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1.
Abstract The Pseudomonas aeruginosa antibiotic supersusceptible mutant Z61 was 50–400-fold more susceptible than its wild-type parent K799 to 5 hydrophobic antibiotics. The strain Z61 outer membrane also demonstrated enhanced permeability towards a hydrophobic fluorescent probe. Strain Z61 cells had an altered cell surface, as revealed by phase-partitioning experiments, a lower amount of Lipid A phosphate, and a reduction in the number of Mg2+ binding sites in Lipid A, as demonstrated by dansyl polymyxin competition experiments. An antibiotic permation pathway directly through the outer membrane bilayer, rather than through porin proteins, is proposed for strain Z61.  相似文献   

2.
Abstract: We investigated the regulation by intracellular Ca2+ of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3H]mepyramine binding sites on intact cells accessible to the hydrophilic H1-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [3H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca2+/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H1-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of Gq protein-coupled receptors is transiently inhibited by Ca2+/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.  相似文献   

3.
The effect of aluminum on dimorphic fungi Yarrowia lipolytica was investigated. High aluminum (0.5–1.0 mM AlK(SO4)2) inhibits yeast–hypha transition. Both vanadate-sensitive H+ transport and ATPase activities were increased in total membranes isolated from aluminum-treated cells, indicating that a plasma membrane H+ pump was stimulated by aluminum. Furthermore, Al-treated cells showed a stronger H+ efflux in solid medium. The present results suggest that alterations in the plasma membrane H+ transport might underline a pH signaling required for yeast/hyphal development. The data point to the cell surface pH as a determinant of morphogenesis of Y. lipolytica and the plasma membrane H+-ATPase as a key factor of this process.  相似文献   

4.
Abstract: Calmodulin was isolated as an electrophoretically homogeneous protein from bovine posterior pituitary glands. The yield indicated that this gland is a particularly rich source. Purified bovine posterior pituitary calmodulin and bovine brain calmodulin had identical electrophoretic mobilities on 10% and 12% polyacrylamide gels. The protein was further identified by molecular weight determination and by amino acid analysis which showed that it contained trimethyllysine, one residue per molecule. Bovine posterior pituitary calmodulin was found to activate a preparation of calmodulin-deficient phosphodiesterase from bovine heart. In addition, pituitary calmodulin stimulated Ca2++ Mg2+-ATPase activity associated with a purified nerve ending plasma membrane fraction. This dependence could only be demonstrated after successive washing of the membranes with EGTA buffers, a procedure designed to remove endogenous calmodulin.  相似文献   

5.
Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca2+ concentration ([Ca2+]i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca2+ influx brought about by K+ depolarization. Similar effects were obtained with the application of SS analogues, such as d -Trp8-SS, d -Trp8- d -Cys14-SS, CGP-23996, and SMS-201995. In addition, treatment with cyclo-SS, a SS antagonist, did not alter [Ca2+]i. Experiments with selective blockers of different voltage-dependent Ca2+ channels, such as methoxyverapamil (D600) and Ω-conotoxin GVIA, demonstrated that the effects of SS on [Ca2+]i were mediated by voltage-dependent Ca2+ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca2+ influx.  相似文献   

6.
Calcium ions play an important role in the regulation of stomatal movement and the mechanism underlying this action is yet to be determined. It is suggested that guard cell plasma membrane ATPase is a target for calcium action and that this effect is mediated by calmodulin. In this study, the effects of calcium and two calmodulin antagonists on ATPase activity in a crude homogenate of Commelina communis L. guard cell protoplasts were examined. The homogenate contained Mg2+-dependent, K+-simulated ATPase activity, which was inhibited by CaCl2 while stimulated by the calmodulin antagonists, compound 48/80 and chlorpromazine. The calmodulin antagonists partially reversed the inhibitory effect of calcium ions. The results support the possibility of calmodulin involvement in the regulation of guard cell ATPase activity by calcium ions.  相似文献   

7.
Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca2+ dependence to M-phase onset. A potential target of this Ca2+ dependence may be CaM kinase-II (the multifunctional [type II] Ca2+/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34cdc2, but again only after fertilization. These changes in CaMK-II activity and p34cdc2-association after fertilization may ensure that Ca2+ signals are targeted to the M-phase machinery at the appropriate developmental times.  相似文献   

8.
Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H+-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na+ and Cl content, but no significant changes in K+ and Ca2+. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H+-pumping activities, as well as a lower passive permeability to H+ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H+-ATPase activity in salt tolerance of tomato calli is discussed.  相似文献   

9.
Calmodulin and calmodulin-mediated processes in plants   总被引:11,自引:3,他引:8  
Abstract. The Ca2+ -binding protein calmodulin is found in all plants investigated so far. The comparison of the biochemical and functional properties reveals that it is structurally conserved and functionally preserved throughout the plant and animal kingdom. Among the plant enzymes so far known to be dependent on the Ca2+ -calmodulin complex are NAD kinase(s), Ca2+ -transport ATPase, quinate: NAD+ oxidoreductase, soluble and membrane bound protein kinases, and H+ -transport ATPase. Calmodulin may play also an important role in the regulation of other cellular reactions, such as hormone-mediated processes, secretion of enzymes, and contractile mechanisms. On the basis of the NAD kinase and its regulation by light and Ca2+ -calmodulin, it is suggested that changes in the cellular, free Ca2+ concentration following stimulation may alter the metabolism of a plant cell. According to this suggestion free Ca2+ may act as a second messenger in plants much as it does in animal cells.  相似文献   

10.
The effect of phospholipids on the activity of the plasma membrane (PM) Ca2+-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca2+ concentrations (10 μ M ) acidic phospholipids did not affect the Ca2+-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca2+-ATPase activity on free Ca2+ concentration showed that acidic phospholipids increased Vmax and lowered the apparent Km for free Ca2+ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent Km for free Ca2+ of the Ca2+-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca2+-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca2+-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.  相似文献   

11.
ABSTRACT. We have determined the DNA sequence of the gene encoding the protein of the plasma membrane Ca2+-ATPase in Paramecium tetraurelia . The predicted amino acid sequence of the plasma membrane Ca2+-ATPase shows homology to conserved regions of known plasma membrane Ca2+-ATPases and contains the known binding sites for ATP (FITC), acylphosphate formation, and calmodulin, as well as the "hinge" region: all characteristics common to plasma membrane Ca2+-ATPases. The deduced molecular weight for this sequence is 131 kDa. The elucidation of this gene will assist in the studies of the mechanisms by which this excitable cell removes calcium entering through voltage gated calcium channels and the pump functions in chemosensory signal transduction.  相似文献   

12.
Abstract: The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca2+-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca2+/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca2+-induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca2+-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca2+-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca2+-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca2+/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism.  相似文献   

13.
Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA3 and VtB, at a final concentration of 10 µ M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 µ M of VtA3 and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 µ M , which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA3 and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA3 to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA3 produces rapid changes in fungal membrane permeability. It also induces H2O2 production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.  相似文献   

14.
The regulation of the H+-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H+-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H+ extrusion in the incubation medium. The osmotica-induced H+ extrusion was (1) inhibited by the inhibitor of plasma membrane H+-ATPase, erythrosin B (EB), (2) dependent on the external K+ concentration, (3) associated with a net K+ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H+-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H+ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H+-ATPase activity differ at least in some step(s)  相似文献   

15.
Ca2+ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca2+/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca2+/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca2+/calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca2+/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca2+/calmodulin-independent binding whereas it allows the Ca2+/calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca2+-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.  相似文献   

16.
Fluorescence investigations of calmodulin hydrophobic sites   总被引:3,自引:0,他引:3  
Calmodulin activation of target enzymes depends on the interaction between calmodulin hydrophobic regions and some enzyme areas. The Ca2+ induced exposure of calmodulin hydrophobic sites was studied by means of 2-p-toluidinylnaphthalene-6-sulfonate, a fluorescent probe. Scatchard and Job plots showed that the calmodulin-Ca42+ complex bound two molecules of this hydrophobic probe, with KD congruent to 1.4 X 10(-4) M. These sites are not totally exposed until calmodulin has bound four Ca2+ per molecule, so the conformational change is not over before the four specific Ca2+ - binding sites are saturated with Ca2+.  相似文献   

17.
Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca2+-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca2+-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca2+-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).  相似文献   

18.
Hyperforin, an extract of the medicinal plant hypericum perforatum (also named St John's wort), possesses antidepressant properties. Recent data showed that it elevates the intracellular concentration of Ca2+ by activating diacylglycerol-sensitive C-class of transient receptor potential (TRPC6) channels without activating the other isoforms (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC7). This study was undertaken to further characterize the cellular neuronal responses induced by hyperforin. Experiments conducted on cortical neurons in primary culture and loaded with fluorescent probes for Ca2+ (Fluo-4) and Zn2+ (FluoZin-3) showed that it not only controls the activity of plasma membrane channels but it also mobilizes these two cations from internal pools. Experiments conducted on isolated brain mitochondria indicated that hyperforin, like the inhibitor of oxidative phosphorylation, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), collapses the mitochondrial membrane potential. Furthermore, it promotes the release of Ca2+ and Zn2+ from these organelles via a ruthenium red-sensitive transporter. In fact, hyperforin exerts complex actions on CNS neurons. This antidepressant not only triggers the entry of cations via plasma membrane TRPC6 channels but it displays protonophore-like properties. As hyperforin is now use to probe the functions of native TRPC6 channels, our data indicate that caution is required when interpreting results obtained with this antidepressant.  相似文献   

19.
Plasma membrane vesicles (ca 40% inside-out, after one freeze-thaw cycle) were extracted and purified from the shoots of oat ( Avena sativa L. ) and chickweed ( Stellaria media L.) using the two-phase aqueous polymer technique. In the presence of ATP or GTP, a rapid uptake of 45Ca2+ occurred (0.77 and 0.62 nmol Ca2+ mg-1 protein, for ATP and GTP, respectively, in oat, and 0.53 and 0.51 nmol Ca2+ mg-1 protein, for ATP and GTP, respectively, in chickweed). Nucleotide-dependent Ca2+-transport was sensitive to 1 μ M Erythrosin B (with ATP. inhibited by 52% in oat and in chickweed by 72%; with GTP, inhibition was similar in both species at ca 67%); ATP-dependent uptake was greater in oat than in chickweed, but not stimulated by calmodulin. Addition of the calcium ionophore A-23187 resulted in the release of label from the vesicles (41% and 63% release with ATP, and 24% and 52% release with GTP, in oat and chickweed, respectively). The results obtained suggest that Ca2+-transport is independent of the proton pump. In oat, kinetic data indicate a discontinuity in the absorption isotherm at 10 μ M free calcium.  相似文献   

20.
Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca2+ influx through voltage-gated Ca2+ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca2+ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K+ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca2+ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K+ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca2+ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.  相似文献   

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