DOI: 10.1002/cm.970210208. Characterization of tetramethylrhodaminyl‐phalloidin binding to cellular F‐actin

Characterization of tetramethylrhodaminyl‐phalloidin binding to cellular F‐actin

10.1002/cm.970210208

Crossref journal-article

Wiley

Cell Motility (311)

Abstract

AbstractFluorescent derivatives of phallcidin 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 tetramethylrhodaminy (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 equi librium measurements, the affinity of TRITC‐phalloidin binding to rabbit skeletal muscle F‐actin (pyrene labeled) or to PMN lysate F‐actin was 1–4 × 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 ± 10−5 sec−1. The affinity calculated from the kinetic measurements. (2 ± 1 × 10−7 M) agreed well with that obtained by equilibrium measurements. The rate with which 0.6 μM TRITC‐phalloidin inhibited 0.1 μM pyrenyl F‐actin depolymerization (90% inhibition in 10 sec) was much faster than the rate of binding to pyrenyl F‐actin (<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.

Bibliography

Cano, M. L., Cassimeris, L., Joyce, M., & Zigmond, S. H. (1992). Characterization of tetramethylrhodaminylâphalloidin binding to cellular Fâactin. Cell Motility, 21(2), 147â158. Portico.

Authors 4

Manuel L. Cano (first)
Lynne Cassimeris (additional)
Michael Joyce (additional)
Sally H. Zigmond (additional)

References 44 Referenced 42

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Dates

Type	When
Created	20 years, 6 months ago (Feb. 23, 2005, 9:52 p.m.)
Deposited	1 year, 10 months ago (Oct. 24, 2023, 4:33 a.m.)
Indexed	3 months ago (June 2, 2025, 10:06 p.m.)
Issued	33 years, 8 months ago (Jan. 1, 1992)
Published	33 years, 8 months ago (Jan. 1, 1992)
Published Online	20 years, 6 months ago (Feb. 4, 2005)
Published Print	33 years, 8 months ago (Jan. 1, 1992)

Funders 0

None

BibTeX

@article{Cano_1992, title={Characterization of tetramethylrhodaminyl‐phalloidin binding to cellular F‐actin}, volume={21}, ISSN={0886-1544}, url={http://dx.doi.org/10.1002/cm.970210208}, DOI={10.1002/cm.970210208}, number={2}, journal={Cell Motility}, publisher={Wiley}, author={Cano, Manuel L. and Cassimeris, Lynne and Joyce, Michael and Zigmond, Sally H.}, year={1992}, month=jan, pages={147–158} }

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