Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.
The 20,000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 degrees C. The light chain was almost completely exchanged within 30 min under the above conditions. The exchange was markedly inhibited either below 37 degrees C or in the presence of Mg2+ concentrations higher than 10 microM. The 20,000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl)amino]ethyl]amino-naphthalene-1-sulfonic acid (1,5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosin and was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; i.e., actin-activated ATPase activity was dependent on the 20,000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affected either by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20,000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0 = 0.33).(ABSTRACT TRUNCATED AT 250 WORDS)
Morita
J
J
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Takashi
R
R
Ikebe
M
M
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000201:enzymology; D009038:Motion; D009130:Muscle, Smooth / Q000201:enzymology*; D015879:Myosin Subfragments / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D014422:Turkeys
D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D009218:Myosins; D000252:Calcium-Transporting ATPases