Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay.
In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.
Harada
Y
Y
Department of Biophysical Engineering, Osaka University, Japan.
Sakurada
K
K
Aoki
T
T
Thomas
D D
DD
Yanagida
T
T
D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism*; D000818:Animals; D004735:Energy Transfer; D007700:Kinetics; D008854:Microscopy, Electron; D008954:Models, Biological; D009119:Muscle Contraction; D009132:Muscles / Q000502:physiology; D009218:Myosins / Q000378:metabolism / Q000648:ultrastructure; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D014335:Tropomyosin; D000205:Actomyosin; D009218:Myosins