Binding of pig plasma gelsolin to F-actin and partial fractionation into calcium-dependent and calcium-independent forms.
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 microM actin and 2-10 nM 125I-gelsolin, but only 40-50% bound in 1 mM EGTA. Addition of more F-actin to the EGTA supernatant 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.
D000199:Actins / Q000097:blood*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000097:blood*; D002499:Centrifugation, Density Gradient; D003720:Densitometry; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin; D008840:Microfilament Proteins / Q000097:blood*; D008854:Microscopy, Electron; D011485:Protein Binding; D013552:Swine; D014783:Viscosity
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D004533:Egtazic Acid; D002118:Calcium