Actin-gelsolin interactions. Evidence for two actin-binding sites.
We have used a fluorescence enhancement of actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-actin) to study the interactions between rabbit skeletal muscle G-actin and either purified platelet gelsolin or a 130-kDa binary complex of platelet actin and gelsolin that is stable in EGTA and can be purified from human platelets. We have delineated four binding reactions. The exchange of Mg2+ for Ca2+ on the divalent cation-binding site of NBD-actin gives a small fluorescence increase. Binding of monomeric NBD-actin to the binary complex results in a 2.5-fold increase in the emission at 530 nm in the presence of Ca2+ and a 2-fold increase in the presence of EGTA. Titration experiments show that, under nonpolymerizing conditions, one additional actin is bound to the 130-kDa species to form a ternary complex. This binding is Ca2+-sensitive. Purified gelsolin does not appear to bind to NBD-actin in the presence of EGTA, as determined by fluorescence enhancement, gel filtration, or sedimentation measurements, but the addition of Ca2+ promotes rapid binding with a 1.6-1.7-fold enhancement of the emission intensity. A comparison of the relative fluorescence yields/NBD-actin molecule for a binary complex of gelsolin and one NBD-actin, a ternary complex of gelsolin and two NBD-actin molecules, and a ternary complex with an unlabeled actin in the EGTA-stable site and an NBD-actin in the second site indicates that the first NBD-actin, in the EGTA-stable site, does not give a fluorescence increase on binding but the second one does. Finally, we have demonstrated that one molecule of 45Ca2+ is "trapped" when the binary complex is formed and cannot be removed by EGTA. A summary model for these reactions is presented that indicates the interaction between actin and gelsolin is not a freely reversible Ca2+-controlled reaction.
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000032:analysis; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004533:Egtazic Acid / Q000494:pharmacology; D018260:Gelsolin; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D004533:Egtazic Acid; D009327:4-Chloro-7-nitrobenzofurazan; D002118:Calcium