Characterization of tetramethylrhodaminyl-phalloidin binding to cellular F-actin.
Fluorescent derivatives of phalloidin are widely used to measure filamentous actin (F-actin) levels and to stabilize F-actin. We have characterized the kinetics and affinity of binding of tetramethylrhodaminyl (TRITC)-phalloidin to rabbit skeletal muscle F-actin and to F-actin in lysates of rabbit polymorphonuclear leukocytes (PMNs). We have defined conditions where TRITC-phalloidin can be used to inhibit F-actin depolymerization and to quantify F-actin without prior fixation. By equilibrium measurements, the affinity of TRITC-phalloidin binding to rabbit skeletal muscle F-actin (pyrene labeled) or to PMN lysate F-actin was 1-4 x 10(-7) M. In both cases, the stoichiometry of binding was approximately 1:1. Kinetic measurements of TRITC-phalloidin binding to PMN lysate F-actin resulted in an association rate constant of 420 +/- 120 M-1 sec-1 and a dissociation rate constant of 8.3 +/- 0.9 x 10(-5) sec-1. The affinity calculated from the kinetic measurements (2 +/- 1 x 10(-7) M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 microM TRITC-phalloidin inhibited 0.1 microM pyrenyl F-actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F-actin (less than 1% bound in 10 sec), suggesting that phalloidin binds to filament ends more rapidly than to the rest of the filament. We show that TRITC-phalloidin can be used to measure F-actin levels in cell lysates when G-actin is also present (i.e., in cell lysates at high concentrations) if DNase I is included to prevent phalloidin-induced polymerization.
Cano
M L
ML
Department of Chemical Engineering, University of Pennsylvania, Philadelphia 19104.
Cassimeris
L
L
Joyce
M
M
Zigmond
S H
SH
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000737:chemistry; D009504:Neutrophils / Q000737:chemistry; D010590:Phalloidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism
D000199:Actins; D005456:Fluorescent Dyes; D012235:Rhodamines; D010590:Phalloidine; C041085:tetramethylrhodaminylphalloidine