首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Plasma membrane‐associated Ca2+‐binding protein–2 (PCaP2) of Arabidopsis thaliana is a novel‐type protein that binds to the Ca2+/calmodulin complex and phosphatidylinositol phosphates (PtdInsPs) as well as free Ca2+. Although the PCaP2 gene is predominantly expressed in root hair cells, it remains unknown how PCaP2 functions in root hair cells via binding to ligands. From biochemical analyses using purified PCaP2 and its variants, we found that the N–terminal basic domain with 23 amino acids (N23) is necessary and sufficient for binding to PtdInsPs and the Ca2+/calmodulin complex, and that the residual domain of PCaP2 binds to free Ca2+. In mutant analysis, a pcap2 knockdown line displayed longer root hairs than the wild‐type. To examine the function of each domain in root hair cells, we over‐expressed PCaP2 and its variants using the root hair cell‐specific EXPANSIN A7 promoter. Transgenic lines over‐expressing PCaP2, PCaP2G2A (second glycine substituted by alanine) and ?23PCaP2 (lacking the N23 domain) exhibited abnormal branched and bulbous root hair cells, while over‐expression of the N23 domain suppressed root hair emergence and elongation. The N23 domain was necessary and sufficient for the plasma membrane localization of GFP‐tagged PCaP2. These results suggest that the N23 domain of PCaP2 negatively regulates root hair tip growth via processing Ca2+ and PtdInsP signals on the plasma membrane, while the residual domain is involved in the polarization of cell expansion.  相似文献   

2.
Summary Experiments were performed to obtain information on: (i) the specific properties of Ca2+ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca2+ in yeast. These were obtained mainly by considering actual concentrations of Ca2+ in the medium after substracting the amounts bound to the cell. Ak m of 1.9 m and aV max of 1.2 nmol (100 mg·3 min)–1 were calculated.The effects of some inhibitors and other cations on Ca2+ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca2+ uptake in yeast.The effects of Mg2+ on the uptake of Ca2+ agree with the existence of a single transport system for both divalent cations.The actions of Na+ and K+ on the transport of Ca2+ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.  相似文献   

3.
The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.  相似文献   

4.
The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B‐domain of AKAP79 were determined using a biosensor‐based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (KD = 20 n m ) and calmodulin (KD = 30 n m ), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one‐step model, but was only detected in the presence of Ca2+. Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca2+, but with different kinetic rate constants. The data are consistent with a similar initial Ca2+‐dependent binding step for the two proteins. For caldendrin, a second Ca2+‐independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of protein–protein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function. Copyright © 2012 John Wiley & Sons, Ltd.  相似文献   

5.
The binding of ATP and Ca2+ by the Ca2+ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca2+ pump protein has been found to contain one specific ATP and two specific Ca2+ binding sites per phosphorylation site. ATP binding is dependent on Mg2+ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca2+. In the presence of Mg2+ and the absence of Ca2+, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca2+ binding. In the absence of ATP, Ca2+ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg2+, Sr2+ and La3+, in that order, decrease Ca2+ binding by the Ca2+ pump protein. The affinity of the Ca2+ pump protein for both ATP and Ca2+ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca2+ pump protein-MgATP2− and Ca2+ pump protein-calcium complexes are approx. 0.25 and 0.5 μM−1, respectively. The unphosphorylated Ca2+ pump protein does not contain a Mg2+ binding site with an affinity comparable to those of the ATP and Ca2+ binding sites.The affinity of the Ca2+ pump protein for Ca2+ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca2+ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca2+. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca2+.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca22+-MgATP2− complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca2+ and probably Mg2+.  相似文献   

6.
Calmodulin (CAM) is a modulatory protein that regulates cellular activity by binding to a large number of proteins. Key elements in the Ca2+-dependent mechanism of interaction between CAM and the proteins it activates are the selectivity for Ca2+ ions and the requirement for Ca2+-dependent conformational changes. We report on results from a series of molecular dynamics simulations that identified discrete steps in the mechanism of structural rearrangement of CAM. The findings implicate the side chains of arginine residues in the bending of the central alpha helix. Structural and energetic considerations point to a dynamic hydrogen bonding pattern around the arginine residues as a ratcheting-type mechanism, causing the kinking of the central helix in consecutive steps stabilized by each new pattern of hydrogen bonds. Initial model building studies to locate potential binding sites of ligands such as trifluoperazine (TFP) indicate that the compaction of CAM results in several structural changes, that explain the selective binding of molecules such as TFP in the N-terminal domain. The present studies identify specific residues involved in the process of compaction and point to specific CAM residues involved in the binding of the ligand. These insights lead directly to propositions for experimental engineering of the molecular structure of CAM in order to probe the hypotheses and their consequences for the function of this important protein.  相似文献   

7.
The Na+/Ca2+ exchange system is the primary Ca2+ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are reponsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na+ or the absence of cytosolic Ca2+ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca2+ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca2+-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca2+-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.  相似文献   

8.
Abstract: Activation of the Ca2+/Mg2+ ATPase associated with highly purified Torpedo synaptic vesicles results in 45Ca2+ uptake. The accumulated 45Ca2+ is released by hypoosmotic buffer and by the Ca2+ ionophore A23187. Density-gradient centrifugation and permeation chromatography reveal that vesicular acetylcholine and the membrane-bound 45Ca2+ co-migrate, thus implying that 45Ca2+ is transported into cholinergic vesicles. ATP-dependent 45Ca2+ uptake follows saturation kinetics, with KmCa2+= 50 μM, KmATP= 5 μM, and Vmax= 3 ± 0.3 nmol Ca2+/mg protein/min. Treatment of the vesicles with mersalyl, dicyclohexyl-carbodiimide, and quercetin leads to inactivation of the Ca2+/Mg2+ ATPase and to comparable inhibition of 45Ca2+ transport. Ruthenium red and ouabain have no effect on either of these activities. Nigericin in the presence of external K+ is a potent inhibitor of 45Ca2+ translocation, whereas gramicidin activates transport. The proton translocator carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and FCCP + the ionophore valinomycin partially inhibit 45Ca2+ transport. By contrast, the above ionophores do not affect Ca2+/Mg2+ ATPase activity. Tentative mechanisms for ATP-dependent Ca2+ transport into cholinergic synaptic vesicles and the physiological significance of this process are discussed.  相似文献   

9.
The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca2+‐binding region in C2A domain is longer than classical C2 domains and a novel Ca2+ binding site in the vWA domain. The structure of BON1 bound to Mn2+ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca2+. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.  相似文献   

10.
We report the effects of Ca2+ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca2+‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca2+‐induced chemical shifts perturbations yields similar KD values of 16–24 μM for the two CBD2‐AD Ca2+‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca2+‐induced chemical shift changes, we performed a comparative analysis of eight Ca2+‐binding proteins that revealed large differences between different protein folds. The CBD2 15N relaxation data show the CBD2‐AD Ca2+ coordinating loops to be more rigid in the Ca2+‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca2+ and are much more dynamic relative to both the Ca2+‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S2 order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca2+‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca2+‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca2+‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc.  相似文献   

11.
The universal role of calcium (Ca2+) as a second messenger in cells depends on a large number of Ca2+‐binding proteins (CBP), which are able to bind Ca2+ through specific domains. Many CBPs share a type of Ca2+‐binding domain known as the EF‐hand. The EF‐hand motif has been well studied and consists of a helix‐loop‐helix structural domain with specific amino acids in the loop region that interact with Ca2+. In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF‐hand motif. The majority of these genes have not been characterized. We report the characterization of two EF‐hand motif‐containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca2+ measurements showed that Δtg216620 cells did not respond to extracellular Ca2+ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca2+ influx while TgGT1_280480 may be playing a different role in the rhoptries.  相似文献   

12.
Previously it demonstrated that in the absence of Ca2+ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca2+] i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217–220.). These studies designate Ca2+ entry as opposed to [Ca2+] i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca2+ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein–protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J.; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J. 15:4100–4110; Wiser, O., Trus, M., Hernandez, A., Renström, E., Barg, S., Rorsman, P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248–253). The kinetics of Cav1.2 (Lc-type) and Cav2.2 (N-type) Ca2+ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin I, a vesicle-associated protein, accelerated the activation kinetics indicating Cav2.2 coupling to the vesicle. The unique modifications of Cav1.2 and Cav2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Cav1.2 cytosolic domain Lc753–893, acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca2+ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca2+-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca2+-dependent CAV (RRP) release, initiated by the binding of Ca2+ to the channel, upstream to intracellular Ca2+ sensor thus establishing the Ca2+ channel as the Ca2+ sensor of neurotransmitter release.  相似文献   

13.
The distribution of the putative protein products of gene hcs2 in giant command neurons of the parietal ganglia of the terrestrial snail Helix lucorum has been studied using light- and electron-microscopic immunocytochemistry. The product of the hcs2 gene is a hybrid protein belonging to the EF-hand family of Ca2+-binding proteins and is a precursor of several neuropeptides. Polyclonal antibodies to neuropeptides CNP3 and CNP4 and the C-terminal Ca2+-binding domain of the precursor protein have been used to determine their intracellular localization. The targets for all three types of antibodies have been found in cytoplasmic secretory granules. The label (colloidal gold) density in the secretory granules is two times higher in the case of neuropeptides CNP3 and CNP4 than in the case of the Ca2+-binding domain. Thus, a specific association between the putative products of the hcs2 gene and the cell secretory apparatus has been demonstrated. This agrees with the earlier hypothesis that hcs2 products may serve as neurotransmitters or neuromodulators.  相似文献   

14.
Coagulation factor IX/coagulation factor X binding protein from the venom of Agkistrodon halys Pallas (AHP IX/X-bp) is a unique coagulation factor IX/coagulation factor X binding protein (IX/X-bp). Among all IX/X-bps identified, only AHP IX/X-bp is a Ca2+- and Zn2+-binding protein. The binding properties of Ca2+ and Zn2+ ions binding to apo-AHP IX/X-bp and their effects on the stability of the protein have been investigated by isothermal titration calorimetry, fluorescence spectroscopy, and differential scanning calorimetry. The results show that AHP IX/X-bp has two metal binding sites, one specific for Ca2+ with lower affinity for Zn2+ and one specific for Zn2+ with lower affinity for Ca2+. The bindings of Ca2+ and Zn2+ in the two sites are entropy- and enthalpy-driven. The binding affinity of AHP IX/X-bp for Zn2+ is 1 order of magnitude higher than for Ca2+ for either high-affinity binding or low-affinity binding, which accounts for the existence of one Zn2+ in the purified AHP IX/X-bp. Guanidine hydrochloride (GdnHCl)-induced and thermally induced denaturations of Ca2+–Ca2+-AHP IX/X-bp, Zn2+–Zn2+-AHP IX/X-bp, and Ca2+–Zn2+-AHP IX/X-bp are all a two-state processes with no detectable intermediate state(s), indicating the Ca2+/Zn2+-induced tight packing of the protein. Ca2+ and Zn2+ increase the structural stability of AHP IX/X-bp against GdnHCl or thermal denaturation to a similar extent. Although Ca2+ and Zn2+ have no obvious effect on the secondary structure of AHP IX/X-bp, they induce different rearrangements in local conformation. The Zn2+-stabilized specific conformation of AHP IX/X-bp may be helpful to its recognition of the structure of coagulation factor IX. This work suggests that in vitro, Ca2+ plays a structural rather than an active role in the anticoagulation of AHP IX/X-bp, whereas Zn2+ plays both structural and active roles in the anticoagulation. In blood, Ca2+ binds to AHP IX/X-bp and stabilizes its structure, whereas Zn2+ cannot bind to AHP IX/X-bp owing to the low Zn2+ concentration. AHP IX/X-bp prolongs the clotting time in vivo through its binding only with coagulation factor X/activated coagulation factor X.  相似文献   

15.
The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca2+ specifically binds to the Ca2+-binding region, facilitating PtdIns(4,5)P2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P2 binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P2 is hydrolyzed to generate diacylglycerol and IP3, an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.  相似文献   

16.
Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca2+-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca2+ overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain–binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca2+ channel activity at the plasma membrane, resulting in the inhibition of Ca2+-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.  相似文献   

17.
Summary Active calcium transport by cardiac sarcoplasmic reticulum assumes a central role in the excitation-concentration coupling of the myocardium, in that Ca2+-dependent ATPase (mol.wt. 100 000) of cardiac sarcoplasmic reticulum serves as an energy transducer and a translocator of Ca2+ across the membrane. During the translocation of Ca2+, the ATPase undergoes a complex series of reactions during which the phosphorylated intermediate EP is formed. We documented how the elementary steps of the ATPase reaction are coupled with calcium translocation, and provided evidences to indicate that two key steps of ATPase correspond to the conformational change of the enzyme, and appear to alter the affinity of the enzyme for Ca2+.A line of evidence also indicated that Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum is regulated by a specific protein named phospholamban (mol.wt. 22 000), which serves as a substrate for cyclic AMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation of phospholamban resulted in a marked increase in the rate of turnover of the ATPase, by enhancing the rates of the key elementary steps, i.e. the steps at which the intermediate EP is formed and decomposed. Thus phospholamban is putatively thought to serve as a modulator of Cat2+-dependent ATPase of cardiac sarcoplasmic reticulum. A working model was proposed to interpret the mechanism. Also documented is a possibility that another protein kinase activatable by Ca2+ and calmodulin is functional in regulating the phospholamban-ATPase system, thus suggesting the existence of a dual control system, in which both cyclic AMP- and calmodulin-dependent phosphorylation are in control of the Cat2+-dependent ATPase.Such a control mechanism may provide the interpretation, at the cellular level, that catecholamines exert actions on myocardial contractility. Thus, catecholamine-mediated increases in intracellular cyclic AMP could enhance calcium fluxes across the membrane of sarcoplasmic reticulum, thus resulting in the increased rates of relaxation and, at the same time, the increased rate and extent of contraction. Such a mechanism could also be operational in the tissues, other than the myocardium, in which catecholamines and other hormones serve as the first messenger, producing intracellular cyclic AMP as the second messenger.  相似文献   

18.
Classical protein kinase C (PKC) enzymes are known to be important factors in cell physiology both in terms of health and disease. They are activated by triggering signals that induce their translocation to membranes. The consensus view is that several secondary messengers are involved in this activation, such as cytosolic Ca2+ and diacylglycerol. Cytosolic Ca2+ bridges the C2 domain to anionic phospholipids as phosphatidylserine in the membrane, and diacylglycerol binds to the C1 domain. Both diacylglycerol and the increase in Ca2+ concentration are assumed to arise from the extracellular signal that triggers the hydrolysis of phosphatidylinositol-4,5-bisphosphate. However, results obtained during the last decade indicate that this phosphoinositide itself is also responsible for modulating classical PKC activity and its localization in the plasma membrane.  相似文献   

19.
Measurements of intracellular Ca2+ in adrenal medullary cells suggest that a transient rise in Ca2+ leads to a transient secretory response, the rise in Ca2+ being brought about by an influx through voltage-sensitive Ca channels which subsequently inactivate. The level of Ca2+ observed is much smaller than the Ca2+ needed to trigger secretion when introduced directly into the cell. The discrepancy is removed by the presence of diacylglycerot, which increases the sensitivity of the secretory process to Ca2+. The site of action of Ca2+ and diacylglycerol is probably protein kinase C, and tile different secretory responses to increases of Ca2+ and diacylglycerol can be modelled in terms of a preferential order of binding of these two substrates to the enzyme. ATP is needed for secretion: one role is possibly to confer stability to the secretory apparatus; another may involve phosphorylation of some key protein. The kinetics of secretion suggest that if Ca2+ regulates phosphorylation or dephosphorylation, then it is therate of change of phosphorylation that controls secretion rather than theextent of phosphorylation or dephosphorylation. Guanine nucleotide-binding proteins may play a role not only at the level of signal transduction coupling, but also at or near the site of exocytosis, and the mechanism by which some Botulinum toxins inhibit secretion may be associated with these proteins.  相似文献   

20.
OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca2+] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca2+. NMR dynamics data for OsCaM61 indicate that the two lobes of the CaM domain act together unlike the independent behavior of the lobes seen in mammalian CaM and soybean CaM4. Also, data demonstrate that the positively charged nuclear localization signal region in the tail in apo-OsCaM61 is helical, whereas it becomes flexible in the Ca2+-saturated protein. The extra helix in apo-OsCaM61 provides additional interactions in the C-lobe and increases the structural stability of the closed apo conformation. This leads to a decrease in the Ca2+ binding affinity of EF-hands III and IV in OsCaM61. In Ca2+-OsCaM61, the basic nuclear localization signal cluster adopts an extended conformation, exposing the C-terminal extension for prenylation or enabling OsCaM61 to be transferred to the nucleus. Moreover, Ser172 and Ala173, residues in the tail, interact with different regions of the protein. These interactions affect the ability of OsCaM61 to activate different target proteins. Altogether, our data show that the tail is not simply a linker between the prenyl group and the protein but that it also provides a new regulatory mechanism that some plants have developed to fine-tune Ca2+ signaling events.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号