Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation

Pope, Brian; Gooch, John; Hinssen, Horst; Weeds, Alan G.

doi:10.1016/0014-5793(89)81524-8

Cited by 9 publications

(2 citation statements)

References 17 publications

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“…Gelsolin was isolated from pig stomach smooth muscle ( , ) and stored as an ammonium sulfate precipitate in liquid nitrogen. Before use, the precipitate was dissolved in PSAM buffer (10 mM imidazole, 0.5 mM EGTA, 0.2 mM DTT, 2 mM NaN 3 , pH 7.0) and dialyzed against the same buffer.…”

Section: Methodsmentioning

confidence: 99%

Calcium-Induced Conformational Changes in the C-Terminal Half of Gelsolin Stabilize Its Interaction with the Actin Monomer

2004

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The basic mechanism for the nucleating effect of gelsolin on actin polymerization is the formation of a complex of gelsolin with two actin monomers. Probably due to changes in the C-terminal part of gelsolin, a stable ternary complex is only formed at [Ca(2+)] >10(-5) M [Khaitlina, S., and Hinssen, H. (2002) FEBS Lett. 521, 14-18]. Therefore, we have studied the binding of actin monomer to the isolated C-terminal half of gelsolin (segments 4-6) over a wide range of calcium ion concentrations to correlate the conformational changes to the complex formation. With increasing [Ca(2+)], the apparent size of the C-terminal half as determined by gel filtration was reduced, indicating a transition into a more compact conformation. Moreover, Ca(2+) inhibited the cleavage by trypsin at Lys 634 within the loop connecting segments 5 and 6. Though the inhibitory effect was observed already at [Ca(2+)] of 10(-7) M, it was enhanced with increasing [Ca(2+)], attaining saturation only at >10(-4) M Ca(2+). This indicates that the initial conformational changes are followed by additional molecular transitions in the range of 10(-5)-10(-4) M [Ca(2+)]. Consistently, preformed complexes of actin with the C-terminal part of gelsolin became unstable upon lowering the calcium ion concentrations. These data provide experimental support for the role of the type 2 Ca-binding sites in gelsolin segment 5 proposed by structural studies [Choe et al. (2002) J. Mol. Biol. 324, 691]. We assume that the observed structural transitions contribute to the stable binding of the second actin monomer in the ternary gelsolin-actin complex.

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Section: Methodsmentioning

confidence: 99%

Calcium-Induced Conformational Changes in the C-Terminal Half of Gelsolin Stabilize Its Interaction with the Actin Monomer

2004

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“…Cytoplasmic gelsolin was purified from pig stomach smooth muscles and human platelets; plasma gelsolin was purified from pig and human blood plasma by methods described earlier [17,18] and stored as an ammonium sulphate precipitate in liquid nitrogen. Before use, the precipitate was dissolved and dialysed against PSAM buffer (10 mM imidazole, 0.5 mM EGTA, 0.2 mM dithiothreitol and 2 mM NaN 3 , pH 7.0).…”

Section: Preparation Of Proteins and Tissue Extractsmentioning

confidence: 99%

Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface

Fock

Jockusch

Schubert

et al. 2005

Biochemical Journal

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The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

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Differential effects of gelsolins on tissue culture cells

Huckriede

Füchtbauer

Hinssen

et al. 1990

Cell Motil. Cytoskeleton

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Gelsolins, prepared from a number of different sources, showed similar severing activity on F-actin in vitro or on stress fibers of detergent-extracted cells but differed in their effects on actin in stress fibers of microinjected cells. When human gelsolin isolated from plasma was injected into cells in a Ca(++)-containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared. These effects were particularly striking when the Ca(++)-insensitive N-terminal proteolytic fragment of this gelsolin was injected. By contrast, Ca(++)-sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity. Furthermore, the Ca(++)-independent N-terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity. Most striking was the finding that human plasma gelsolin expressed in E. coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N-terminal half of human gelsolin expressed in Escherichia coli. The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations. It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin. Such discrimination could be achieved as a result of post-translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E. coli be reconciled.

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Loss of calcium sensitivity of plasma gelsolin is associated with the presence of calcium ions during preparation

Cited by 9 publications

References 17 publications

Calcium-Induced Conformational Changes in the C-Terminal Half of Gelsolin Stabilize Its Interaction with the Actin Monomer

Calcium-Induced Conformational Changes in the C-Terminal Half of Gelsolin Stabilize Its Interaction with the Actin Monomer

Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface

Differential effects of gelsolins on tissue culture cells

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