Calcium-dependent regulation of actin filament bundling by lipocortin-85.
Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels.
Ikebuchi
N W
NW
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Waisman
D M
DM
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D017302:Annexins; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D002417:Cattle; D003599:Cytoskeleton / Q000648:ultrastructure*; D006023:Glycoproteins / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D008854:Microscopy, Electron; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012542:Scattering, Radiation
D000199:Actins; D017302:Annexins; D002135:Calcium-Binding Proteins; D006023:Glycoproteins; D002118:Calcium