ISSN:
0886-1544
Keywords:
depolymerization
;
DNase I
;
association rate constant
;
dissociation rate constant
;
polymor-phonuclear leukocyte
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
Fluorescent 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.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970210208
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