TY - JOUR
T1 - Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine
AU - Tait, Jonathan F.
AU - Frieden, Carl
N1 - Funding Information:
’ This study was supported in part by Grant AM 13332 from the National Institutes of Health. J.F.T. was supported by National Institutes of Health Research Service Award GM 07200, Medical Scientist, from the National Institute of General Medical Sciences. ‘Author to whom correspondence should be addressed.
PY - 1982/6
Y1 - 1982/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0020434119&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(82)90197-7
DO - 10.1016/0003-9861(82)90197-7
M3 - Article
C2 - 7103505
AN - SCOPUS:0020434119
SN - 0003-9861
VL - 216
SP - 133
EP - 141
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -