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1.

Purpose

To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated.

Method

Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg2+ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg2+ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca2+ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca2+ turnover was observed under a laser scanning confocal microscope.

Results

The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca2 + fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes.

Conclusion

GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.  相似文献   

2.
Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg2+ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg2+ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE.  相似文献   

3.
目的:探讨线粒体CB1受体(mitochondrial cannabinoid receptor1,mtCB1)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法:原代培养新生的Wistar大鼠海马神经元,将培养至第8天的海马神经元采用随机数字表分为5组(n=60):正常组(N组):正常培养,不做任何处理;缺氧复氧组(H/R组):采用氧糖剥夺法构建海马神经元缺氧复氧损伤模型,缺氧6h,复氧20 h;缺氧复氧组+ACEA+AM251组(H/R+ACEA+AM251组):缺氧6 h结束后立即加入ACEA和AM251,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+ACEA+Hemopressin(H/R+ACEA+Hemo组):缺氧6h结束后立即加入ACEA和Hemopressin,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+赋形剂组(H/R+V组):同样于缺氧6h结束后立即加入二甲基亚砜(DMSO),终浓度0.1%,复氧20 h。使用激光共聚焦显微镜检测细胞内Ca~(2+)的浓度,流式细胞仪检测细胞凋亡率,Western blot检测凋亡诱导因子(AIF)、线粒体分裂相关蛋白Drp1、Fis1,细胞凋亡相关蛋白细胞色素C(Cytc)和Rho相关的卷曲蛋白激酶1(ROCK1)的表达。结果:与N组相比,H/R组、H/R+ACEA+AM251组、H/R+ACEA+Hemo组和H/R+V组的细胞内Ca~(2+)浓度、细胞凋亡率、以及AIF、Drp1、Fis1、Cytc、ROCK1蛋白的表达水平均明显增加(P0.05);与H/R组相比,H/R+ACEA+Hem组上述各检测指标明显降低(P0.05),H/R+ACEA+AM251组和H/R+V组各指标比较差异无统计学意义(P0.05)。结论:线粒体CB1受体(mtCB1受体)可能通过降低细胞内ROS的含量来减少细胞内Ca~(2+)浓度和ROCK1的表达,进而抑制线粒体分裂,并最终减轻海马神经元缺氧复氧损伤。  相似文献   

4.
When suspended in media lacking free Mg2+, chloroplasts from young pea plants (Pisum sativum CV Progress No. 9) lose 25 to 75% of their stromal Mg2+ content to the medium, without breakage. This effect amounts for the inhibition of protein synthesis in the dark by ATP in excess of the Mg2+ provided, since free ATP chelates Mg2+. The rate of loss is from 1 to 4.5 microgram-atoms Mg2+/milligram Chl/hour; and depleted chloroplasts take up Mg2+ from the medium at even faster rates, to a total amount not much more than that present originally (0.8 to 1.8 microgram-atoms/milligram Chl with an average of 1.33 ± 0.32 μg-atoms/mg Chl). Leakage is completely prevented by 0.25 to 0.40 millimolar external Mg2+. Addition of Mg2+ at a level sufficient to prevent leakage from intact chloroplasts results in approximately 20% stimulation in light-driven protein synthesis.  相似文献   

5.
The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21(DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45°C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15–20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45°C and was completely inactivated after incubation at 65°C for 1 h.  相似文献   

6.
When samples of pea tendril tissue were incubated in the Wachstein-Meisel medium for the demonstration of adenosine triphosphatases, deposits of lead reaction product were localized between the membranes of the chloroplast envelope. The presence of Mg2+ was necessary for adenosine triphosphatase activity, and Ca2+ could not substitute for this requirement. Varying the pH of incubation to 5.5 or 9.4 inhibited enzyme activity, as did the addition of p-chloromercuribenzoic acid or N-ethylmaleimide. The adenosine triphosphatase was apparently inactivated or degraded when the plants were grown in the dark for 24 hours prior to incubation. The enzyme was substrate-specific for adenosine triphosphate; no reaction was obtained with adenosine diphosphate, uridine triphosphate, inosine triphosphate, p-nitrophenyl phosphate, and sodium β-glycerophosphate. Sites of nonspecific depositions of lead are described. The adenosine triphosphatase on the chloroplast envelope may be involved in the light-induced contraction of this organelle.  相似文献   

7.
分离出一周SD乳鼠海马组织,进行离体海马细胞培养;在培养基中加入神经营养素-6成熟肽片段,通过神经元特异性烯醇化酶免疫组化染色,计数存活海马神经元数量,与对照组比较,观察神经营养素-6对神经元存活的促进作用。结果显示,神经营养素-6实验组海马神经元存活数目显著高于对照组。表明人源性神经营养素-6可以促进体外培养神经元的存活。  相似文献   

8.
Calcium ions (Ca2+) are important second messengers in neurons. Ketamine (KETAM) is an anesthetic and analgesic, with psychotomimetic effects and abuse potential. KETAM modulates the entry of Ca2+ in neurons through glutamate receptors, but its effect on transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) channels has not been clarified. This study investigated the short-term effects of KETAM on oxidative stress and TRPM2 and TRPV1 channel gating in hippocampal and dorsal root ganglion (DRG) neurons of rats. Freshly isolated hippocampal and DRG neurons were incubated for 24 h with KETAM (0.3 mM). The TRPM2 channel antagonist, N-(p-amylcinnamoyl)anthranilic acid (ACA), inhibited cumene hydroperoxide and ADP-ribose-induced TRPM2 currents in the neurons, and capsazepine (CPZ) inhibited capsaicin-induced TRPV1 currents. The TRPM2 and TRPV1 channel current densities and intracellular free calcium ion concentration of the neurons were lower in the neurons exposed to ACA and CPZ compared to the control neurons, respectively. However, the values were not further decreased by the KETAM + CPZ and KETAM + ACA treatments. KETAM decreased lipid peroxidation levels in the neurons but increased glutathione peroxidase activity. In conclusion, short-term KETAM treatment decreased oxidative stress levels but did not seem to influence TRPM2- and TRPV1-mediated Ca2+ entry.  相似文献   

9.
Although magnesium (Mg2+) is the most abundant divalent cation in plant cells, little is known about the mechanism of Mg2+ uptake by plant roots. Here, we report a key function of Magnesium Transport6 (MGT6)/Mitochondrial RNA Splicing2-4 in Mg2+ uptake and low-Mg2+ tolerance in Arabidopsis thaliana. MGT6 is expressed mainly in plant aerial tissues when Mg2+ levels are high in the soil or growth medium. Its expression is highly induced in the roots during Mg2+ deficiency, suggesting a role for MGT6 in response to the low-Mg2+ status in roots. Silencing of MGT6 in transgenic plants by RNA interference (RNAi) resulted in growth retardation under the low-Mg2+ condition, and the phenotype was restored to normal growth after RNAi plants were transferred to Mg2+-sufficient medium. RNAi plants contained lower levels of Mg2+ compared with wild-type plants under low Mg2+ but not under Mg2+-sufficient conditions. Further analysis indicated that MGT6 was localized in the plasma membrane and played a key role in Mg2+ uptake by roots under Mg2+ limitation. We conclude that MGT6 mediates Mg2+ uptake in roots and is required for plant adaptation to a low-Mg2+ environment.  相似文献   

10.
Isolated hepatocytes converted exogenous [α-32P]ATP to cyclic [32P]AMP at high rates. This system was used for kinetic studies of the effects of glucagon, fluoride, free magnesium and free ATP4? on adenylate cyclase. In the absence or presence of glucagon, free Mg2+ activated adenylate cyclase by decreasing the Km for MgATP2? without changing V. Free ATP4? was not a potent inhibitor of adenylate cyclase and the only effect of glucagon was to increase V.Fluoride also increased the V of adenylate cyclase, but, in contrast to the results obtained with glucagon, the effect increased as the concentration of free Mg2+ increased. One explanation of the effect of fluoride, consistent with the idea that free Mg2+ activates adenylate cyclase and free ATP is not an inhibitor, is that fluoride increases the affinity of the enzyme for Mg2+. Weak inhibition of adenylate cyclase by ATP4? in the presence of fluoride cannot be excluded.  相似文献   

11.
Magnesium (Mg2+) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg2+ deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg2+ restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg2+ (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg2+ deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg2+-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP+) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg2+ deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg2+ deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg2+ deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg2+. In conclusion, Mg2+ deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg2+ deficient patients.  相似文献   

12.
Mg2+, Ca2+, Mn2+, Zn2+, and Cu content of neurons from chick embryo cortex cultivated in chemically defined serum free growth medium was determined by energy dispersive X-ray fluorescence and atomic absorption spectroscopy. The intracellular volume of cultured neurons was determined to be 2.73 l/mg. Intracellular Mn2+, Fe2+, Zn2+, and Cu2+ in the cultivated neurons were 100–200 times the concentrations in the growth medium: Mg2+ and Ca2+ were 0.9 and 1.7 mM respectively, around 20 fold higher than in growth medium. Mg2+, Fe2+, Cu2+ and Zn2+ concentrations in neurons were in the range of ca. 300–600 M, approximately 2–3 times the values previously reported in glial cells; Ca2+ and Mn2+ content of the neurons were higher by 5 and 10 fold respectively compared to glial cells. In neurons, the subcellular distribution of Fe2+, Cu2+, and Mn2+ follows the rank order: cytosol>microsomes>mitochondria; for Zn2+ the distribution differs as following: cytosol >mitochondria>microsomes. Determination of the superoxide dismutase activities in the cultivated neurons indicated that the Mn2+ linked activity predominates whereas, the Cu-Zn dependent enzyme is dominant in glial cells. Enrichment of the culture medium with Mn2+ to 2.5 M enhanced the Mn-SOD by approximately 33% but Cu2+–Zn2+ enzyme activity was not modified. The high Mn2+ content, the capacity to accumulate Mn2+, and the predominancy of the Mn–SOD form observed in neurons is in accord with a fundamental functional role for this metal ion in this type of brain cells.  相似文献   

13.
Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA3) with or without 5 millimolar CaCl2 shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca2+. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca2+. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA3 with 5 millimolar CaCl2 and layers incubated in GA3 alone. RNA isolated from layers incubated for 12 hours in GA3 with and without Ca2+ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca2+ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca2+-deprived aleurone layers was translatable. Ca2+ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.  相似文献   

14.
Microbial mineralization of carbonate is a research subject widely studied in the past decades. The magnesium ions (Mg2+), present in water systems, are a key determinant in biomineralization process of carbonate and they are widely found in calcium-based biominerals as an accessory component. However, the crystallization mechanism and morphological change of carbonate polymorphs in the presence of Mg2+ ions has not been clarified sufficiently. In this report, a series of culture experiments were performed for 50 days using the Arthrobacter sp. strain MF-2 in a M2 culture medium using Mg/Ca molar ratios (R) of 0, 1.5, 3, 6, 9, and 12 in solution. And the roles of Mg2+ ions on the crystal growth and morphological change of biogenic carbonate were investigated. Experimental results show: (1) MF-2 could induce aragonite, high-Mg calcite, and Ca-dolomite formation in M2 culture media with different R values. The increased stability of amorphous calcium carbonate suggests Mg2+ ions inhibit carbonate crystallization. (2) The mineral morphologies were varied (rod-shaped, dumbbell-shaped, cauliflower-like, spherical, etc.) in the medium with R = 1.5, but they became simple (spherical and lamellar) in high Mg2+ concentrations (Mg > 0.15 M, R > 3). (3) The increased ionic strength of Mg2+ ions in the environment has an influence on the polymorphs and morphologies of carbonate formed by controlling the metabolism of strain MF-2 and the activity of carbonic anhydrase.  相似文献   

15.
Lieberman M  Wang SY 《Plant physiology》1982,69(5):1150-1155
The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca2+, Mg2+, or Ca2+ plus Mg2+ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca2+, Mg2+, or Ca2+ plus Mg2+ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca2+, Mg2+, and Ca2+ plus Mg2+. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca2+ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.  相似文献   

16.
The effect of Mg2+ ions on inducing pyrophosphatase activity of germinating cotton (Gossypium hirsutum L.) seeds was investigated. The presence of Mg2+ ions in the germination medium markedly shortened time for the attainment of the pyrophosphatase maximum activity (T max). In the absence of Mg2+ ions in the nutrient medium, T max comprised 6.0–6.5 days, whereas in the presence of 3–5 mM Mg2+, T max was decreased to 3–4 days. An increase in the concentration of Mg2+ ions in the medium up to 5 mM resulted in an increase in pyrophosphatase activity. The effect of Mg2+ ions on the activity of a purified pyrophosphatase preparation isolated from three-day-old cotton seedlings was investigated. Mg2+ ions did not affect the rate of attainment of a maximum pyrophosphatase activity, but decreased the value of the Michaelis-Menten constant.  相似文献   

17.
This study investigated the effects of extracellular Mg2+ ([Mg2+]o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca2+ ([Ca2+]i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg2+]o, ACh evoked significant increases in amylase secretion and [Ca2+]i. Either low (0 mM) or elevated (5 and 10 mM) [Mg2+]o attenuated ACh-evoked responses. In a nominally Ca2+ free medium, elevated [Mg2+]o attenuated basal and ACh-evoked amylase secretion and [Ca2+]i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg2+]o, ACh evoked a gradual decrease in [Mg2+]i. These results indicate that the ACh-evoked Mg2+ efflux is an active process since Mg2+ has to move against its gradient. Either lidocaine, amiloride, N-methyl-d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg2+]i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg2+]i but the response was less pronounced with either [Na+]o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca2+ and Mg2+ signalling in parotid acinar cells and that ACh-evoked Mg2+ transport was not dependent upon [Na+]o.  相似文献   

18.
Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1–100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.  相似文献   

19.
Enhancement of NMDA-mediated responses by cyanide   总被引:2,自引:0,他引:2  
The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion.Abbreviations NMDA N-Methyl-D-Aspartate - EAA excitatory amino acid - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate  相似文献   

20.
The sarcoplasmic reticulum Ca2+-ATPase was reacted with vanadate in the presence of Mg2+ and EGTA, and the effect of Ca2+, Mg2+ and ATP on the kinetics of vanadate release from the enzyme vanadate complex was studied after dilution with vanadate-free media. Ca2+ increased, whereas ATP decreased the rate of vanadate release. In absence of free Mg2+ in the release media ATP was bound to the vanadate-reacted Ca2+-ATPase with high affinity (Kd 4–5 μM), and full saturation with ATP resulted in complete inhibition of vanadate release. In media containing free Mg2+, where ATP predominantly was present as MgATP, binding of the nucleotide to vanadate-reacted Ca2+-ATPase occurred with low apparent affinity. Mg2+ alone did not affect the rate of vanadate release. At saturating ATP concentrations the release rate in the presence of free Mg2+ was less inhibited than in its absence. These results indicate that uncomplexed ATP interacts with the same Mg2+ at the catalytic site, which is involved in formation of the enzyme-vanadate complex (EMgV), and thereby hinders dissociation of vanadate. Destabilization of the complex by free Mg2+ may be caused by the presence of an additional magnesium ion in the catalytic site together with ATP.  相似文献   

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