Binding of pig plasma gelsolin to F‐actin and partial fractionation into calcium‐dependent and calcium‐independent forms

Abstract

The interaction of pig plasma gelsolin with F‐actin has been studied by a sedimentation assay using 125I‐gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F‐actin in 0.1 mM CaCl2 in experiments using 24 μM actin and 2–10 nM 125I‐gelsolin, but only 40–50% bound in 1 mM EGTA. Addition of more F‐actin to the EGTA supernantant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F‐actin that has been prepared in the absence of calcium ions. F‐actin prepared from G‐actin in calcium or pretreated with calcium, binds 125I‐gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium. Further evidence for partial calcium sensitivity in the interactions between gelsolin and F‐actin has been obtained by other methods, including viscometry and electron microscopy. The gelsolin present in the EGTA supernatant is complexed to G‐actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F‐actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain. The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F‐actin and one that does not. Both bind to G‐actin. Partial separation has been achieved using actin‐Sepharose. Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin‐Sepharose do not correspond to specific isoelectric species.

The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover : pH sensitivity of F‐actin binding by human ADF, but not of Acanthamoeba actophorin

“…Sedimentation assays. Binding of actophorin and ADF to F-actin was analysed as previously reported [7] by the method of Pope and Weeds [24]. Actin and either actophorin or ADF at various pH values in buffer F were centrifuged at 386 000 g for 15 min in a Beckman TL100 bench-top ultracentrifuge.…”

F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)

“…F-actin and ADF (both 10 M) were incubated for 20 min in 12 mM Tris⅐HCl (pH 7.0), 50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 0.2 mM CaCl 2 , and 1 mM NaN 3 . After sedimentation at 100,000 ϫ g for 15 min in a Beckman TLA100 centrifuge (40), aliquots from the supernatant and pellet, resuspended in a volume of buffer equal to the supernatant, were analyzed by SDS͞PAGE as described in ref. 41.…”

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.

“…F-actin binding was measured by sedimentation at 100,000 x g for 15 min in a Beckman TLA100 centrifuge (41). F-actin and ZmABP3 protein were incubated together (10 ,uM) for 20 min in 12 mM Tris HCl (pH 7.0), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 0.2 mM CaCl2, and 1 mM NaN3.…”