Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers.
We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)
Stein
R A
RA
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Ludescher
R D
RD
Dahlberg
P S
PS
Fajer
P G
PG
Bennett
R L
RL
Thomas
D D
DD
D000818:Animals; D004801:Eosine Yellowish-(YS) / Q000378:metabolism / Q000031:analogs & derivatives; D008163:Luminescent Measurements; D009119:Muscle Contraction; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits
C033531:eosin maleimide; D009218:Myosins; D004801:Eosine Yellowish-(YS)