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Journal of Physiology (1991), 437, pp. 751-763 With 6 figures Printed in Great Britain
MEASUREMENT OF WORK DONE BY ATP-INDUCED SLIDING BETWEEN RABBIT MUSCLE MYOSIN AND ALGAL CELL ACTIN CABLES IN VITRO
BY K. OIWA, S. CHAEN AND H. SUGI From the Department of Physiology, School of Medicine, Teikyo University Itabashi-ku, Tokyo 173, Japan (Received 6 September 1990) SUMMARY
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 04-0-6PO, 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 0O4-06Po. 5. These results are discussed in connection with the basic properties of the actin-myosin sliding in muscle contraction. INTRODUCTION
In spite of considerable progress in directly studying the ATP-dependent actin-myosin interaction with in vitro movement assay systems (Sheetz & Spudich, 1983; Shimmen & Yano, 1984; Kron & Spudich, 1986; Harada, Noguchi, Kishino & Yanagida, 1987), it has been impossible to relate the results to the basic characteristics of muscle contraction, since in these systems the actin-myosin sliding takes place only under unloaded conditions. To overcome this difficulty, we have recently developed an in vitro assay system in which a myosin-coated glass needle of a known elastic coefficient slides along the actin filament arrays (actin cables) in the internodal cell of an alga Nitellopsis in the presence of ATP, so that both the force MS 8781
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and the movement due to the ATP-dependent actin-myosin sliding can be measured simultaneously (Chaen, Oiwa, Shimmen, Iwamoto & Sugi, 1989). With this force-movement assay system, we obtained a convex upwards force-velocity curve analogous to that of single muscle fibres in the auxotonic condition (Iwamoto, Sugaya & Sugi, 1990), indicating that the assay system retains a basic characteristic of muscle contraction. In the present experiments, we measured the amount of work done by the actin-myosin sliding, which was induced by ionophoretic applications of ATP at various baseline forces. It will be shown that the work versus baseline force relation obtained is bell-shaped like the well-known work versus load relation in contracting muscle (Fenn, 1923). METHODS
Experimental arrangement Figure 1 shows the experimental arrangement. The internodal cell strip preparation (P) (ca 0 7 x 7 mm), prepared from the internodal cell of a green alga Nitellopsis obtusa (Chaen et al. 1989), was mounted flat with inner surface up in the experimental chamber (E) filled with ATP-free solution in such a way that the plane of the preparation inclined to the horizontal at ca 40 deg. The ATP-free solution had the following composition (mM): MgCl2, 5; EGTA, 4; dithiothreitol, 1; PIPES, 10; P1-P3-di(adenosine-5')pentaphosphate, 0-225; D-sorbitol, 200 (pH 7 0). In some experiments, the ATP-free solution further contained 50 unit/ml hexokinase and 2 mM-D-glucose. A glass microneedle (M) (tip diameter, ca 10/um; elastic coefficient, 150-170 pN/#m; frequency of oscillation, ca 40 Hz) was coated with myosin prepared from rabbit skeletal muscle (Perry, 1955) at the tip. The needle extended from a drift-free micromanipulator (Goodfellow Technology, UK) horizontally and at right angles with the chloroplast rows of the preparation, so that the myosin molecules on the needle tip were in contact with actin cables. The experimental chamber was mounted on the. mechanical stage of an inverted light microscope (Nikon, Diaphoto-TMD, Japan) to observe the microneedle with a Nikon 40 x dry objective (NA 0 55). Further details of the method appear elsewhere (Chaen et al. 1989). Ionophoretic application of ATP The myosin molecules on the needle tip were activated to interact with the actin cables by ionophoretic application of ATP. A glass capillary microelectrode (ATP electrode, A), filled with a solution containing 100 mM-ATP (resistance, 100-600 MO) and mounted on a separate drift-free manipulator, was placed close to the myosin-coated needle. The distance from the tip of the ATP electrode to that of the needle was ca 50 pum, unless otherwise stated. The application of ATP to the needle tip was performed by applying negative current pulses (intensity, 5-85 nA; duration, 0 5-10 s) from an electronic stimulator (Nihon Kohden, SEN-3301, Japan) to the electrode through a current-clamp circuit. To inhibit spontaneous release of ATP from the ATP electrode, a positive DC current (2-5 nA) was continuously applied to the electrode. Care was taken to apply current pulses at constant intervals, so that the DC currents were applied for a constant period before each application of current pulse. The relation between the amount of electrical charge passed through the ATP electrode and that of ionophoretically released ATP from the electrode was examined by measuring the amount of released ATP with a luminometer utilizing the luciferin-luciferase reaction (Analytical Luminescence Laboratory, Monolight 2010, USA; lower limit of detection, < 10-13 mol ATP). A linear relation was always obtained between the electrical charge and the released ATP in five different ATP electrodes used, the slope being 10 +0-5 x 10"15mol ATP/nC (S.D.).
Recording of the needle movement To minimize the change in relative position of the myosin-coated microneedle to the ATP electrode as the needle moved past the actin cables, the range of the needle movement for each application of ATP was limited to 1-2 pm by using microneedles with relatively large stiffness. The small movement of the needle in response to ionophoretically applied ATP was recorded by
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splitting the image of the needle tip with a wedge-shaped mirror (W) into two parts, each of which was projected on a photodiode (P1 and P2) (Hamamatsu Photonics, S2386-5K, Japan). The position of the mirror was initially adjusted in such a way that the mirror split the needle tip image into two equal parts. In this condition, there was no difference in the output voltage signal
'~~~~ Fig. 1. Experimental arrangement. The myosin-coated tip of a glass microneedle (M) is put in contact with the inner surface of the internodal cell strip preparation (P) at right angles to the chloroplast rows on which the actin cables are located. The experimental chamber (E) filled with ATP-free solution was mounted on an inverted light microscope. The needle was made to slide along the actin cables by applying negative current pulses to the microelectrode (A) filled with 100 mM-ATP. The needle tip movement was recorded by splitting the needle tip image with the wedge-shaped mirror (W) into two parts, each of which was projected on a photodiode (P1 and P2). For further explanation, see text.
(difference signal) between the two photodiodes. A small movement of the needle tip produced a change in the difference signal between the photodiodes (spatial resolution, ca 4 nm). The difference signal between the photodiodes was divided by the sum of the outputs with an analog divider, so that the difference signal was a linear function of the change in needle position up to ca 5,um irrespective of the brightness of the microscopic field. Since the needle moved against the increasing force of its elastic recoil, the situation was comparable to a muscle shortening by pulling a Hookean spring (auxotonic condition). Experimental procedure and data analysis The myosin-coated needle was made to slide on the actin cables repeatedly by ionophoretically applied ATP at constant intervals of 20-60 s. Before the total distance of needle movement exceeded the linear range of the recording system, the position of the wedge-shaped mirror was adjusted (with a manipulator carrying the mirror) to bring the difference signal between the photodiodes back to zero. After a series of needle movements, the maximum 'isometric' force (P0) generated by the myosin molecules on the needle was calculated from the needle position at which the needle showed no further movement in response to applied ATP. The value of Po ranged from 500 to 1500 pN. During the course of repeated application of ATP, the difference signal between the photodiodes could be returned to zero after the ATP-induced needle movement by moving the
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mechanical stage carrying the experimental chamber. Thus the needle was restored to its original position while kept attached to the actin cables. It was also possible to change the baseline force by moving the mechanical stage. After each needle movement in a series of movements, the position of the ATP electrode was adjusted to keep its distance from the needle unchanged. The needle movement records were stored in a microcomputer (NEC, PC98XL, Japan) through an analog-to-digital converter of 12-bit resolution together with the current pulse records, and were displayed on an X-Y recorder or processed with the microcomputer for data analysis. The microcomputer was also used for simulating the time course of diffusion of ionophoretically released ATP from the ATP electrode to the needle tip (see Fig. 20). All experiments were performed at room temperature (20-23 °C). RESULTS
Nature of the ATP-induced movement of a myosin-coated microneedle along the actin cables In the ATP-free solution, the myosin-coated tip of the microneedle attached firmly to the actin cables due to rigor linkages between the myosin molecules on the needle and the actin cables. In response to a negative current pulse applied to the ATP electrode (ATP current pulse), the myosin-coated needle started moving along the actin cable as a result of the actin-myosin interaction induced by the ionophoretically applied ATP; the needle movement took place in one direction determined by the polarity of the actin cables (Chaen et al. 1989). As shown in Fig. 2A and B, the time course of the ATP-induced needle movement was sigmoidal, so that the velocity of movement first increased with time to reach a maximum and then decreased with time. In the preliminary experiments, in which the spontaneous release of ATP from the electrode was not inhibited by a positive current, the needle did not stop after the termination of an ATP current pulse but showed a slow continuous movement (Fig. 2A), indicating a continuous ATPactivated actin-myosin interaction. In the later experiments, such continuous needle movement was completely eliminated by applying positive DC current (2-5 nA) to the ATP electrode. Then the needle eventually stopped after termination of the ATP current pulse (Fig. 2B), indicating that, when the applied ATP is no longer available for the actin-myosin sliding, the myosin molecules on the needle again form rigor linkages with the actin cables, thus inhibiting the elastic recoil of the bent needle. In Fig. 2 C are shown the calculated time courses of ATP concentration changes produced by 2 s ATP current pulses of various intensities at a point 50 ,tm distant from the ATP electrode, i.e. at the position of the myosin-coated needle. The simulation was made by numerically solving the three-dimensional differential equation for the diffusion of ionophoretically released ATP, using a diffusion constant of 7 1 x 10-6 cm2/s (Bowen & Martin, 1964). After the onset of a 2 s ATP current pulse, the calculated ATP concentration around the needle rises until the termination of the current pulse with a time course depending on the current intensity, and then decreases slowly. The ATP hydrolysis by the myosin molecules on the needle may not significantly affect the time course of ATP concentration changes, if the limited number of myosin molecules involved (250-750, see Discussion) and their actin-activated turnover rate (10 s-', Wagner & Weeds, 1979) are taken into consideration.
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Stepwise needle movement caused by repeated application of ATP A myosin-coated needle, which had stopped after an ATP-induced movement, could be made to move again by another ATP current pulse, starting from the baseline force attained by the previous movement, and eventually stopped at a new A
B
~L
F|T10nnA
I
120 nA
0.1Pm
10.2gm 5s
C
140onA
5s
Fig. 2. Nature of the ATP-induced needle movement. A, example of needle movement without application of positive DC current eliminating spontaneous ATP release from the ATP electrode. B, example of needle movement with positive DC current. In both A and B, the upper and lower traces show ATP current pulse and needle movement respectively. C, calculated time course of ATP concentration changes around the microneedle (lower traces) in response to 2 s ATP current pulses of various intensities (upper traces), assuming an electrical charge versus released ATP relation of 1-0 x 10-15 mol ATP/nC.
position (Fig. 3A). Such summation of needle movement could be repeated many times by repeated application of ATP current pulses, thus building up a stepwise needle movement record. If, however, the force of elastic recoil of the bent needle exceeded 0-8-0-9PO, the ATP-induced needle movement was sometimes preceded by a small backward movement, recognized as an initial dip on the movement record (Fig. 3B). The initial dip appeared only with ATP current pulses of more than 2 s. When the force exerted by the needle eventually reached PO, the needle showed no further movement in response to an ATP current pulse except for the occasional appearance of an initial dip followed by recovery to the initial position (Fig. 3C). Since rigor actin-myosin linkages break rapidly in the presence of ATP (Lymn & Taylor, 1971; Abe, Yamada, Takahashi & Sugi, 1989), the conversion of the myosin molecules from the rigor state to force-generating state may be expected to complete very quickly. This implies that, immediately after the ATP-induced breaking of rigor linkages, the myosin molecules can start moving the needle almost simultaneously, as they generate force (of less than 0-8PO) to resist the elastic recoil of the bent
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needle. This view is supported by the result that the interval between the onset of ATP current pulse and that of needle movement showed little variation (0O4-0-8 s), and did not change appreciably with the amount of baseline force when this was less than 08Po (Fig. 5). The occasional appearance of a dip in the needle movement (Fig.
A|
5 nA
J 0-5 jum
5s Bl
J
120 nA
__
10 s
120 nA
0.5 ,m
10 s
Fig. 3. Examples of the ATP-induced needle movements starting from different baseline forces. A, stepwise needle movement record caused by two successive 1 s ATP current pulses. The first movement started from zero baseline force, while the second movement started from a baseline force of ca O-lPO, which had been attained by the previous movement. B, appearance of the initial dip in the needle movement starting from the baseline force of 08Po. C, disappearance of ATP-induced needle movement except for the initial dip followed by recovery to the initial position.
3B and C) might result from an instability of the balance between the forces exerted by the myosin molecules and the bent needle when the myosin molecules are activated for a long period under a high baseline force. Relation between the baseline force and the amount of work done by the actin-myosin sliding with a constant amount of ATP application The stepwise needle movement records produced by repeated applications of ATP allowed us to study the relation between the amount of baseline force, against which the needle started moving, and the amount of work done by the myosin molecules on the needle in response to a constant amount of ionophoretically applied ATP. The
WORK BY ATP-INDUCED ACTIN-MYOSIN SLIDING 757 amount of work (W) done by the myosin molecules during the course of an ATPinduced needle movement from position xl to position X2 along the actin cables is: W = f oX2~~~~~~~X F(x)dx = { Kxdx = K 2
X
D/2,
where F(x) is the force exerted by the bent needle and K is the elastic coefficient of the needle. 0.1
0
3005[-0
0~~~~~~~~
0
0-5 Po Po Baseline force Fig. 4. Relation between the amount of work done (in fJ) by the needle movement induced by a constant amount of ATP application and the amount of baseline force (relative to PO). Needle movements were induced by 1 s ATP current pulses (15 nA) from various baseline forces in a random order. The curve was drawn by eye. Note a finite value of work done for zero baseline force, because the needle movement took place in the auxotonic condition.
In Fig. 4, the amount of work done during the course of a needle movement induced by a 1 s ATP current pulse (15 nA) is plotted against the amount of baseline force from which the needle started moving. Initially we expected that the amount of work done during the course of a needle movement would be more or less the same irrespective of the amount of baseline force, since a constant amount of ATP was supplied for each needle movement. Contrary to our expectation, however, the amount of work done in response to a constant amount of ATP application first increased with increasing amount of baseline force from zero to 04-O-6PO, and then decreased with further increasing baseline force, reaching zero when the baseline force was P0. The dependence of the amount of work produced by a constant amount of ATP on the amount of baseline force remained unchanged when force exerted by the bent needle was varied in a random order, without detaching the needle from the actin cables, by moving the position of the mechanical stage carrying the experimental chamber. Thus, the work versus baseline force relation was bell-shaped. Similar results were obtained with eleven different myosin-coated needles moving on the actin cables from eight different internodal cell preparations.
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Effect of the baseline force on the time course of needle movement with a constant amount of applied ATP During the course of experiments for studying the work versus baseline force relation shown in Fig. 4, it was recognized that the needle movement with a constant
A
B
/ _
| ~~~~~~~~~1 PM 5s
C
Fig. 5. Changes in the time course of the needle movements starting from different baseline forces. In each movement record, downward arrow indicates time of onset of 2 s ATP current pulse (34 nA), and upward arrow indicates approximate time of termination of needle movement. The amount of baseline force was zero in A, 0 45PO in B and 072Po in C.
amount of ATP application changed its time course systematically depending on the baseline force. In Fig. 5 are shown three needles movement records starting from different baseline forces. The duration of needle movement was longest when it started from zero baseline force (Fig. 5A), and decreased as the baseline force was increased from zero to 0-4Y-6Po (Fig. 5B). On the other hand, the maximum velocity of needle movement did not change appreciably over the range of baseline forces from zero to 0-4 t6Po. The duration of needle movement further decreased when the baseline force was increased to 07-O-8PO, but the needle movement was sometimes followed by a slow backward movement lasting for 15 s or more (Fig. 5C).
The ATP-induced needle movement in the presence of hexokinase and D-glucose Myosin molecules on the needle were also repeatedly activated to interact with the actin cables from the same baseline force by applying various amounts of ATP, the initial needle position being restored after completion of each needle movement. The relation between work and amount of applied ATP was obtained at two to three different baseline forces using the same myosin-coated needle. To avoid accumulation of ATP in the experimental chamber after applying a number of ATP current pulses, hexokinase (50 units/ml) and D-glucose (2 mM) were added to ATP-free solution. A typical result is shown in Fig. 6A. As can be seen in the inset, the needle
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movement stopped completely in 1-2 s after the termination of the ATP current pulse, no subsequent slow needle movement being observed without any positive DC current. This indicates that hexokinase is effective in quickly reducing ATP concentration in the experimental chamber after the termination of an ATP current A 0.3
100 nA .61
0 L0.4 0 upm
0.2
0-1 0.1
Time (s)
A
P0
0
O~~~~~2 C40*-1 PoP
0~~~~~ 0
50 100 150 Electrical charge (nC)
B 0.4
0.3
02 o0.1
0
0-5 P0 Po Baseline force Fig. 6. The ATP-induced needle movement in the presence of hexokinase and D-glucose. A, relation between the amount of work done (in fJ) by the ATP-induced needle movement and the amount of electrical charge (nC) passed through the ATP electrode. The lines are drawn by eye. Needle movements were induced from three different baseline forces: zero (D), 0-31Po (0) and 0-61Po (A). Inset shows a typical needle movement in the presence of hexokinase and D-glucose. B, relation between the amount of work done (in fJ) by the needle movement induced by 2 s ATP current pulse (85 nA) and the amount of baseline force (relative to PO). Needle movements were induced from various baseline forces in a random order. 0
pulse. The slope of the line relating the amount of work done by the actin-myosin sliding with the amount of electrical charge passed through the ATP electrode (except for the range of work close to zero) was found to be steeper as the baseline force was increased from zero to 04-06Po. Similar results were obtained with five
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different needles on the actin cables from three different cell preparations. This may be taken to indicate, together with the results in Fig. 4, that the efficiency with which the ionophoretically applied ATP is used for producing work by the actin-myosin sliding is dependent on the initial baseline force from which the actin-myosin sliding takes place. A constant amount of ATP was also repeatedly applied to the myosin-coated needle at various baseline forces in the presence of hexokinase and D-glucose. As shown in Fig. 6B, a bell-shaped work versus baseline force relation similar to that in Fig. 4 was obtained, indicating that the bell-shaped relation remains the same irrespective of whether the ATP concentration around the needle decreases gradually after the termination of an ATP current pulse (Fig. 2C) or sharply by the action of hexokinase. As with the experiments without hexokinase and D-glucose (Fig. 5), the maximum velocity of needle movement did not change appreciably in the range of baseline forces from zero to 0A4-0-6Po. DISCUSSION
Comparison of the present assay system with stimulated muscle In the present study, we have successfully combined the technique of ionophoretic application of ATP with the in vitro force-movement assay system, in which both the force and the movement of a myosin-coated needle on the actin cables can be simultaneously recorded (Chaen et al. 1989). The myosin molecules on the needle were activated to interact with the actin cables for a limited period determined by the ATP current pulse (Fig. 2C). The ATP-induced actin-myosin sliding in the present assay system may be comparable to contraction of stimulated muscle, except that in the latter the initiation of the actin-myosin sliding is mediated by Caa2+ released from the sarcoplasmic reticulum. After the termination of an ATP current pulse, the needle eventually stopped at a new position due to the formation of rigor actin-myosin linkages inhibiting the elastic recoil of the bent needle (Figs 2 and 3), in contrast with stimulated muscle which relaxes completely after the end of stimulation due to the reuptake of Ca2+ by the sarcoplasmic reticulum. The value of PO in the present assay system ranged from 500 to 1500 pN. If the maximum isometric force generated by each myosin head is assumed to be ca 1 pN (Hill, 1974), it follows that the number of myosin molecules responsible for the needle movement is only 250-750. These figures are at most 1/106 of the number of myosin molecules within a half-sarcomere of a single muscle fibre generating a PO of ca 1-5 mN. The extremely limited number of myosin molecules involved in the present assay system seems to provide a favourable condition to obtain information about the properties of individual myosin molecules. The myosin molecules would detach rapidly from the actin cables when the ATP concentration around the needle rises above a critical value, starting to interact with the actin cables almost simultaneously to move the needle. This view is supported by the constant interval between the onset of the ATP current pulse and that of needle movement irrespective of the initial baseline force (Fig. 5). In stimulated muscle, on the other hand, the isometric force rises with time after the onset of stimulation, so that the interval between the onset of stimulation and the onset of after-loaded shortening increases with
WORK BY ATP-INDUCED ACTIN-MYOSIN SLIDING 761 increasing after-load, indicating that a finite time is necessary to activate a huge number of myosin molecules in muscle. Dependence of the apparent efficiency of the ATP-induced actin-myosin sliding on the .baseline force The present experiments have shown that, when a constant amount of ATP is applied to the myosin-coated needle on the actin cables, the amount of work done by the ATP-induced actin-myosin sliding increases with increasing baseline force from zero to 04-06PO, and then decreased with further increasing baseline force (Fig. 4). In accordance with the ascending part of the bell-shaped work versus baseline force relation (Fig. 4), the amount of work done by the actin-myosin sliding increases with increasing amount of ATP application more steeply as the baseline force is increased from zero to 04-06Po (Fig. 6A), indicating an increase in the apparent efficiency with which the actin-myosin sliding produces work using the ionophoretically applied ATP. In this connection, it is of interest that, in contracting muscle, the amount of work done increases as the amount of external load is increased from zero to a certain value, thus giving a well-known bell-shaped work versus load relation (Fenn, 1923) analogous to the bell-shaped work versus baseline force relation in Fig. 4, indicating that the present force-movement assay system retains well the basic characteristic of contracting muscle in spite of random orientation of myosin molecules on the needle. In the present assay system, a very limited number of myosin molecules are almost simultaneously activated to interact with the actin cables. On this basis, it seems possible that the above dependence of the apparent efficiency of the actin-myosin sliding on the baseline force results from the ability of individual myosin molecules to produce work with various apparent efficiencies depending on the baseline force, rather than the change in the number of myosin molecules involved in the sliding. There is, however, an opposite possibility that the change in the apparent efficiency is due to the change in the number of myosin molecules involved; if an increased baseline force pulls some rigor actin-myosin linkages to bring them into a more favourable configuration for starting the active sliding on delivery of ATP, more and more myosin molecules would be involved in the sliding as the baseline force is increased. In contracting muscle, it has been well known that the bell-shaped work versus load relation is associated with the similar bell-shaped relation between the amount of heat produced and the load (Fenn, 1923). Though it is not possible to measure heat production or ATP hydrolysis in our present assay system, the increase in the apparent efficiency of ATP-induced actin-myosin sliding is likely to be associated with an increased rate of ATP hydrolysis. This point should be made clear by future experimental work. A most basic characteristic of contracting muscle is the steady-state hyperbolic force-velocity relation (Hill, 1938). Since the power is the product of shortening velocity and force (= load), the power versus load relation during steady shortening is directly obtained from the force-velocity curve. The relation obtained in this way is bell-shaped, as the power is zero when the load is zero or P0. Recently, Oiwa, Chaen, Kamitsubo, Shimmen & Sugi (1990) have shown with a centrifuge microscope
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that, when a myosin-coated polystyrene bead moving on the actin cables is subjected centrifugal forces serving as external loads, the steady-state relation is also hyperbolic in shape except for a high-force region. This force-velocity that the implies power versus load relation in this assay system is also bell-shaped. In this connection, it is of interest that the bell-shaped work versus baseline force relation has been obtained on the ATP-induced actin-myosin sliding both in the to various constant
absence (Fig. 4) and in the presence of hexokinase and D-glucose (Fig. 6B). Since the duration of the needle movement is nearly constant in the presence of hexokinase and D-glucose (inset of Fig. 6), the average power versus baseline force relation (comparable with the power versus load relation) is also bell-shaped. Since the maximum velocity of needle movement does not change appreciably in the ascending part of the bell shaped work versus baseline force curve (Fig. 5), it is suggested that the power output of the actin-myosin sliding is primarily determined by the force in each actin-myosin linkage rather than the velocity of actin-myosin sliding (Huxley, 1957). We wish to thank Sir Andrew F. Huxley and Dr Manuel F. Morales for valuable criticisms of this work. This work was supported by the Science Research Promotion Fund from the Japan Private School Promotion Foundation (H.S.) and by Grant-in-Aid for Scientific Research 62300017 from the Ministry of Education, Science and Culture of Japan (H. S.).
REFERENCES ABE,O., YAMADA, T., TAKAHASHI, K. & SUGI, H. (1989). Initiation of active force development in a molluscan smooth muscle by laser photolysis of caged ATP. Proceedings of the Japan Academy 65B, 165-168.
W. J. & MARTIN, H. L. (1964). The diffusion of adenosine triphosphate through aqueous BOWEN, solutions. Archives of Biochemistry and Biophysics 107, 30-36. CHAEN, S.,OIWA, K., SHIMMEN, T., IWAMOTO, H. & SUGI, H. (1989). Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro. of the National Academy of Sciences of the USA 86, 1510-1514. Proceedings A W. 0. between the energy liberated and the work
FENN, (1923). quantitative comparison performed by the isolated sartorius muscle of the frog. Journal of Physiology 58, of175-203. HARADA, Y., NoGUCHI, A., KISHINO, A. & YANAGIDA, T. (1987). Sliding movement single actin filaments on one-headed myosin filaments. Nature 326, 605-608. HILL, A. V. (1938). The heat of shortening and the dynamic constants of muscle. Proceedings of the Royal Society B 126, 136-195. HILL, T. L. (1974). Theoretical formalism for the sliding filament model of contraction of striated muscle. Part I. Progress in Biophysics and Molecular Biology 28, 267-340. HUXLEY, A. F. (1957). Muscle structure and theories of contraction. Progress in Biophysics and Biophysical Chemistry 7, 255-318. IWAMOTO, H., SUGAYA, R. & SUGI, H. (1990). Force-velocity relation of frog skeletal muscle fibres shortening under continuously changing load. Journal of Physiology 422, 185-202. KRON, S. J. & SPUDICH, J. A. (1986). Fluorescent actin filaments move on myosin fixed to a glass surface. Proceedings of the National Academy of Sciences of the USA 83, 6272-6276. (1971). Mechanism of adenosine triphosphate hydrolysis by LYMN, R. W. & TAYLOR, E. W.4617-4624. actomyosin. Biochemistry 10, OIWA, K., CHAEN, S., KAMITSUBO, E., SHIMMEN, T. & SUGI, H. (1990). Steady-state force-velocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro studied with a centrifuge microscope. Proceedings of the National Academy of Sciences of the USA 87, 7893-7897. PERRY, S. V. (1955). Myosin adenosinetriphosphatase. In Methods in Enzymology, vol. II, ed. COLOWICK, S. P. & KAPLAN, N. O., pp. 582-588. Academic Press, New York.
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SHEETZ, M. P. & SPUDICH, J. A. (1983). Movement of myosin-coated fluorescent beads on actin cables in vitro. NVature 303, 31-35. SHIMMEN, T. & YANO, M. (1984). Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma 121, 132-137. WAGNER. P. D. & WEEDS, A. G. (1979). Determination of the association of myosin subfragment 1 with actin in the presence of ATP. Biochemistry 18, 2260-2266.
Journal of Physiology (1991), 437, pp. 751-763 With 6 figures Printed in Great Britain
MEASUREMENT OF WORK DONE BY ATP-INDUCED SLIDING BETWEEN RABBIT MUSCLE MYOSIN AND ALGAL CELL ACTIN CABLES IN VITRO
BY K. OIWA, S. CHAEN AND H. SUGI From the Department of Physiology, School of Medicine, Teikyo University Itabashi-ku, Tokyo 173, Japan (Received 6 September 1990) SUMMARY
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 04-0-6PO, 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 0O4-06Po. 5. These results are discussed in connection with the basic properties of the actin-myosin sliding in muscle contraction. INTRODUCTION
In spite of considerable progress in directly studying the ATP-dependent actin-myosin interaction with in vitro movement assay systems (Sheetz & Spudich, 1983; Shimmen & Yano, 1984; Kron & Spudich, 1986; Harada, Noguchi, Kishino & Yanagida, 1987), it has been impossible to relate the results to the basic characteristics of muscle contraction, since in these systems the actin-myosin sliding takes place only under unloaded conditions. To overcome this difficulty, we have recently developed an in vitro assay system in which a myosin-coated glass needle of a known elastic coefficient slides along the actin filament arrays (actin cables) in the internodal cell of an alga Nitellopsis in the presence of ATP, so that both the force MS 8781
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and the movement due to the ATP-dependent actin-myosin sliding can be measured simultaneously (Chaen, Oiwa, Shimmen, Iwamoto & Sugi, 1989). With this force-movement assay system, we obtained a convex upwards force-velocity curve analogous to that of single muscle fibres in the auxotonic condition (Iwamoto, Sugaya & Sugi, 1990), indicating that the assay system retains a basic characteristic of muscle contraction. In the present experiments, we measured the amount of work done by the actin-myosin sliding, which was induced by ionophoretic applications of ATP at various baseline forces. It will be shown that the work versus baseline force relation obtained is bell-shaped like the well-known work versus load relation in contracting muscle (Fenn, 1923). METHODS
Experimental arrangement Figure 1 shows the experimental arrangement. The internodal cell strip preparation (P) (ca 0 7 x 7 mm), prepared from the internodal cell of a green alga Nitellopsis obtusa (Chaen et al. 1989), was mounted flat with inner surface up in the experimental chamber (E) filled with ATP-free solution in such a way that the plane of the preparation inclined to the horizontal at ca 40 deg. The ATP-free solution had the following composition (mM): MgCl2, 5; EGTA, 4; dithiothreitol, 1; PIPES, 10; P1-P3-di(adenosine-5')pentaphosphate, 0-225; D-sorbitol, 200 (pH 7 0). In some experiments, the ATP-free solution further contained 50 unit/ml hexokinase and 2 mM-D-glucose. A glass microneedle (M) (tip diameter, ca 10/um; elastic coefficient, 150-170 pN/#m; frequency of oscillation, ca 40 Hz) was coated with myosin prepared from rabbit skeletal muscle (Perry, 1955) at the tip. The needle extended from a drift-free micromanipulator (Goodfellow Technology, UK) horizontally and at right angles with the chloroplast rows of the preparation, so that the myosin molecules on the needle tip were in contact with actin cables. The experimental chamber was mounted on the. mechanical stage of an inverted light microscope (Nikon, Diaphoto-TMD, Japan) to observe the microneedle with a Nikon 40 x dry objective (NA 0 55). Further details of the method appear elsewhere (Chaen et al. 1989). Ionophoretic application of ATP The myosin molecules on the needle tip were activated to interact with the actin cables by ionophoretic application of ATP. A glass capillary microelectrode (ATP electrode, A), filled with a solution containing 100 mM-ATP (resistance, 100-600 MO) and mounted on a separate drift-free manipulator, was placed close to the myosin-coated needle. The distance from the tip of the ATP electrode to that of the needle was ca 50 pum, unless otherwise stated. The application of ATP to the needle tip was performed by applying negative current pulses (intensity, 5-85 nA; duration, 0 5-10 s) from an electronic stimulator (Nihon Kohden, SEN-3301, Japan) to the electrode through a current-clamp circuit. To inhibit spontaneous release of ATP from the ATP electrode, a positive DC current (2-5 nA) was continuously applied to the electrode. Care was taken to apply current pulses at constant intervals, so that the DC currents were applied for a constant period before each application of current pulse. The relation between the amount of electrical charge passed through the ATP electrode and that of ionophoretically released ATP from the electrode was examined by measuring the amount of released ATP with a luminometer utilizing the luciferin-luciferase reaction (Analytical Luminescence Laboratory, Monolight 2010, USA; lower limit of detection, < 10-13 mol ATP). A linear relation was always obtained between the electrical charge and the released ATP in five different ATP electrodes used, the slope being 10 +0-5 x 10"15mol ATP/nC (S.D.).
Recording of the needle movement To minimize the change in relative position of the myosin-coated microneedle to the ATP electrode as the needle moved past the actin cables, the range of the needle movement for each application of ATP was limited to 1-2 pm by using microneedles with relatively large stiffness. The small movement of the needle in response to ionophoretically applied ATP was recorded by
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splitting the image of the needle tip with a wedge-shaped mirror (W) into two parts, each of which was projected on a photodiode (P1 and P2) (Hamamatsu Photonics, S2386-5K, Japan). The position of the mirror was initially adjusted in such a way that the mirror split the needle tip image into two equal parts. In this condition, there was no difference in the output voltage signal
'~~~~ Fig. 1. Experimental arrangement. The myosin-coated tip of a glass microneedle (M) is put in contact with the inner surface of the internodal cell strip preparation (P) at right angles to the chloroplast rows on which the actin cables are located. The experimental chamber (E) filled with ATP-free solution was mounted on an inverted light microscope. The needle was made to slide along the actin cables by applying negative current pulses to the microelectrode (A) filled with 100 mM-ATP. The needle tip movement was recorded by splitting the needle tip image with the wedge-shaped mirror (W) into two parts, each of which was projected on a photodiode (P1 and P2). For further explanation, see text.
(difference signal) between the two photodiodes. A small movement of the needle tip produced a change in the difference signal between the photodiodes (spatial resolution, ca 4 nm). The difference signal between the photodiodes was divided by the sum of the outputs with an analog divider, so that the difference signal was a linear function of the change in needle position up to ca 5,um irrespective of the brightness of the microscopic field. Since the needle moved against the increasing force of its elastic recoil, the situation was comparable to a muscle shortening by pulling a Hookean spring (auxotonic condition). Experimental procedure and data analysis The myosin-coated needle was made to slide on the actin cables repeatedly by ionophoretically applied ATP at constant intervals of 20-60 s. Before the total distance of needle movement exceeded the linear range of the recording system, the position of the wedge-shaped mirror was adjusted (with a manipulator carrying the mirror) to bring the difference signal between the photodiodes back to zero. After a series of needle movements, the maximum 'isometric' force (P0) generated by the myosin molecules on the needle was calculated from the needle position at which the needle showed no further movement in response to applied ATP. The value of Po ranged from 500 to 1500 pN. During the course of repeated application of ATP, the difference signal between the photodiodes could be returned to zero after the ATP-induced needle movement by moving the
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mechanical stage carrying the experimental chamber. Thus the needle was restored to its original position while kept attached to the actin cables. It was also possible to change the baseline force by moving the mechanical stage. After each needle movement in a series of movements, the position of the ATP electrode was adjusted to keep its distance from the needle unchanged. The needle movement records were stored in a microcomputer (NEC, PC98XL, Japan) through an analog-to-digital converter of 12-bit resolution together with the current pulse records, and were displayed on an X-Y recorder or processed with the microcomputer for data analysis. The microcomputer was also used for simulating the time course of diffusion of ionophoretically released ATP from the ATP electrode to the needle tip (see Fig. 20). All experiments were performed at room temperature (20-23 °C). RESULTS
Nature of the ATP-induced movement of a myosin-coated microneedle along the actin cables In the ATP-free solution, the myosin-coated tip of the microneedle attached firmly to the actin cables due to rigor linkages between the myosin molecules on the needle and the actin cables. In response to a negative current pulse applied to the ATP electrode (ATP current pulse), the myosin-coated needle started moving along the actin cable as a result of the actin-myosin interaction induced by the ionophoretically applied ATP; the needle movement took place in one direction determined by the polarity of the actin cables (Chaen et al. 1989). As shown in Fig. 2A and B, the time course of the ATP-induced needle movement was sigmoidal, so that the velocity of movement first increased with time to reach a maximum and then decreased with time. In the preliminary experiments, in which the spontaneous release of ATP from the electrode was not inhibited by a positive current, the needle did not stop after the termination of an ATP current pulse but showed a slow continuous movement (Fig. 2A), indicating a continuous ATPactivated actin-myosin interaction. In the later experiments, such continuous needle movement was completely eliminated by applying positive DC current (2-5 nA) to the ATP electrode. Then the needle eventually stopped after termination of the ATP current pulse (Fig. 2B), indicating that, when the applied ATP is no longer available for the actin-myosin sliding, the myosin molecules on the needle again form rigor linkages with the actin cables, thus inhibiting the elastic recoil of the bent needle. In Fig. 2 C are shown the calculated time courses of ATP concentration changes produced by 2 s ATP current pulses of various intensities at a point 50 ,tm distant from the ATP electrode, i.e. at the position of the myosin-coated needle. The simulation was made by numerically solving the three-dimensional differential equation for the diffusion of ionophoretically released ATP, using a diffusion constant of 7 1 x 10-6 cm2/s (Bowen & Martin, 1964). After the onset of a 2 s ATP current pulse, the calculated ATP concentration around the needle rises until the termination of the current pulse with a time course depending on the current intensity, and then decreases slowly. The ATP hydrolysis by the myosin molecules on the needle may not significantly affect the time course of ATP concentration changes, if the limited number of myosin molecules involved (250-750, see Discussion) and their actin-activated turnover rate (10 s-', Wagner & Weeds, 1979) are taken into consideration.
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Stepwise needle movement caused by repeated application of ATP A myosin-coated needle, which had stopped after an ATP-induced movement, could be made to move again by another ATP current pulse, starting from the baseline force attained by the previous movement, and eventually stopped at a new A
B
~L
F|T10nnA
I
120 nA
0.1Pm
10.2gm 5s
C
140onA
5s
Fig. 2. Nature of the ATP-induced needle movement. A, example of needle movement without application of positive DC current eliminating spontaneous ATP release from the ATP electrode. B, example of needle movement with positive DC current. In both A and B, the upper and lower traces show ATP current pulse and needle movement respectively. C, calculated time course of ATP concentration changes around the microneedle (lower traces) in response to 2 s ATP current pulses of various intensities (upper traces), assuming an electrical charge versus released ATP relation of 1-0 x 10-15 mol ATP/nC.
position (Fig. 3A). Such summation of needle movement could be repeated many times by repeated application of ATP current pulses, thus building up a stepwise needle movement record. If, however, the force of elastic recoil of the bent needle exceeded 0-8-0-9PO, the ATP-induced needle movement was sometimes preceded by a small backward movement, recognized as an initial dip on the movement record (Fig. 3B). The initial dip appeared only with ATP current pulses of more than 2 s. When the force exerted by the needle eventually reached PO, the needle showed no further movement in response to an ATP current pulse except for the occasional appearance of an initial dip followed by recovery to the initial position (Fig. 3C). Since rigor actin-myosin linkages break rapidly in the presence of ATP (Lymn & Taylor, 1971; Abe, Yamada, Takahashi & Sugi, 1989), the conversion of the myosin molecules from the rigor state to force-generating state may be expected to complete very quickly. This implies that, immediately after the ATP-induced breaking of rigor linkages, the myosin molecules can start moving the needle almost simultaneously, as they generate force (of less than 0-8PO) to resist the elastic recoil of the bent
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needle. This view is supported by the result that the interval between the onset of ATP current pulse and that of needle movement showed little variation (0O4-0-8 s), and did not change appreciably with the amount of baseline force when this was less than 08Po (Fig. 5). The occasional appearance of a dip in the needle movement (Fig.
A|
5 nA
J 0-5 jum
5s Bl
J
120 nA
__
10 s
120 nA
0.5 ,m
10 s
Fig. 3. Examples of the ATP-induced needle movements starting from different baseline forces. A, stepwise needle movement record caused by two successive 1 s ATP current pulses. The first movement started from zero baseline force, while the second movement started from a baseline force of ca O-lPO, which had been attained by the previous movement. B, appearance of the initial dip in the needle movement starting from the baseline force of 08Po. C, disappearance of ATP-induced needle movement except for the initial dip followed by recovery to the initial position.
3B and C) might result from an instability of the balance between the forces exerted by the myosin molecules and the bent needle when the myosin molecules are activated for a long period under a high baseline force. Relation between the baseline force and the amount of work done by the actin-myosin sliding with a constant amount of ATP application The stepwise needle movement records produced by repeated applications of ATP allowed us to study the relation between the amount of baseline force, against which the needle started moving, and the amount of work done by the myosin molecules on the needle in response to a constant amount of ionophoretically applied ATP. The
WORK BY ATP-INDUCED ACTIN-MYOSIN SLIDING 757 amount of work (W) done by the myosin molecules during the course of an ATPinduced needle movement from position xl to position X2 along the actin cables is: W = f oX2~~~~~~~X F(x)dx = { Kxdx = K 2
X
D/2,
where F(x) is the force exerted by the bent needle and K is the elastic coefficient of the needle. 0.1
0
3005[-0
0~~~~~~~~
0
0-5 Po Po Baseline force Fig. 4. Relation between the amount of work done (in fJ) by the needle movement induced by a constant amount of ATP application and the amount of baseline force (relative to PO). Needle movements were induced by 1 s ATP current pulses (15 nA) from various baseline forces in a random order. The curve was drawn by eye. Note a finite value of work done for zero baseline force, because the needle movement took place in the auxotonic condition.
In Fig. 4, the amount of work done during the course of a needle movement induced by a 1 s ATP current pulse (15 nA) is plotted against the amount of baseline force from which the needle started moving. Initially we expected that the amount of work done during the course of a needle movement would be more or less the same irrespective of the amount of baseline force, since a constant amount of ATP was supplied for each needle movement. Contrary to our expectation, however, the amount of work done in response to a constant amount of ATP application first increased with increasing amount of baseline force from zero to 04-O-6PO, and then decreased with further increasing baseline force, reaching zero when the baseline force was P0. The dependence of the amount of work produced by a constant amount of ATP on the amount of baseline force remained unchanged when force exerted by the bent needle was varied in a random order, without detaching the needle from the actin cables, by moving the position of the mechanical stage carrying the experimental chamber. Thus, the work versus baseline force relation was bell-shaped. Similar results were obtained with eleven different myosin-coated needles moving on the actin cables from eight different internodal cell preparations.
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Effect of the baseline force on the time course of needle movement with a constant amount of applied ATP During the course of experiments for studying the work versus baseline force relation shown in Fig. 4, it was recognized that the needle movement with a constant
A
B
/ _
| ~~~~~~~~~1 PM 5s
C
Fig. 5. Changes in the time course of the needle movements starting from different baseline forces. In each movement record, downward arrow indicates time of onset of 2 s ATP current pulse (34 nA), and upward arrow indicates approximate time of termination of needle movement. The amount of baseline force was zero in A, 0 45PO in B and 072Po in C.
amount of ATP application changed its time course systematically depending on the baseline force. In Fig. 5 are shown three needles movement records starting from different baseline forces. The duration of needle movement was longest when it started from zero baseline force (Fig. 5A), and decreased as the baseline force was increased from zero to 0-4Y-6Po (Fig. 5B). On the other hand, the maximum velocity of needle movement did not change appreciably over the range of baseline forces from zero to 0-4 t6Po. The duration of needle movement further decreased when the baseline force was increased to 07-O-8PO, but the needle movement was sometimes followed by a slow backward movement lasting for 15 s or more (Fig. 5C).
The ATP-induced needle movement in the presence of hexokinase and D-glucose Myosin molecules on the needle were also repeatedly activated to interact with the actin cables from the same baseline force by applying various amounts of ATP, the initial needle position being restored after completion of each needle movement. The relation between work and amount of applied ATP was obtained at two to three different baseline forces using the same myosin-coated needle. To avoid accumulation of ATP in the experimental chamber after applying a number of ATP current pulses, hexokinase (50 units/ml) and D-glucose (2 mM) were added to ATP-free solution. A typical result is shown in Fig. 6A. As can be seen in the inset, the needle
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movement stopped completely in 1-2 s after the termination of the ATP current pulse, no subsequent slow needle movement being observed without any positive DC current. This indicates that hexokinase is effective in quickly reducing ATP concentration in the experimental chamber after the termination of an ATP current A 0.3
100 nA .61
0 L0.4 0 upm
0.2
0-1 0.1
Time (s)
A
P0
0
O~~~~~2 C40*-1 PoP
0~~~~~ 0
50 100 150 Electrical charge (nC)
B 0.4
0.3
02 o0.1
0
0-5 P0 Po Baseline force Fig. 6. The ATP-induced needle movement in the presence of hexokinase and D-glucose. A, relation between the amount of work done (in fJ) by the ATP-induced needle movement and the amount of electrical charge (nC) passed through the ATP electrode. The lines are drawn by eye. Needle movements were induced from three different baseline forces: zero (D), 0-31Po (0) and 0-61Po (A). Inset shows a typical needle movement in the presence of hexokinase and D-glucose. B, relation between the amount of work done (in fJ) by the needle movement induced by 2 s ATP current pulse (85 nA) and the amount of baseline force (relative to PO). Needle movements were induced from various baseline forces in a random order. 0
pulse. The slope of the line relating the amount of work done by the actin-myosin sliding with the amount of electrical charge passed through the ATP electrode (except for the range of work close to zero) was found to be steeper as the baseline force was increased from zero to 04-06Po. Similar results were obtained with five
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different needles on the actin cables from three different cell preparations. This may be taken to indicate, together with the results in Fig. 4, that the efficiency with which the ionophoretically applied ATP is used for producing work by the actin-myosin sliding is dependent on the initial baseline force from which the actin-myosin sliding takes place. A constant amount of ATP was also repeatedly applied to the myosin-coated needle at various baseline forces in the presence of hexokinase and D-glucose. As shown in Fig. 6B, a bell-shaped work versus baseline force relation similar to that in Fig. 4 was obtained, indicating that the bell-shaped relation remains the same irrespective of whether the ATP concentration around the needle decreases gradually after the termination of an ATP current pulse (Fig. 2C) or sharply by the action of hexokinase. As with the experiments without hexokinase and D-glucose (Fig. 5), the maximum velocity of needle movement did not change appreciably in the range of baseline forces from zero to 0A4-0-6Po. DISCUSSION
Comparison of the present assay system with stimulated muscle In the present study, we have successfully combined the technique of ionophoretic application of ATP with the in vitro force-movement assay system, in which both the force and the movement of a myosin-coated needle on the actin cables can be simultaneously recorded (Chaen et al. 1989). The myosin molecules on the needle were activated to interact with the actin cables for a limited period determined by the ATP current pulse (Fig. 2C). The ATP-induced actin-myosin sliding in the present assay system may be comparable to contraction of stimulated muscle, except that in the latter the initiation of the actin-myosin sliding is mediated by Caa2+ released from the sarcoplasmic reticulum. After the termination of an ATP current pulse, the needle eventually stopped at a new position due to the formation of rigor actin-myosin linkages inhibiting the elastic recoil of the bent needle (Figs 2 and 3), in contrast with stimulated muscle which relaxes completely after the end of stimulation due to the reuptake of Ca2+ by the sarcoplasmic reticulum. The value of PO in the present assay system ranged from 500 to 1500 pN. If the maximum isometric force generated by each myosin head is assumed to be ca 1 pN (Hill, 1974), it follows that the number of myosin molecules responsible for the needle movement is only 250-750. These figures are at most 1/106 of the number of myosin molecules within a half-sarcomere of a single muscle fibre generating a PO of ca 1-5 mN. The extremely limited number of myosin molecules involved in the present assay system seems to provide a favourable condition to obtain information about the properties of individual myosin molecules. The myosin molecules would detach rapidly from the actin cables when the ATP concentration around the needle rises above a critical value, starting to interact with the actin cables almost simultaneously to move the needle. This view is supported by the constant interval between the onset of the ATP current pulse and that of needle movement irrespective of the initial baseline force (Fig. 5). In stimulated muscle, on the other hand, the isometric force rises with time after the onset of stimulation, so that the interval between the onset of stimulation and the onset of after-loaded shortening increases with
WORK BY ATP-INDUCED ACTIN-MYOSIN SLIDING 761 increasing after-load, indicating that a finite time is necessary to activate a huge number of myosin molecules in muscle. Dependence of the apparent efficiency of the ATP-induced actin-myosin sliding on the .baseline force The present experiments have shown that, when a constant amount of ATP is applied to the myosin-coated needle on the actin cables, the amount of work done by the ATP-induced actin-myosin sliding increases with increasing baseline force from zero to 04-06PO, and then decreased with further increasing baseline force (Fig. 4). In accordance with the ascending part of the bell-shaped work versus baseline force relation (Fig. 4), the amount of work done by the actin-myosin sliding increases with increasing amount of ATP application more steeply as the baseline force is increased from zero to 04-06Po (Fig. 6A), indicating an increase in the apparent efficiency with which the actin-myosin sliding produces work using the ionophoretically applied ATP. In this connection, it is of interest that, in contracting muscle, the amount of work done increases as the amount of external load is increased from zero to a certain value, thus giving a well-known bell-shaped work versus load relation (Fenn, 1923) analogous to the bell-shaped work versus baseline force relation in Fig. 4, indicating that the present force-movement assay system retains well the basic characteristic of contracting muscle in spite of random orientation of myosin molecules on the needle. In the present assay system, a very limited number of myosin molecules are almost simultaneously activated to interact with the actin cables. On this basis, it seems possible that the above dependence of the apparent efficiency of the actin-myosin sliding on the baseline force results from the ability of individual myosin molecules to produce work with various apparent efficiencies depending on the baseline force, rather than the change in the number of myosin molecules involved in the sliding. There is, however, an opposite possibility that the change in the apparent efficiency is due to the change in the number of myosin molecules involved; if an increased baseline force pulls some rigor actin-myosin linkages to bring them into a more favourable configuration for starting the active sliding on delivery of ATP, more and more myosin molecules would be involved in the sliding as the baseline force is increased. In contracting muscle, it has been well known that the bell-shaped work versus load relation is associated with the similar bell-shaped relation between the amount of heat produced and the load (Fenn, 1923). Though it is not possible to measure heat production or ATP hydrolysis in our present assay system, the increase in the apparent efficiency of ATP-induced actin-myosin sliding is likely to be associated with an increased rate of ATP hydrolysis. This point should be made clear by future experimental work. A most basic characteristic of contracting muscle is the steady-state hyperbolic force-velocity relation (Hill, 1938). Since the power is the product of shortening velocity and force (= load), the power versus load relation during steady shortening is directly obtained from the force-velocity curve. The relation obtained in this way is bell-shaped, as the power is zero when the load is zero or P0. Recently, Oiwa, Chaen, Kamitsubo, Shimmen & Sugi (1990) have shown with a centrifuge microscope
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that, when a myosin-coated polystyrene bead moving on the actin cables is subjected centrifugal forces serving as external loads, the steady-state relation is also hyperbolic in shape except for a high-force region. This force-velocity that the implies power versus load relation in this assay system is also bell-shaped. In this connection, it is of interest that the bell-shaped work versus baseline force relation has been obtained on the ATP-induced actin-myosin sliding both in the to various constant
absence (Fig. 4) and in the presence of hexokinase and D-glucose (Fig. 6B). Since the duration of the needle movement is nearly constant in the presence of hexokinase and D-glucose (inset of Fig. 6), the average power versus baseline force relation (comparable with the power versus load relation) is also bell-shaped. Since the maximum velocity of needle movement does not change appreciably in the ascending part of the bell shaped work versus baseline force curve (Fig. 5), it is suggested that the power output of the actin-myosin sliding is primarily determined by the force in each actin-myosin linkage rather than the velocity of actin-myosin sliding (Huxley, 1957). We wish to thank Sir Andrew F. Huxley and Dr Manuel F. Morales for valuable criticisms of this work. This work was supported by the Science Research Promotion Fund from the Japan Private School Promotion Foundation (H.S.) and by Grant-in-Aid for Scientific Research 62300017 from the Ministry of Education, Science and Culture of Japan (H. S.).
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W. J. & MARTIN, H. L. (1964). The diffusion of adenosine triphosphate through aqueous BOWEN, solutions. Archives of Biochemistry and Biophysics 107, 30-36. CHAEN, S.,OIWA, K., SHIMMEN, T., IWAMOTO, H. & SUGI, H. (1989). Simultaneous recordings of force and sliding movement between a myosin-coated glass microneedle and actin cables in vitro. of the National Academy of Sciences of the USA 86, 1510-1514. Proceedings A W. 0. between the energy liberated and the work
FENN, (1923). quantitative comparison performed by the isolated sartorius muscle of the frog. Journal of Physiology 58, of175-203. HARADA, Y., NoGUCHI, A., KISHINO, A. & YANAGIDA, T. (1987). Sliding movement single actin filaments on one-headed myosin filaments. Nature 326, 605-608. HILL, A. V. (1938). The heat of shortening and the dynamic constants of muscle. Proceedings of the Royal Society B 126, 136-195. HILL, T. L. (1974). Theoretical formalism for the sliding filament model of contraction of striated muscle. Part I. Progress in Biophysics and Molecular Biology 28, 267-340. HUXLEY, A. F. (1957). Muscle structure and theories of contraction. Progress in Biophysics and Biophysical Chemistry 7, 255-318. IWAMOTO, H., SUGAYA, R. & SUGI, H. (1990). Force-velocity relation of frog skeletal muscle fibres shortening under continuously changing load. Journal of Physiology 422, 185-202. KRON, S. J. & SPUDICH, J. A. (1986). Fluorescent actin filaments move on myosin fixed to a glass surface. Proceedings of the National Academy of Sciences of the USA 83, 6272-6276. (1971). Mechanism of adenosine triphosphate hydrolysis by LYMN, R. W. & TAYLOR, E. W.4617-4624. actomyosin. Biochemistry 10, OIWA, K., CHAEN, S., KAMITSUBO, E., SHIMMEN, T. & SUGI, H. (1990). Steady-state force-velocity relation in the ATP-dependent sliding movement of myosin-coated beads on actin cables in vitro studied with a centrifuge microscope. Proceedings of the National Academy of Sciences of the USA 87, 7893-7897. PERRY, S. V. (1955). Myosin adenosinetriphosphatase. In Methods in Enzymology, vol. II, ed. COLOWICK, S. P. & KAPLAN, N. O., pp. 582-588. Academic Press, New York.
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SHEETZ, M. P. & SPUDICH, J. A. (1983). Movement of myosin-coated fluorescent beads on actin cables in vitro. NVature 303, 31-35. SHIMMEN, T. & YANO, M. (1984). Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma 121, 132-137. WAGNER. P. D. & WEEDS, A. G. (1979). Determination of the association of myosin subfragment 1 with actin in the presence of ATP. Biochemistry 18, 2260-2266.
