Isolation and characterization of gelsolin from cultured BHK cells.
The Ca2+-dependent actin-polymerization nucleating protein of the cytoplasmic fraction of Baby hamster kidney (BHK) C13 cells has been isolated by anion-exchange, hydroxyapatite and gel-filtration chromatography. This protein has been identified as a cytoplasmic gelsolin by the following criteria: molecular mass of 90 kDa on SDS-PAGE, immunocrossreactivity with pig plasma gelsolin and similar actin-binding properties to gelsolins purified from other sources. BHK gelsolin forms a 1:2 ternary complex with rabbit muscle actin that is dependent on the presence of Ca2+. The ternary complex is dissociated on chelation of Ca2+ with EGTA to a binary complex and free actin. BHK gelsolin nucleates the polymerization of pyrene-labelled G-actin in a Ca2+-dependent manner. The proportion of unpolymerized monomer is increased in the presence of BHK gelsolin by an amount consistent with capping of the positive filament ends. The rate of actin depolymerization induced by diluting F-actin to below its critical concentration (Cc) is unaffected by the presence of BHK gelsolin in EGTA. However, in the presence of Ca2+ the rate of depolymerization is increased indicating that BHK gelsolin severs actin filaments in a Ca2+-dependent manner.
Edgar
A J
AJ
Department of Biophysics, King's College, London, U.K.
D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002478:Cells, Cultured; D006224:Cricetinae; D018260:Gelsolin; D007668:Kidney / Q000378:metabolism*; D008840:Microfilament Proteins / Q000302:isolation & purification*; D008970:Molecular Weight
D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins