Use of a Chaotropic Anion Iodide in the Purification of Z-Line Proteins: Isolation of CapZ from Fish White Muscle

Papa, Iris; Astier, Catherine; Kwiatek, Olivier; Lebart, Marie‐Christine; Raynaud, Fabrice; Benyamin, Yves; Roustan, Claude

doi:10.1006/prep.1999.1124

Cited by 6 publications

(4 citation statements)

References 31 publications

(25 reference statements)

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“…The difference in the densities of the heavy and classical (light) DRMs could be obviously due to a higher protein-to-lipid ratio in the former, and therefore the integrity of the heavy DRMs could be more dependent on proteinprotein interactions. Thus, we tested the resistance of both types of DRMs to the chaotropic agent 0.6 M KI (24), which is known to be a mild disruptor of protein-protein interactions (27). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

A New Type of Membrane Raft-Like Microdomains and Their Possible Involvement in TCR Signaling

Otáhal

Angelisová

Hrdinka

et al. 2010

The Journal of Immunology

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Membrane rafts and signaling molecules associated with them are thought to play important roles in immunoreceptor signaling. Rafts differ in their lipid and protein compositions from the rest of the membrane and are relatively resistant to solubilization by Triton X-100 or similar detergents, producing buoyant, detergent-resistant membranes (DRMs) that can be isolated by density gradient ultracentrifugation. One of the key signaling molecules present in T cell DRMs is the transmembrane adaptor protein LAT (linker for activation of T cells). In contrast to previous results, a recent study demonstrated that a LAT construct not present in the buoyant DRMs is fully able to support TCR signaling and development of T cells in vivo. This finding caused doubts about the real physiological role of rafts in TCR signaling. In this study, we demonstrate that these results can be explained by the existence of a novel type of membrane raft-like microdomains, producing upon detergent solubilization “heavy DRMs” containing a number of membrane molecules. At a moderate level of expression, LAT supported TCR signaling more efficiently than constructs targeted to the microdomains producing heavy DRMs or to nonraft membrane. We suggest that different types of membrane microdomains provide environments regulating the functional efficiencies of signaling molecules present therein.

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Section: Resultsmentioning

confidence: 99%

A New Type of Membrane Raft-Like Microdomains and Their Possible Involvement in TCR Signaling

Otáhal

Angelisová

Hrdinka

et al. 2010

The Journal of Immunology

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“…Gelsolin G2 and G4–6 domains were labeled by FITC as described elsewhere [29]. Biotinylation of G2 domain by BACNHS was performed as reported previously [30]. Excess reagent was eliminated by chromatography on a PD10 column (Pharmacia) in 0.1 m NaHCO 3 buffer pH 8.6.…”

Section: Proteins and Peptidesmentioning

confidence: 99%

Co‐operation of domain‐binding and calcium‐binding sites in the activation of gelsolin

Lagarrigue

Maciver

Fattoum

et al. 2003

European Journal of Biochemistry

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Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a Ôhelical-latchÕ at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (K d ¼ 15 lM). We also report a calcium dependent binding site for the C-terminus (G4-6) within G2 and delimit this further to a specific region formed by residues 203-225 and 159-193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4-6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.

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“…Gelsolin G4–6 domain was labeled by FITC or Oregon green 488 isothiocyanate as described elsewhere [30]. Biotinylation of the G2 domain by biotinamidocaproate N ‐hydroxyl‐succinimide ester was performed as reported previously [31]. Excess reagents were eliminated by chromatography on a PD10 column (Pharmacia) in 0.1 m NaHCO 3 buffer pH 8.6.…”

Section: Proteins and Peptidesmentioning

confidence: 99%

The activation of gelsolin by low pH

Lagarrigue

Ternent

Maciver

et al. 2003

European Journal of Biochemistry

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Gelsolin is a multidomain and multifunction protein that nucleates the assembly of filaments and severs them. The activation of gelsolin by calcium is a multistep process involving many calcium binding sites that act to unfold the molecule from a tight structure to a more loose form in which three actin‐binding sites become exposed. Low pH is also known to activate gelsolin, in the absence of calcium and this too results in an unfolding of the molecule. Less is known how pH‐activation occurs but we show that there are significant differences in the mechanisms that lead to activation. Crucially, while it is known that the bonds between G2 and G6 are broken by co‐operative occupancy of calcium binding sites in both domains [Lagarrique, E., Maciver, S. K., Fattoum, A., Benyamin, Y. & Roustan, C. (2003) Eur. J. Biochem. 270, 2236–2243.], pH values that activate gelsolin do not result in a weakening of the G2‐G6 bonds. We report the existence of pH‐dependent conformational changes within G2 and in G4–6 that differ from those induced by calcium, and that low pH overrides the requirement for calcium for actin‐binding within G4–6 to a modest extent so that a Kd of 1 µm is measured, compared to 30–40 nm in the presence of calcium. Whereas the pH‐dependent conformational change in G2 is possibly different from the change induced by calcium, the changes measured in G4–6 appear to be similar in both calcium and low pH.

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Use of a Chaotropic Anion Iodide in the Purification of Z-Line Proteins: Isolation of CapZ from Fish White Muscle

Cited by 6 publications

References 31 publications

A New Type of Membrane Raft-Like Microdomains and Their Possible Involvement in TCR Signaling

A New Type of Membrane Raft-Like Microdomains and Their Possible Involvement in TCR Signaling

Co‐operation of domain‐binding and calcium‐binding sites in the activation of gelsolin

The activation of gelsolin by low pH

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