Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine

Jonathan F. Tait, Carl Frieden

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Rabbit skeletal muscle actin has been labeled with the fluorescent sulfhydryl reagent iodoacetamidotetramethylrhodamine (rhodamine). The label is probably located on Cys-373 because prior treatment of the actin with N-ethylmaleimide prevents incorporation of rhodamine. When the rhodamine-actin is polymerized with MgSO4 or KCl, there is approximately a 1.5-fold increase in fluorescence. The change in fluorescence is correlated with incorporation of monomeric globular actin into polymer and can therefore be used as a quantitative measure of polymerization. Trace quantities of rhodamine-actin can be used to follow the kinetics of polymerization of unlabeled actin without disturbing the sample, and it is shown that the use of a capillary viscometer accelerates the rate of polymerization. Fluorescence photobleaching recovery experiments, which measure the diffusion of rhodamine-labeled actin, show that nondiffusible (apparent diffusion coefficient < 10-10 cm2/s) filaments appear during the polymerization process but that immediately after shearing these filaments are readily diffusible. These results demonstrate the destructive nature of hydrodynamic shear stress on polymerized actin.

Original languageEnglish
Pages (from-to)133-141
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume216
Issue number1
DOIs
StatePublished - Jun 1982

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