Kinetics of gelsolin interaction with phalloidin-stabilized F-actin. Rate constants for binding and severing.
The kinetics of gelsolin interaction with actin filaments have been investigated using two fluorescent probes, tetramethylrhodamine isothiocyanate-labeled phalloidin bound to F-actin and N-(1-pyrenyl)iodoacetamide-labeled actin. We have also analyzed the F-actin severing by gelsolin using an assay for actin filaments which measures the polymerization rate of monomeric actin added to the gelsolin-severed filaments. Phalloidin-stabilized actin filaments were used in order to minimize the depolymerization reaction and thus simplify the kinetic analysis. Because gelsolin activity is Ca(2+)-activated, experiments were conducted in the presence of 0.5 mM CaCl2 to ensure maximal activity. We show that the interaction of gelsolin with F-actin may be separated into two distinct kinetic phases which correspond to binding and severing events. Using a two-step model of gelsolin activity, we have determined that gelsolin binds to F-actin with an association rate constant of 2 x 10(7) M-1 s-1, dissociates with a rate constant in the range 0.4-1.2 s-1, and subsequently severs phalloidin-stabilized F-actin with a first-order rate constant of 0.25 s-1. Characterization of the binding and severing reactions will facilitate further investigation of gelsolin activity and its regulation.
Kinosian
H J
HJ
Department of Physiology & Cell Biology, Albany Medical College, New York 12208, USA.
Selden
L A
LA
Estes
J E
JE
Gershman
L C
LC
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes; D018260:Gelsolin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007460:Iodoacetamide / Q000031:analogs & derivatives; D007700:Kinetics; D008956:Models, Chemical; D010590:Phalloidine / Q000031:analogs & derivatives; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012235:Rhodamines; D013050:Spectrometry, Fluorescence
D000199:Actins; D005456:Fluorescent Dyes; D018260:Gelsolin; D011108:Polymers; D012235:Rhodamines; D010590:Phalloidine; C029358:N-(1-pyrenyl)iodoacetamide; C041085:tetramethylrhodaminylphalloidine; D007460:Iodoacetamide