Simple and rapid purification of brevin

Kurokawa, Hirro; Fujii, Wataru; Ohmi, Kazuhiro; Sakurai, Takashi; Nonomura, Yoshiaki

doi:10.1016/0006-291x(90)92342-w

Cited by 110 publications

(78 citation statements)

References 17 publications

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“…Gelsolin eluted as a single peak, which was collected and concentrated using Centricon concentrators. The final concentration was determined spectrophotometrically to be 10 mg/ml using A 280 ϭ 1.24 (23). Four sample concentrations (10, 5, 2.5, and 1 mg/ml) were made by dilution of the 10 mg/ml stock.…”

Section: Methodsmentioning

confidence: 99%

Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

Ashish

Paine

Perryman

et al. 2007

Journal of Biological Chemistry

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Gelsolin regulates the dynamic assembly and disassembly of the actin-based cytoskeleton in non-muscle cells and clears the circulation of filaments released following cell death. Gelsolin is a six-domain (G1-G6) protein activated by calcium via a multistep process that involves unfolding from a compact form to a more open form in which the three actin-binding sites (on the G1, G2, and G4 subdomains) become exposed. To follow the global structural changes that accompany calcium activation of gelsolin, small-angle x-ray scattering (SAXS) data were collected for full-length human plasma gelsolin at nanomolar to millimolar concentrations of free Ca 2؉ . Analysis of these data showed that, upon increasing free Ca 2؉ levels, the radius of gyration (R g ) increased nearly 12 Å , from 31. , even higher calcium concentrations help to stabilize a more open structure, with increases in R g and D max of ϳ2 and ϳ15 Å , respectively. At these higher calcium levels, the SAXS-based models provide a molecular shape that is compatible with that of the crystal structures solved for Ca 2؉ /gelsolin C-terminal and N-terminal halves ؎ monomeric G-actin. Placement of these crystal structures within the boundaries of the SAXS-based model suggests a movement of the G1/G2 subunits that would be required upon binding to actin.

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Section: Methodsmentioning

confidence: 99%

Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

Ashish

Paine

Perryman

et al. 2007

Journal of Biological Chemistry

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“…ADP-actin was prepared as described (38). Plasma gelsolin was purified from bovine plasma as described (39). Gelsolin (0.375 M) was mixed with freshly gel-filtered (Sephadex G-150 column (37)) monomeric ATP-actin (0.1875 M) in buffer A (8) and PtdIns(3,4,5)P 3 , PtdIns(4,5)P 2 , PtdIns(4)P, or PtdIns sonicated in buffer A at various concentrations for 5 min at 25°C.…”

Section: Methodsmentioning

confidence: 99%

The Small GTP-binding Protein Rac Promotes the Dissociation of Gelsolin from Actin Filaments in Neutrophils

Arcaro

1998

Journal of Biological Chemistry

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Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5-[␥-thio]triphosphate (GTP␥S) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTP␥S-loaded i ␣ 2 and the ␤ 1 ␥ 2 subunits of heterotrimeric G-proteins (G i ␣ 2 and G␤ 1 ␥ 2 ) also triggered actingelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTP␥S-loaded G i ␣ 2 or G␤ 1 ␥ 2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.

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“…Human plasma gelsolin was purified by ammonium sulfate fractionation followed by Chromatographie separation on DE-52 ion exchange matrix as described by Kurokawa et al [20]. Purified gelsolin was dialyzed into 75 mM NaCl, 20 mM Tris, pH 7.4 and 0.2 mM EGTA, rapidly frozen in liquid nitrogen and stored at -80°C.…”

Section: Proteinsmentioning

confidence: 99%

Functional consequences of disulfide bond formation in gelsolin

Allen¹

1997

FEBS Letters

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Gelsolin is an actin monomer binding and filament severing protein synthesized in plasma and cytoplasmic forms differing by an N-terminal amino acid extension and a disulfide bond between Cys-188 and Cys-201. To determine whether this bond altered gelsolin regulation or function, oxidized and reduced plasma gelsolins were assayed for severing, monomer binding and nucleation activity at a variety of rate-limiting calcium concentrations. The results indícate that the disulfide bond in domain 2 of gelsolin influences the transmission of information from C-terminal regulatory sites to functional sites in the Nterminus.

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Simple and rapid purification of brevin

Cited by 110 publications

References 17 publications

Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

Global Structure Changes Associated with Ca2+ Activation of Full-length Human Plasma Gelsolin

The Small GTP-binding Protein Rac Promotes the Dissociation of Gelsolin from Actin Filaments in Neutrophils

Functional consequences of disulfide bond formation in gelsolin

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