Electron Microscopic Recording of Myosin Head Power and Recovery Strokes Using the Gas Environmental Chamber

Despite extensive studies, the amplitude and the mode of the myosin head movement, coupled with ATP hydrolysis, still remain to be a matter for debate and speculation. To obtain direct information about the ATP-coupled myosin head movement, we prepared synthetic myosin filaments (myosin-myosin rod copolymer), in which myosin heads were position-marked with gold particles via antibodies to myosin heads and kept in hydrated, living state in the gas environmental chamber. ATP was applied to the specimen iontophoretically by passing the current to an ATP-containing microelectrode, and the ATP-induced myosin head movement was recorded with an imaging plate system under a magnification of 10,000×, with the following novel findings: (1) In the absence of ATP, myosin heads fluctuate around a definite neutral position. (2) In the absence of actin filaments, myosin heads move away from the bare region of myosin filaments (recovery stroke, average amplitude, 6 nm) on ATP application and return to the neutral position after exhaustion of ATP. (3) In the presence of actin filaments, the ATP-induced myosin head power stroke exhibits two different modes depending on mechanical conditions. (4) Myosin heads determine the direction of ATP-induced movement without being guided by actin filaments

Measurement of Nucleotide Exchange Rate Constants in Single Rabbit Soleus Myofibrils during Shortening and Lengthening Using a Fluorescent ATP Analog

ABSTRACT The kinetics of displacement of a fluorescent nucleotide, 2Ј(3Ј) amido]ethyl]carbamoyl]-adenosine 5Ј-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibril