Kazuhiro Oiwa

Measurement of work done by ATP-induced sliding between rabbit muscle myosin and algal cell actin cables in vitro

1. The basic properties of the ATP‐dependent actin‐myosin interaction responsible for muscle contraction were studied using an in vitro force‐movement assay system, in which a glass microneedle coated with rabbit skeletal muscle myosin was made to slide on the actin filament arrays (actin cables) in the internodal cell of an alga Nitellopsis obtusa with ionophoretic application of ATP. 2. In response to an ATP current pulse (intensity, 5‐85 nA; duration, 0.5‐10 s), the myosin‐coated needle moved for a distance and eventually stopped, indicating reformation of rigor actin‐myosin linkages to prevent elastic recoil of the bent needle. A subsequent ATP current pulse again produced the needle movement starting from the baseline force attained by the preceding needle movement. 3. With a constant amount of ATP application, the amount of work done by the ATP‐induced actin‐myosin sliding first increased with increasing baseline force from zero to 0.4‐0.6P0, and then decreased with further increasing baseline force, thus giving a bell‐shaped work versus baseline force relation. 4. With increasing amount of ATP application, the amount of work done by the actin‐myosin sliding increased more steeply as the baseline force was increased from zero to 0.4‐0.6P0. 5. These results are discussed in connection with the basic properties of the actin‐myosin sliding in muscle contraction.

FORCE-VELOCITY RELATIONS OF RAT CARDIAC MYOSIN ISOZYMES SLIDING ON ALGAL CELL ACTIN CABLES IN VITRO

The difference in kinetic properties between two myosin isozymes (V1 and V3) in rat ventricular myocardium was studied by determining the steady-state force-velocity (P-V) relations in the ATP-dependent movement of V1 and V3-coated polystyrene beads on actin cables of giant algal cells mounted on a centrifuge microscope. The maximum unloaded velocity of bead movement was larger for V1 than for V3. The velocity of bead movement decreased with increasing external load applied by the centrifuge microscope, and eventually reached zero when the load was equal to the maximum isometric force (P0) generated by the myosin heads. The maximum isometric force P0 was less than 10 pN, and did not differ significantly between V1 and V3. The P-V curves consisted of a hyperbolic part in the low force range and a non-hyperbolic part in the high force range. The critical force above which the curve deviated from the hyperbola was much smaller for V1 than for V3. An analysis using a model with an extremely small number of myosin heads involved in the bead movement suggested a marked difference in kinetic properties between V1 and V3.

Relation between magnetically-applied force and velocity in beads coated with rabbit myosin, sliding on actin cables in Nitellopsis cells

SummaryWe have succeeded in controlling the sliding movement of myosin-coated magnetizable beads on actin cables in Nitellopsis cells by the inhomogeneous magnetic field adjacent to a small, strong permanent magnent. The relation between magnetic force acting on the bead and the bead velocity was, in many respects, similar to that obtained from the same system by the use of centrifugal force (Oiwa et al., 1990). In particular, force favouring the motion (negative load) had little effect on the velocity until it was sufficient to pull the bead off the actin, whereas a relatively small positive load caused a reduction in velocity to a plateau value. Although the present method does not allow a good control of force direction, it demonstrates the promise of magnetic force in studying in vitro motility.

Unitary Distance of ATP-Induced Actin-Myosin Sliding Studied with an In Vitro Force-Movement Assay System

We studied the unitary distance of ATP-induced actin-myosin sliding using an in vitro force-movement assay system consisting of a myosin-coated glass microneedle and well organized actin filament arrays (actin cables) in the internodal cell of an alga Nitellopsis obtusa. The number of myosin heads interacting with actin cables was reduced to about 100, as judged from the isometric force of about 100 pN attained in the presence of 2 mM ATP. When the amount of iontophoretically applied ATP was reduced by decreasing the amount of charge passed through the ATP electrode from 80 to 2 nC, the distance of the ATP-induced actin-myosin sliding decreased almost linearly from about 100 to about 10 nm, no detectable sliding being observed with further reduction of charge through the electrode. The sliding distances with small amounts of ATP (7-16 nC) distributed around integral multiples of 10 nm, suggesting the unitary distance of actin-myosin sliding of about 10 nm.

Kinetic Properties of the ATP-Dependent Actin-Myosin Sliding as Revealed by the Force-Movement Assay System with a Centrifuge Microscope

To study the kinetic properties of the ATP-dependent actin-myosin sliding responsible for muscle contraction, we developed an in vitro force-movement assay system, in which centrifugal forces were applied to myosin-coated polystyrene beads sliding along actin cables of giant algal cells in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement (positive loads), the beads moved with constant velocities. The steady-state force-velocity (P-V) curve thus obtained was double-hyperbolic in shape, being analogous to the P-V curve of single muscle fibers. Under constant centrifugal forces in the direction of the bead movement (negative loads), on the other hand, the beads also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads, but decreased markedly (by 20-60%). We also studied the effect of centrifugal forces at right angles with actin cables on the bead movement.

Dependence of the Work Done by ATP-Induced Actin-Myosin Sliding on the Initial Baseline Force: Its Implications for Kinetic Properties of Myosin Heads in Muscle Contraction

The properties of the ATP-dependent actin-myosin sliding responsible for muscle contraction was studied using an in vitro force-movement assay system, in which a myosin-coated glass microneedle was made to slide on actin filament arrays (actin cables) in the giant algal cell with iontophoretic application of ATP. With a constant amount of ATP application, the amount of work done by the actin-myosin sliding increased with increasing baseline force from zero to 0.4-0.6 Po, and then decreased with further increasing baseline force, thus giving a bell-shaped work versus baseline force relation. The result that the maximum actin-myosin sliding velocity did not change appreciably with increasing baseline force up to 0.4-0.6 Po implies, together with the limited number of myosin heads involved, that (1) the rate of power output of actin-myosin sliding is determined primarily by the amount of external load rather than the velocity of actin-myosin sliding, and (2) the bell shaped work versus baseline force relation (and also the hyperbolic force-velocity relation) results from the kinetic properties of individual myosin head rather than the change in the number of myosin heads involved.