Transient kinetic analysis of rhodamine phalloidin binding to actin filaments.
We have characterized the binding of rhodamine phalloidin to actin filaments and actin filaments saturated with either myosin subfragment-1 or tropomyosin in 50 mM KCl, 1 mM MgCl2 buffer at pH 7.0. Direct transient kinetic measurements of rhodamine phalloidin binding to actin filaments indicate an association rate constant of 2.8 x 10(4) M-1 s-1 and a dissociation rate constant of 4.8 x 10(-4) s-1. The ratio of the rate constants yields a dissociation equilibrium constant of 17 nM. From equilibrium measurements, the apparent affinity of rhodamine phalloidin for actin filaments is 116 nM. The difference between the affinities determined by equilibrium and kinetic experiments is attributed to the depolymerization of filaments at low actin concentrations in the equilibrium samples. The binding stoichiometry is one rhodamine phalloidin molecule per actin subunit. When myosin subfragment-1 and tropomyosin are bound to actin filaments, the rate constants for rhodamine phalloidin binding are the same as for actin alone and in agreement with the binding affinities measured in equilibrium experiments. Presumably these proteins stabilize the filaments. Neither substitution of CaCl2 for MgCl2 nor the inclusion of 20 mM phosphate altered the rate or equilibrium constants.
De La Cruz
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EM
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Pollard
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TD
D008841:Actin Cytoskeleton / Q000737:chemistry*; D000199:Actins / Q000737:chemistry*; D000205:Actomyosin / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D010590:Phalloidine / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D011817:Rabbits; D012235:Rhodamines; D014335:Tropomyosin / Q000737:chemistry
D000199:Actins; D010710:Phosphates; D012235:Rhodamines; D014335:Tropomyosin; D010590:Phalloidine; D000205:Actomyosin; D008274:Magnesium; D002118:Calcium