear to be necessary for channel inhibition, as Na+ and Ba2+ are no (or only weak) inhibitors. Mg2+ and possibly other cations may play a role in the regulation of current through these channels.
Received: 25 August 1995/Revised: 16 November 1995 相似文献