DS 25749 PDF

DS 25749 PDF

BOSCO. Roan, calved Aug. 10, l. bred by Janus \V. Singleton. white , calved March 11, , bred and owned by D. S. Hosford & Sons Monticello. BOSCO, Roan, сапы! Aug. 10, bred by Jmnes W. Singleton, Quincy calved March 11, , bred and owned by D. S. Hosford & Sons Monticello. Document Includes User Manual Henseignements é noter par I’ utilisateur Le numéro ds se’rie du monlteur est indiqué a I’arriére de I’apparail.

Author: Shaktilmaran Braramar
Country: Bahamas
Language: English (Spanish)
Genre: Politics
Published (Last): 12 June 2013
Pages: 358
PDF File Size: 15.3 Mb
ePub File Size: 18.99 Mb
ISBN: 333-1-46437-627-1
Downloads: 71882
Price: Free* [*Free Regsitration Required]
Uploader: Juzilkree

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery.

Its cytosolic portion mainly consists of two 257749 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences.

NIKE KOBE AD 922482-005 922482-005 922482-005 GREY SNAKESKIN CHROME RED DS SIZE: 11.5 MENS 25749f

To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic 225749 phosphatidylinositol 4,5-bisphosphate PI 4,5 P 2but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin.

PI 4,5 P 2 binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch.

Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI 4,5 P 2 -enriched plasma membrane. Calcium-dependent secretion 25794 neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission.

The underlying membrane fusion reaction between vesicle and plasma membrane has been intensively studied and found to be promoted by both protein-protein as well as protein-lipid interactions.

Synaptotagmin 1 is anchored vs the membrane of synaptic vesicles via a single 2749 region. Its N-terminal region comprises a short luminal domain, whereas the larger cytoplasmic C-terminal region consists of tandem C2 domains, termed C2A and C2B, tethered to each other via a short linker 7 a schematic outline of the structural features of synaptotagmin 1 is given in Fig.

Several isoforms with similar domain structure have been identified 8. The C2A domain of synaptotagmin 1 was the first C2 domain structure to be determined In subsequent studies other C2 domains, including the C2B domain of synaptotagmin, were shown to exhibit very similar three-dimensional structures.

C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction PKC, phospholipases, phosphatidylinositol 3-kinases, etc.

Calcium ions bind in a cup-shaped depression formed by the N- and C-terminal loops of the C2 key motifs of C2 domains. In canonical C2 domains, this incomplete coordination sphere can be occupied by anionic and neutral 1415 phospholipids, enabling the C2 domain to be attached to the membrane.

In fact, upon rise of the intracellular calcium level, C2 domain-containing enzymes are translocated to the membrane so that the catalytic domains can interact with lipids or membrane-anchored protein substrates Yet synaptotagmin 1 does not contain such a catalytic domain, suggesting that the properties of its tandem C2 domains are the sole key to understanding its molecular function.

So far the multifarious interplay between the SNARE machinery, the two fusing membranes, and synaptotagmin 1 is not well understood.

Can-Am Maverick X3 X DS

Calcium binding to synaptotagmin in the absence of membranes has been studied by NMR. This penetration was corroborated by electro-paramagnetic resonance EPR spectroscopy studies, which also showed that the penetration depth increased when both C2 domains of synaptotagmin 1 were attached to each other 21 as compared with the single domains 22 We employed isothermal titration calorimetry ITC to measure the intrinsic calcium binding affinities of synaptotagmin 1 C2 domains both as isolated domains as well as in the context of the tandem C2AB protein.

Then, we investigated whether the intrinsic calcium affinity is modulated in the presence of lipids using a newly developed fluorescence resonance energy transfer FRET approach. We found that the two C2 domains bind calcium largely independently but cooperate in membrane binding.


Remarkably, in the presence of PI 4,5 P 2drastically lower amounts of calcium were needed for membrane binding. All protein constructs used were from Rattus norvegicus and cloned into the expression vector pET28a. Expression constructs of the isolated C2A domain aa 97—the C2B domain aa —the soluble domain of synaptotagmin aa 97—and of the full-length protein aa 1— have been described before Also the following calcium mutants of the full-length protein and of the soluble domain have been described earlier The single cysteine variant SC was obtained after first removing the single native cysteine CS and then introducing a point mutation at position The protein concentrations were determined using either the Bradford assay or UV absorption.

The single cysteine variant was further labeled with Alexa Fluor C 5 maleimide. The dialyzed protein solution was then incubated with the fluorophore for 2 h at room temperature and separated from the free dye using a Sephadex G50 superfine column. The transmembrane region containing proteins syntaxin 1A — and synaptobrevin 2 1— were purified by ion-exchange chromatography in the presence of 15 m m CHAPS.

Full-length synaptotagmin was purified in the presence of 0. Liposomes were prepared as previously described 24with a few modifications.

These stocks were then mixed in appropriate amounts to obtain the desired PS concentrations. The liposomes were formed by detergent removal using the Fast Desalting PC 3. The PS concentration was calculated from the total phospholipid concentration, which was determined using the total phosphate determination method This was done by first washing the Chelex beads with water and then adding the beads directly to the dialysis buffer for 2 h.

The Chelex beads were removed by filtration through a glass filter. The buffer pH was adjusted and 257749 through a 0. The ITC experiment was done as previously described The protein solution was loaded into the sample cell, and the calcium chloride solution was loaded in the syringe.

The calcium chloride solution, in the syringe, was prepared by diluting a 1 m stock solution with ITC buffer to the appropriate concentration. To obtain the effective heat of binding, the heat of dilution, measured by injecting the calcium chloride solution into buffer, was subtracted. Labeled synaptotagmin 1 C Alexa was used to a final concentration of 0.

The liposome titrations cs done by measuring the donor fluorescence intensity F upon the addition of the labeled liposomes Texas Red phosphatidylethanolamine. The normalized intensity values rs plotted against the PS concentration. The calcium titration was done by mixing 0. Calcium chloride stock solution was then titrated at a number of steps with the donor signal recorded at each of these steps.

Using a similar normalization method as described above in this case, F 0 represents the donor intensity before calcium additionthe donor intensity was then plotted against the free calcium concentrations, which were calculated from the total calcium concentrations de the Igor Pro software. Liposome preparation and fusion experiments were done as previously described To compensate for the lack of PS in synaptobrevin liposomes, a higher ratio of PC 70 was used.

To study the intrinsic calcium binding properties of synaptotagmin 1, we employed ITC, adapting an approach previously used for the C2 domains of classical PKCs 2627 and phospholipases 14 The ITC approach allows for measuring the heat change associated with binding by simply titrating the ligand to the macromolecule.

The heat changes are then integrated and fitted to obtain the entire set of thermodynamic parameters of the interaction.

To test whether binding constants determined by ITC agree with earlier NMR studies 1718we initially performed the titration on the isolated C2A aa 97—; see Fig. Typically, injections of CaCl 2 into solution containing the individual C2 domains of synaptotagmin produced strong heat changes.

Can-Am Maverick X3 X DS

In fact, the best fit of our ITC data for the C2A domain was obtained with a three-site sequential binding model. The best fit of the ITC data for the C2B domain was obtained using a one-site binding model that assumes that one or more ligands can bind independently. Structure of synaptotagmin 1.

Synaptotagmin 1 protein consists of two Es domains, C2A and C2B, that coordinate three and two calcium ions, respectively The acidic residues that coordinate calcium binding is shown schematically, with the residues mutated in the calcium binding mutants i.


The different mutants and constructs used in the study are schematically depicted B.

Calcium binding to the C2 domain of synaptotagmin 1 measured by ITC. The upper panels show the raw titration data, and the lower panels show the integrated heat changes after subtracting the heat of dilution. Interestingly, we observed that the two C2 domains of synaptotagmin adopt a thermodynamically divergent mechanism in calcium binding.

The C2A domain bound calcium via an endothermic reaction, whereas the C2B domain exhibited an exothermic profile. The thermodynamic dz of calcium binding are summarized in Table 1. The thermodynamic parameters of calcium binding to the different synaptotagmin constructs are listed in Table 1. These affinities are very similar to the values obtained by earlier NMR work. We next titrated calcium to d wild-type C2AB fragment of synaptotagmin 1 aa 97— to test whether calcium binding is cooperative between the two C2 domains.

Notably, because of the opposing enthalpic changes observed for the two C2 domains, the overall recorded heat changes were much smaller for the 255749 C2AB protein compared with the individual C2 domains Fig.

The 2749 isotherm for the C2AB was fitted using a four-site sequential model, again assuming that the two calcium binding sites of the C2B domain bound with similar affinities. These values only slightly deviate from the results obtained for the individual domains, suggesting that no major cross-talk between the calcium binding sites 257749 the two C2 domains exists.

Thermodynamic parameters of calcium binding to different synaptotagmin constructs assessed by ITC. We also made use of previously described calcium binding mutants for either of the two domains as well as a double mutant that abolishes calcium binding in both C2 domains 24 The calcium binding experiments enabled us to establish the intrinsic binding properties of the two C2 domains of synaptotagmin 1 for calcium in solution.

The rs question we had was how the intrinsic calcium binding properties are modulated when lipids are present. We attempted to carry out ITC titrations of calcium to synaptotagmin in the presence of liposomes. However, because of technical difficulties, possibly caused 257749 aggregation, the data we obtained were not sufficiently reliable to be fitted. We, therefore, developed a robust FRET-based assay for the interaction of the soluble portion of synaptotagmin with membranes in vitro.

For this assay, a variety of single cysteine variants was generated and tested for liposome binding data not shown. Each single cysteine variant was specifically labeled with the donor fluorophore Alexa As shown in Rs.

A novel FRET assay allows the monitoring of synaptotagmin 1 binding to liposomes. Binding was studied using FRET between synaptotagmin 1 labeled with the donor dye, Alexaat position on the C2B domain and liposomes containing phosphatidylethanolamine labeled with Texas Red as acceptor dye. Initially the spectrum was determined for the labeled synaptotagmin 0. This normalization was done for the different synaptotagmin mutants with liposomes containing different compositions of lipids i.

To compare membrane binding at different conditions, quenching of donor fluorescence intensity was normalized as described in the legend to Fig.

We found that wild-type C2AB did not bind in the absence of anionic phospholipids PS and PI 4,5 P ddconfirming that negatively charged lipids are essential for the binding of synaptotagmin to the dds. The binding strength increased in direct proportion with 25479 PS concentrations Fig. Compared with wild-type C2AB, both calcium mutants exhibited clearly reduced binding to all tested liposome compositions.

To gain more insight into the affinity of membrane binding, we titrated labeled liposomes 25794 synaptotagmin-containing solution in the presence of 2 m m calcium chloride.

Even at high concentrations of lipids only marginal binding was detected. Because of much tighter binding of wild-type synaptotagmin, i.

The two C2 domains of synaptotagmin 1 cooperate for membrane binding.