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Effect of calcium ions on the flexibility of reconstituted thin filaments of muscle studied by quasielastic scattering of laser light
J. Mol. Biol. (1972) 68, 511422
Effect of Calcium Ions on the Flexibility of Reconstituted Thin Filaments of Muscle Studied by Quasielastic Scattering of Laser Light t SHIN’ICHI
ISHIWATA
AND SATORU FUJIME
Department of Physics, Faculty of Science, Nqoya Nagoya 464, Japan
University
(Received 9 December 1971, and in revised form 21 March 1972) Quasielastic scattering of laser light was used to examine the effect of calcium ions on the flexibility of a reconstituted thin filament, namely, an F-actin/ tropomyosin/troponin complex of striated muscle. The results showed that the flexibility of a reconstituted thin filament changed reversibly at about 1 pM of free calcium ions (i.e. the physiological concentration). Below 1 pM-Caa+ a tropomyosin/troponin system suppressed the bending motion of F-a&n. This suppression was removed by calcium ions above 1 pM, and the F-actin flexibility became almost the same as that of an F-actin/tropomyosin complex. A quantitative study showed that calcium ions affected the reconstituted thin filament in two ways at the free calcium ion concentrations of about 1 ~111: and 20 PM, corresponding, respectively, to the two different calcium binding constants of troponin. An over-all conformational change of a reconstituted thin filament is brought about by calcium ions. It is suggested that the effect of calcium ions on troponin can be transferred to F-actin by altering the binding between tropomyosin and several monomer units in F-actin. The possible role of the dynamic nature of F-actin in muscle contraction is discussed.
1. Introduction Since a tropomyosin/troponin (or relaxing protein) and calcium ion system was discovered by Ebashi and his co-workers, much attention has been paid to the regulatory mechanism of muscular contraction at molecular level (Ebashi & Endo, 1968). From the results of the investigations carried out before this present work, the scheme of regulation of muscular contraction and relaxation is as follows (Ebashi & Endo, 1968): calcium ions released from the reticulum to the contractile system by the depolarization of the membrane, which is caused by the action potential, triggers the interaction between thin and thick filaments to induce their relative sliding. Conversely, when calcium ions are recaptured by the reticulum as a result of the cessation of the depolarization of the membrane, the interaction between two filaments for sliding vanishes and muscle relaxation follows. Ebashi and coworkers have clearly demonstrated that, without the tropomyosin/troponin complex, the actomyosin and Mg”’ -ATP system can not respond to calcium ions at physiological, i.e. about micromolar, concentrations and that troponin is the sole calcium-receptive protein of the contractile system. Further, they proposed a model of the thin filament composed t Paper I in this series is Fujime
& Ishiwata,
1971. 511
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S. ISHIWATA
AND
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of F-actin, tropomyosin and troponin and suggested that tropomyosin might be a mediator transmitting the effect of calcium ions from troponin to F-actin (Ebashi, Endo & Ohtsuki, 1969). Therefore, to elucidate the role of thin filaments in muscular contraction, it is very important to study characteristics of an F-actin/tropomyosin/ troponin complex in vitro at about micromolar concentrations of free calcium ions. Recently Tonomura, Watanabe & Morales (1969) studied the effect of calcium ions on the F-actin/tropomyosin/troponin complex by electron spin resonance, using spinlabelled actin, tropomyosin and troponin. According to their results, information about the binding of calcium ions to troponin was transferred to F-actin probably across tropomyosin. This result supports the Ebashi theory (for example, Ebashi t Endo, 1968). We have studied the effect of calcium ions on the dynamic nature of the complex of F-actin, tropomyosin and troponin by using a newly developed technique, namely, quasi-elastic scattering of laser light. This gives information on the flexibility of bending motion of the complex. That is, by this technique we can detect an over-all conformational change of the complex, in contrast to the spin-label technique, which gives information about a local change. A preliminary note on the present work has been published (Ishiwata & Fujime, 1971a).
2. Materials and Methods All the musole proteins used in the present work were extracted from the rabbit back and leg striated muscle. (a) F-A&n Dry rabbit muscle was prepared by almost the same method as that of Straub (1943), except that new proteins discovered by Ebashi and his co-workers (Ebashi, Ebashi & Maruyama, 1964; Ebashi & Ebsshi, 1966) were carefully removed before the acetone treatment of the myosin-extracted minced muscle. G-a&n was extracted with 20 ml. of distilled water/g of the acetone powder at low temperature (2°C). For polymerization, KC1 was added to the extracted solution to a final concentration of 25 mM. The F-a&in thus obtained was sedimented by ultraoentrifugation at 78,000 g for 3.5 hr. The sedimented pellet was dissolved in 0.4 mM-ATP at pH 8.3 in the absence of salt and depolymerized. Dialysis and sonication were carried out for complete depolymerization. The cycle of polymerization and depolymerization was repeated twice. The concentration of F-a&n was determined by measuring the flow birefringence (Kasai, Kawashima & Oosawa, 1960). (b) Troprny&n
and tropmin
Tropomyosin and troponin were prepared from the muscle residue after extraction
of myosin and were isolated by acid precipitation at pH 4.6 (Ebashi, Wakabayashi & Ebashi, 1971). The precipitate at pH 4.6 was dissolved in 0.4 r.r-LiCl, from which tropomyosin was fractionated at pH 7 with ammonium sulphate (34 to 36 g/dl.). Troponin was obtained from the supernatant fraction at pH 4.5 by fractionation at pH 7 with ammonium sulphate (30 to 36 g/dl.). For further purification of these proteins, the acid precipitation at pH 4.5 was repeated in O-4 Irr-LiCl (or 1 M-KC?). Tropon in thus obtained increased the viscosity of the solution of tropomyosin, as Ebashi BEKodama (1966) have shown. In order to investigate the physiological activity of troponin, a functional assay was performed by observing superprecipitation (Ebashi, Kodama t Ebsshi, 1968), i.e. the effect of calcium ions on the turbidity change of synthetic actomyosin containing tropomyosin and troponin at 650 nm after the addition of ATP. From these experiments we can say that our troponin definitely passed the functional assay. The concentrations of tropomyosin and troponin were determined by measuring the optical density. Eg,& of 1% tropomyosin and E&‘&, of 1% troponin solutions were found to be 2.6 (Ooi, 1967) and 4-5, respectively, from the nitrogen contents determined by a micro-Kjeldahl method.
DYNAMIC
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OF F-ACTIN
(c) My&n
A
Myosin A was prepared by a method similar to that described by A. Szent-Gyorgyi (1961) with slight modifications. The concentration was determined by a biuret method (Gornall, Bardawill & David, 1949). ATP was purchased from Sigma Chemical Co. and other chemicals from Katayama Chemical Co. (Osaka). (d) Preparations of solutions When F-a&in, tropomyosin and troponin were mixed at room temperature, the complex showed remarkable thixotropio properties and very intense forward light scattering, indicating the presence of very large aggregates of the order of several microns in diameter. These aggregates, which were observed also by viscosity (Ebashi & Kodama, 1966) and flow birefringence (Maruyama & Ebaahi, 1970) measurements, rotation viscometry and light scattering, gave disturbances of sutllcient magnitude to allow the study of the physicochemical properties of the complex by optical methods. However, once the solution of the complex was heated at 45°C for 10 mm after mixing, thixotropic properties as well ss intense forward light scattering were considerably diminished; that is, the dispersed state of the complex was obtained. Physiological activity of the “annealed” complex was confirmed by observing the effect of calcium ions on the superprecipitation of the suspension composed of myosin A and the annealed complex (Table 1). (Details of this experiment will be published elsewhere in the near future.) Therefore we used the solutions annealed at 45°C for 10 min throughout the present work. TABLE
1
Fuactimal assay of tropmyosin/troponin
system
Relaxing index (Y/X) Control (AM) AM+TM+TN
AM + TM + TN (46°C for 10 min) AM + TM + TN (20°C for 13 hr)
1.5 8 5 17
Abbreviations used: AM, actomyosin; (myosin, 160 &ml. 8nd F-ectin, 60 &ml.); TM, tropomyosin, 26 &ml.; TN, troponin, 26 &ml. Solvent, 3Omx-KCl, 201m4-Tris . IICI (pH 8*3), 0.6 mna-MgCl,, and 480 @r-GaGI, or 240 an-EGTA was further added. ATP was added at 0.6 mn. For a measure of relaxing aotivity, the ratio y/z, was used, where z and y are, reqeotively, the half times of the inorease of ebsorbance 8t 660 nm after adding ATP in the presence and absenoe of oaloium ions (Ebashi et al.. 1968). Since we did not take any special precaution to remove contamination by calcium ions during the preparation of muscle proteins, free calcium ions of about 30 F were usually present iu the solution of the F-actin/tropomyosin/troponin complex except when EGTA (ethylene glycol bis @-amino ethylether)N,N’-tetraacetic acid) or a Ca/EGTA buffer wae added.
(e) Apparatus Flow birefringence was determined by a Rae-type home-made apparatus. A Zeiss spectrophotometer was used to measure ultraviolet absorption and turbidity at 360 and 660 nm. The apparatus used in the quasi-elastic scattering experiment was essentially the same as that used previously (Fujime, 1970a,b; Fujime & Ishiwata, 1971). A 6328 A light beam from a He-Ne laser (N 15 mW) was passed through the solution. The soattered light was received by a photomultiplier tube and was analysed with a home-made apparatus of the
614
S. ISHIWATA
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self-beat (or homodyne) type, the bandwidth of which was 3 Hz. All measurements were made at room temperature (23°C). Spectral densities of scattered light were satisfactorily andysed with a single Lorentzian :
SK W)OCWW~+ PI,
(1)
end the half width at half height is r = 2DKa
+ l/1 (in Hz units),
(2)
where w is the frequency difference (in Hz units) between incident and scattered light, D and 7 are, respectively, the translational diffusion coefficient and the average relaxation time of bending motion of s, long polymer such as F-actin, and K is the scattering vector, the absolute value of which is defined by K = (4?r/Qsin(#2), (where h is the wavelength of incident light in a medium and 4 the scattering angle). Spectral densities were measured at various scattering angles between 40 and GO”, and Ka-dependence of r was obtained. As shown in Figure 1, the slope and the intercept with the ordinate of the straight line (equation (2)) give, respectively, the values of 20 and l/7. The decrease of I/T corresponds to the increase of the chain flexibility. For details, see Fujime (1970a,b) and Fujime & Ishiwata (1971).
3. Results In previous work (Ishiwata t Fujime, 1971b; Fujime & Ishiwata, 1971), we studied the change in flexibility of the F-actin/tropomyosin complex at various weight ratios of tropomyosin to F-a&in and at various salt concentrations. It was concluded that there are two types of binding of tropomyosin to F-ectin: i.e. tropomyosin can bind directly to F-actin up to a weight ratio of 1:6 and in addition up to 1:6 indirectly, in the sense that only directly bound tropomyosin molecules can make F-a&in stiffer. In this present work, we measured at first the spectral densities of scattered light from solutions of the F-actin/tropomyosin/troponin system at various weight ratios of tropomyosin to F-a&in. A weight ratio of 2 of tropomyosin to troponin was maintained throughout. Figure 1 shows the results of the analysis of the spectral densities. From this Figure, two effects of the tropomyosin/troponin complex on F-actin are clearly seen. One is that the presence of troponin caused the decrease in D; such a decrease in D was not observed in the F-actin/tropomyosin complex without troponin. The second effect was that by adding tropomyosin and troponin, the intercept of the r versus K2 line with the ordinate (i.e. I/T) gradually shifted to the higher frequency side and the extent of this shift was slightly larger than in the case of the F-actin/tropomyosin complex without troponin (Ishiwata & Fujime, 1971b; Fujime & Ishiwata, 1971). To sum up, the effects of troponin on the F-actin/tropomyosin complex (in the presence of free calcium ions of about 30 pM) are the apparent decreases of both D and of the flexibility. To investigate the effect of calcium ions qualitatively, EGTA was added to a solution of F-actin, tropomyosin and troponin at a weight ratio of 6 : 1: 0.5. This ratio was chosen on the basis of the fact that tropomyosin can bind directly to I”-actin up to a weight ratio of tropomyosin to F-ectin of 1:6 (Ishiwata & Fujime, 1971b; Fujime & Ishiwata, 1971), which is nearly equal to the composition of the thin filament in a living muscle (Ebashi et al., 1969; Hartshorne & Pyun, 1971). As shown in Figure 2, the addition of EGTA in sufficient quantity to remove free calcium ions increased the slope and the intercept of the r versus K2 line. Then, the addition of
DYNAMIC
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515
FIG. 1. Half width at half height (r) wersu(I K2 at various weight ratios of tropomyosin to Factin. Weight rmtio of tropomyosin to troponin w&s kept 2. F-s&n, 3 mg/ml. Solvent, 100 mM-KCl, 5 rnM-Tris . HCl (pH 8.3) and 0.5 mM-ATP. (0) 0; (0) 1:30;(A) 1:lO; (A) 1:6; (0) 1:3.
150 1 (al
L.$
0
~ I
4 K’x 10-‘0cm-2
+
Fm. 2. Half width at half height (r) uer%ue Ka for a weight ratio of 1: 6. (a) (0) As prepared; (0) 200 FM-EUTA added; (0) further addition of 300 pM-cac1,. (b) (0) Aa prepared; (0) 60 q-EGTA added; (0) further addition of CmClp less than 50 PM; (m) further addition of 60 PM-EGTA. Other conditions, the same ES in the legend to Fig. 1. Arabic numermla attached to arrows indicate the order of measurements.
516
S. ISHIWATA
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excess amount of calcium ions to cancel the effect of added EGTA decreased the and the intercept to the initial values (Fig. 2(a)). When calcium ions nearly equivalent to added EGTA were added, however, the intercept of the r versus K2 line decreased to the initial value but the slope did not change (Fig. 2(b)). The addition of EGTA to this solution increased the intercept again. Thus, there are at least three states of the complex of F-actin, tropomyosin and troponin, depending on the concentration of free calcium ions ; at high concentrations of calcium ions, the complex has low D and l/r values ; in the absence of calcium ions, D and l/r are high; and at an intermediate concentration of calcium ions, D is high and l/r is low. All changes in D and l/7 were reversible. No appreciable change of the total intensity of scattered light was observed. At a low tropomyosin and troponin content, the complex was so well dispersed that the annealing treatment was not necessary. A complex of F-actin, tropomyosin and troponin at a weight ratio of 30 : 1: 0.5 was also examined to see the effect of annealing treatment. The result (Fig. 3) was similar to that shown in Figure 2. The annealing treatment did not have any effect on the change of flexibility of the complex by calcium ions. an
slope
FIQ. 3. Hmlf width at helf height (P) vema K2 for a weight ratio of 1: 30. Conditions and symbols am the same as in the legend to Fig. 2(e). In this cam, there was no annealing treatment.
FIQ. 4. Half width at half height (r) VW.TUSKa for control experiments. (0) F-a&in only; (O,.) F-actin/troponin; (A, A) F-actin/tropomyosin. Addition of 200 @-CsClp (open symbols) or 200 PM-EGTA (slled symbols) did not affeot the results. F-a&in, 3 mg/ml.; tropomyosin, 0.5 mg/ml.; troponin, @26 mg/ml. Other conditions, the same .ss in the legend to Fig. 1.
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617
Weight ratio of tropamyosin to F-win
FIG. 5. Summary of the qualitative results. l/7 (Hz) and the slope proportional to D (arbitrary unit) uemua weight ratios of tropomvosin to F-a&in. ( x ) F-a&in only (or F-a&in/troponin); (0) F-c&in/tropomyosin; (0) F-aatin/tropomyosin/troponin (a~ prepared); (0) F-aatin/tropomyoein/troponin &a 200 PM-EQTA. Conditions are the same aa in the legends to Figs 1, 2 and 3.
Figure 4 shows results of control experiments, in which it was confirmed that, in the case of F-a&in only, F-a&in and tropomyosin or F-a&in and troponin, the presence or the absence of calcium ions did not affect the P versu.s K2 relation. Only in the case of the three component complex of F-actin, tropomyosin and troponin, did calcium ions have the above-mentioned effect. In Figure 6, the qualitative aspect of the effect of troponin on the F-actin/tropomyosin complex described above is clearly shown. II(I-
i; I
lo( >-
-t: - 9(I-
8()-
’ Concentration
F-e&in, Solvent.
of EGTA
’
140
’
’
160
( pM 1
FIO. 6. 11~ ( 0) and the slope (0) uererBzu) added EGTA. 3 me/ml.; tropomyosin, 0.6 m&L; troponin, 0.26 nag/ml. 100 mar-KCl, 6 mM-Tris . HCl (pH 8.3), O-5 mm-ATP and 100 PM-C&l,.
S. ISHIWATA
618
AND
S. FUJIME
The order of the flexibility of the complexes w&s as follows: > F-actin/tropomyosin N (F-actin/tropomyosin/tro F-actin N F-actin/troponin ca2+. ponin),,a + > (F-actin/tropomyosin/troponin),,
-
I;;t100 0“O------l
90
I.0 80
-++k
s
-il!;1J05i P
05:
v)
i $ ! 0 20 40 Concentrotlon of EGTA
Conditions
68 (pM)
FIQ. 7. l/r (0) and the slope (0) vereua added EGTA. are the same as in the legend to Fig. 6, except that no CaCl, was added.
A solution of F-actin, tropomyosin and troponin at a weight ratio of 6 : 1: 0.5 was prepared with 100 EM-CaCl, at pH 8.3. EGTA was added to this solution step by step and the frequency spectra of scattered light measured at each step. Since the association constant between EGTA and calcium ions at pH 8.3 is very large (~10~ M-l), the concentration of free calcium ions decreased in proportion to the EGTA added, so long as the concentration of calcium ions was higher than that of EGTA. After the
I 8
I 7
I 6
I 5
I, 4,+
FIU. 8. l/r (open symbols) and the slope (filled symbols) ver8u8 pCe. The numerical Figs 6 and 7 are rewritten in terms of pCa. For details, sea text.
values in
DYNAMIC
STUDY
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F-ACTIM
519
concentration of EGTA exceeded that of calcium ions, the concentration of free calcium ions fell to 10-’ M or lower. As shown in Figure 6, at 120 PM-EGTA, the trenslational diffusion coefficient D suddenly increased. At about 145 PM-EGTA, the value of l/r suddenly increased. As described previously, there are two effects of the presence of calcium ions on the complex of F-actin, tropomyosin and troponin. The results of an experiment in which no calcium ions were ad&d beforehand are given in Figure 7 and are essentially similar to those shown in Figure 6. In the experiments shown in Figures 6 and 7 it was difficult to determine precisely the concentration of free calcium ions, because the concentration of contaminated calcium ions in the original sample solutions was not known. However, an attempt was made to translate the abscissa of these Figures into approximate values of the concentration of free calcium ions (Fig. 8). If this can be done, data of Figures 6 and 7 must coincide with each other. To obtain such a coincidence, the value of the concentration of contaminated calcium ions was adjusted by iteration, on the assumption that the binding constant between EGTA and calcium ions at pH 8.3 is about
PC0
Fm. 9. l/s (O), the slope (0) and the mean amplitude of bending motion (A) ver8218 pCa. (a) The concentration of Caa+ ions was controlled by a Ca/EGTA buffer solution. F-act& 3 mg/ml.; tropomyosin, @6 mg/ml.; troponin, 0.25 mg/ml. Solvent, 100 mrd-KCI, 10 mna-Tris * maleate (pH 6.8) and 0.6 mrtx-ATP. (b) The mean amplitude (6) of bending motion of an F-actiu/tropomyosiu/troponin complex of 1 II in lenath (L) under the same conditions ae above (a), w&s estimated accordk~ to the equation SAL3 -, ‘1 p = 3n’ 2‘n (Fujime & Ishiwata, 1971; Fujime, 1971), where the value of the (?a + 11214 parame&r A WA det&kned by using the relaxation time (7) shown in (8). 34
520
S. ISHIWATA
AND
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1.6 x lo0 ~-l. Then it was found that the concentration of contaminated calcium ions was about 37 PM and that the concentration of free calcium ions when a change in l/~ was observed was about O-7 pM. The concentration when a change in D was observed is found to be about 20 PM. Finally, to determine quantitatively the calcium ion concentration when I/T changed, the concentration of free calcium ions was controlled by using a Ca/EGTA buffer to allow one to ignore the contamination of calcium ions. 10 mM-Tris * maleate, pH 6.8, instead of 5 mM-Tris * HCl, pH 8.3, was used as a pH buffer because it was convenient to prepare the Ca buffer. The binding constant between EGTA and calcium ions was assumed to be 2 x lo6 M-l at pH 6.8 (Schwarzenbach, Senn & Anderegg, 1957). As shown in Figure 9, the change of l/r occurred at around 1 PMCa2+ . This was not influenced by the addition of 2 mM-Mg2 + . The mean amplitude of bending motion of an F-actin/tropomyosin/troponin complex, the contour length of which is 1 pm, was also estimated (Fig. 9) according to the equations derived in previous papers (Fujime & Ishiwata, 1971; Fujime, 1971). The difference between the concentrations of free calcium ions where l/r changed at pH 8.3 and 6.8 was reasonable, if the pH dependence of the binding constant of troponin with calcium ions was taken into account (Fuoks, Reddy & Briggs, 1971).
4. Discussion (a) Trandationul
diffu.sion
coeficient
In the absence of free calcium ions, the translational diffusion coefficient D is nearly the same as that of F-actin only. This value of D can be interpreted as that of a single F-a&in filament of a few microns in length (Fujime, 1970a). Therefore, it is reasonable to consider that, under this condition, 7 gives the relaxation time of spontaneous bending motion of the single filament. Similarly, the increase of 7 found at about 1 p*M-Ca2+means the increase of the flexibility of the single filament, because the value of D did not change. At a concentration of calcium ions greater than 20 pM, the value of D decreased considerably, whereas the value of l/r changed only a little. This decrease in D is probably due to the interaction between filaments. However, this does not indicate the formation of large aggregates, because the turbidity at 350 run did not change. The observed decrease in D can be explained by loose coupling among a few filaments, which would have no appreciable effect on the intrafilament movement. At first sight, the change in D at about 20 pM4-Ca2+seems to have no connection with the contraction mechanism in the well-organized striated muscle. However, this phenomenon means that the concentration of calcium ions reversibly controls a “sol (a dispersed state of filaments) + gel (an aggregated state)” transformation. It is expected that such a sol-gel transformation would have some connection with the control of the motility of lower organisms such as Myxomycete plasmodia (for example, Hatano, 1970). (b) Binding
between troponin
and calcium ions
According to Ebashi et al. (1968), troponin contains about 4 moles of calcium ions per lo5 g protein. In the presence of 4 mM-MgCl, at pH 6.8, the binding constant of half of the sites is 1.3 x lo6 ~-l and that of the remaining half 5 x lo4 M-l. Fucks $ Briggs (1968) also reported that the binding constants are 2.1 x lo6 iwe (class 1)
DYNAMIC
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F-ACTIN
621
and 3 x lo4 r.r-l (olass 2) in the presence of 2 rn&MgCl, at pH 7-O. On the other hand, apparent binding constants deduoed from Figures 8 and 9 are about 1 x lo8 at-’ and 5 x lo4 M-I. These values are in good agreement with those of Ebashi et al. (1968) and Fucks $ Briggs (1968), if the differenoes in solvent conditions are taken into account. Comparing these results, it can be concluded that troponin has et least two different effects corresponding to the degree of occupation of the binding sites by calcium ions, i.e. one half of the binding sites (lo6 M-‘) are related to the regulation of the flexibility chsnge of an F-actin/tropomyosin/troponin complex, and the other half ( lo4 M-I) to the change in the D vslue of the complex. (0) Supplementa y diwu88ion When calcium ions bind to troponin in a micromolar range of calcium ion concentration, a conformational change is expected to occur in troponin (Wakabayashi & Ebashi, 1968). This conformational change is considered to have an effect on the state of F-e&in through tropomyosin (Tonomura et al., 1969). A similar conclusion was reached from the present investigation: when troponin binds caloium ions, 8 transitional change of the flexibility of F-actin occurs only in the presence of tropomyosin. This is a typical example of “sequential control” in the biological system ; “ . . . --f membrane depolarization + calcium ion release + troponin + tropomyosin + actin 3 (acto)myosin + . . .“. The present results not only prove that calcium ions bound to troponin can regulate the state of F-actin in the presence of tropomyosin, but also suggest that the thin filament is not merely a rigid rod supporting tension developed in muscle, and that the dynamic nature of F-actin may have an important role in muscular contraction. What role can be given to the over-all flexibility change of the thin filament? One possibility is that, since there is a certain distance between the thin filament and the thick filament in muscle, the increase of the amplitude of spontaneous bending motion of the former may be required for the interaction between the two filaments (cf. Fig. 9). Even if the cross-bridge from myosin had a flexible hinge at the end, its interaction with actin might be made possible only by the help of flexibility of the thin filament,. Tropomyosin molecules are believed to bind to F-a&in along grooves between two strands of F-actin (Ebashi et aZ., 1969). Troponin binds not directly to F-actin but to tropomyosin, and is distributed periodically along the thin filament (Endo, Nonomum, Maseki, Ohtsuki t Ebeshi, 1966; Ohtsuki, Mesaki, Nonomura $ Ebashi, 1967). Each tropomyosin molecule covers more than half a pitch of the helix of F-a&in. When a conformational change of troponin induced by calcium ions is transmitted to tropomyosin, influences may extend to plural actin monomers in F-actin. Thus, troponin having no calcium ion, when bound to tropomyosin, makes the complex most rigid probably by strengthening the parallel binding between F-actin and tropomyosin. This effect is eliminated when troponin binds calcium ions. A recent preliminary study by the same method (Ishiwata $ Fujime, 1971c) showed that the increase in flexibility of F-actin by heavy meromyosin (Fujime & Ishiwata, 1971) can be regulated by the tropomyosin/troponin system, depending on the presence of free calcium ions at about micromolar concentrations, In the presence of calcium ions, the flexibility of the complex of F-a&in, tropomyosin and troponin is very much increased by binding of heavy meromyosin at a certain range of the molar ratio. In the absence of calcium ions, however, the flexibility of the complex is not changed by heavy meromyosin. It is well known that, in the presence of calcium ions,
822
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AND
S. FUJIME
the complex of F-actin, tropomyosin and troponin activates the Mg-ATPase of (heavy mero)myosin, whereas in the absence of calcium ions, activation does not occur (e.g. Kominz, 1966; Eisenberg & Kielley, 1970). The ATPase activation may be a result of the local interaction of the filament with myosin (Ebashi $ Endo, 1968). The apparent parallelism between results of light scattering and of the ATPase measurements strongly suggests that the F-actin flexibility observed here is a direct reflection of rather local interactions, although light scattering indeed measures average properties of the whole filament. We intend to extend further this kind of study on the interaction between thin filaments and myosin in the presence of ATP, bearing in mind the plausible hypothesis that the dynamic nature of the thin filament may have an essential role in muscular contraction and in other cellular movements (e.g. Oosawa, Asakura & Ooi, 1961). We thank Professor Fumio Oosawa for his guidance and encouragement throughout the present work. We are indebted to Professor Setsuro Ebashi and Dr Fumiko Ebashi of the University of Tokyo for valuable discussions and advice on the preparation of troponin. We also wish t,o thank all the members of our laboratory for stimulating discussions. REFERENCES Ebashi, S. & Ebashi, F. (1965). J. Biochem., Tokyo, 58, 7. Ebashi, S., Ebashi, F. & Maruyama, K. (1964). Nature, 203, 645. Ebashi, S. & Endo, M. (1968). In Progress in Biophysics a,nd Molecular Biology, vol. 18, p. 123. Oxford: Pergamon Press, Ltd. Ebashi, S., Endo, M. & Ohtsuki, I. (1969). Quart. Rev. Biophys. 2, 351. Ebashi, S. & Kodama, A. (1965). J. Biochem., Tokyo, 58, 107. Ebashi, S. & Kodama, A. (1966). J. Biochem.., Tokyo, 59, 425. Ebashi, S., Kodama, A. & Ebashi, F. (1968). J. Biochem., Tokyo, 64, 465. Ebashi, S., Wakabayashi, T. & Ebashi, F. (1971). J. Biochem., Tokyo, 69, 441. Eisenberg, E. & Kielley, W. W. (1970). Biochem. Biophys. Rea. Comm. 40, 50. Endo, M., Nonomura, Y., Masaki, T., Ohtsuki, I. & Ebashi, 6. (1966). J. Biochem., Tokyo, 60, 605. Fucks, F. & Briggs, F. N. (1968). J. Gen. PhysioZ. 51, 655. Fucks, F., Reddy, Y. & Briggs, F. N. (1971). Biochim,. biophys. Acttr, 221, 407. Fujime, S. (1970a). J. Phys. Sot. Japan, 29, 751. Fujime, S. (19706). J. Phys. Sot. Japan, 29, 416. Fujime, S. (1971). J. Phys. Sot. Japan, 31, 1805. Fujime, S. & Ishiwata, S. (1971). J. Mol. Biol. 62, 251. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949). J. Biol. Chem. 177, 751. Hartshorne, I). J. & Pyun, H. Y. (1971). Biochim. biophy.s. Acta, 229, 698. Hatano, S. (1970). Exp. CeZE Res. 61, 199. Ishiwata, S. & Fujime, S. (1971a). J. Phys. Sot. Japan, 30, 303. Ishiwata, S. & Fujime, S. (1971b). J. Phys. Sot. Jwpan, 30, 302. Ishiwata, S. & Fujime, S. (1971c). J. Phys. Sot. Japan, 31, 1601. Kasai, M., Kawashima, H. & Oosawa, F. (1960). J. Polymer Sci. 44, 51. Kominz, D. R. (1966). Arch. Biochem. Biophys. 115, 583. Maruyama, K. & Ebashi, 8. (1970). Sci. Papers CoZ. Gen. Educ., Univ. Tokyo, 20, 171. Ohtsuki, I., Masaki, T., Nonomura, Y. & Ebashi, S. (1967). J. Biochem., Tokyo, 61, 817. Ooi, T. (1967). Biochemistry, 6, 2433. Oosawa, F., Asakura, S. & Ooi, T. (1961). Progr. Theoret. Phys., Kyoto, Suppl. 17, 14. Schwarzenbach, G., Senn, H. & Anderegg, G. (1957). HeZv. chim. Acta, 40, 1886. Straub, F. B. (1943). Studies Inst. Med. Chem.. Univ. Szeged. 3, 23. Szent-GyGrgyi, A. (1951). Chemistry of MzLscu.Zar Contraction, 2nd edn., p. 146. London: Academic Press Inc. Tonomura, Y., Watanabe, S. & Morales, M. F. (1969). Biochemistry, 8, 217 1. Wakabayashi, T. & Ebashi, S. (1968). J. Biochem., Tokyo, 64, 731.
Effect of Calcium Ions on the Flexibility of Reconstituted Thin Filaments of Muscle Studied by Quasielastic Scattering of Laser Light t SHIN’ICHI
ISHIWATA
AND SATORU FUJIME
Department of Physics, Faculty of Science, Nqoya Nagoya 464, Japan
University
(Received 9 December 1971, and in revised form 21 March 1972) Quasielastic scattering of laser light was used to examine the effect of calcium ions on the flexibility of a reconstituted thin filament, namely, an F-actin/ tropomyosin/troponin complex of striated muscle. The results showed that the flexibility of a reconstituted thin filament changed reversibly at about 1 pM of free calcium ions (i.e. the physiological concentration). Below 1 pM-Caa+ a tropomyosin/troponin system suppressed the bending motion of F-a&n. This suppression was removed by calcium ions above 1 pM, and the F-actin flexibility became almost the same as that of an F-actin/tropomyosin complex. A quantitative study showed that calcium ions affected the reconstituted thin filament in two ways at the free calcium ion concentrations of about 1 ~111: and 20 PM, corresponding, respectively, to the two different calcium binding constants of troponin. An over-all conformational change of a reconstituted thin filament is brought about by calcium ions. It is suggested that the effect of calcium ions on troponin can be transferred to F-actin by altering the binding between tropomyosin and several monomer units in F-actin. The possible role of the dynamic nature of F-actin in muscle contraction is discussed.
1. Introduction Since a tropomyosin/troponin (or relaxing protein) and calcium ion system was discovered by Ebashi and his co-workers, much attention has been paid to the regulatory mechanism of muscular contraction at molecular level (Ebashi & Endo, 1968). From the results of the investigations carried out before this present work, the scheme of regulation of muscular contraction and relaxation is as follows (Ebashi & Endo, 1968): calcium ions released from the reticulum to the contractile system by the depolarization of the membrane, which is caused by the action potential, triggers the interaction between thin and thick filaments to induce their relative sliding. Conversely, when calcium ions are recaptured by the reticulum as a result of the cessation of the depolarization of the membrane, the interaction between two filaments for sliding vanishes and muscle relaxation follows. Ebashi and coworkers have clearly demonstrated that, without the tropomyosin/troponin complex, the actomyosin and Mg”’ -ATP system can not respond to calcium ions at physiological, i.e. about micromolar, concentrations and that troponin is the sole calcium-receptive protein of the contractile system. Further, they proposed a model of the thin filament composed t Paper I in this series is Fujime
& Ishiwata,
1971. 511
612
S. ISHIWATA
AND
5. FUJIME
of F-actin, tropomyosin and troponin and suggested that tropomyosin might be a mediator transmitting the effect of calcium ions from troponin to F-actin (Ebashi, Endo & Ohtsuki, 1969). Therefore, to elucidate the role of thin filaments in muscular contraction, it is very important to study characteristics of an F-actin/tropomyosin/ troponin complex in vitro at about micromolar concentrations of free calcium ions. Recently Tonomura, Watanabe & Morales (1969) studied the effect of calcium ions on the F-actin/tropomyosin/troponin complex by electron spin resonance, using spinlabelled actin, tropomyosin and troponin. According to their results, information about the binding of calcium ions to troponin was transferred to F-actin probably across tropomyosin. This result supports the Ebashi theory (for example, Ebashi t Endo, 1968). We have studied the effect of calcium ions on the dynamic nature of the complex of F-actin, tropomyosin and troponin by using a newly developed technique, namely, quasi-elastic scattering of laser light. This gives information on the flexibility of bending motion of the complex. That is, by this technique we can detect an over-all conformational change of the complex, in contrast to the spin-label technique, which gives information about a local change. A preliminary note on the present work has been published (Ishiwata & Fujime, 1971a).
2. Materials and Methods All the musole proteins used in the present work were extracted from the rabbit back and leg striated muscle. (a) F-A&n Dry rabbit muscle was prepared by almost the same method as that of Straub (1943), except that new proteins discovered by Ebashi and his co-workers (Ebashi, Ebashi & Maruyama, 1964; Ebashi & Ebsshi, 1966) were carefully removed before the acetone treatment of the myosin-extracted minced muscle. G-a&n was extracted with 20 ml. of distilled water/g of the acetone powder at low temperature (2°C). For polymerization, KC1 was added to the extracted solution to a final concentration of 25 mM. The F-a&in thus obtained was sedimented by ultraoentrifugation at 78,000 g for 3.5 hr. The sedimented pellet was dissolved in 0.4 mM-ATP at pH 8.3 in the absence of salt and depolymerized. Dialysis and sonication were carried out for complete depolymerization. The cycle of polymerization and depolymerization was repeated twice. The concentration of F-a&n was determined by measuring the flow birefringence (Kasai, Kawashima & Oosawa, 1960). (b) Troprny&n
and tropmin
Tropomyosin and troponin were prepared from the muscle residue after extraction
of myosin and were isolated by acid precipitation at pH 4.6 (Ebashi, Wakabayashi & Ebashi, 1971). The precipitate at pH 4.6 was dissolved in 0.4 r.r-LiCl, from which tropomyosin was fractionated at pH 7 with ammonium sulphate (34 to 36 g/dl.). Troponin was obtained from the supernatant fraction at pH 4.5 by fractionation at pH 7 with ammonium sulphate (30 to 36 g/dl.). For further purification of these proteins, the acid precipitation at pH 4.5 was repeated in O-4 Irr-LiCl (or 1 M-KC?). Tropon in thus obtained increased the viscosity of the solution of tropomyosin, as Ebashi BEKodama (1966) have shown. In order to investigate the physiological activity of troponin, a functional assay was performed by observing superprecipitation (Ebashi, Kodama t Ebsshi, 1968), i.e. the effect of calcium ions on the turbidity change of synthetic actomyosin containing tropomyosin and troponin at 650 nm after the addition of ATP. From these experiments we can say that our troponin definitely passed the functional assay. The concentrations of tropomyosin and troponin were determined by measuring the optical density. Eg,& of 1% tropomyosin and E&‘&, of 1% troponin solutions were found to be 2.6 (Ooi, 1967) and 4-5, respectively, from the nitrogen contents determined by a micro-Kjeldahl method.
DYNAMIC
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OF F-ACTIN
(c) My&n
A
Myosin A was prepared by a method similar to that described by A. Szent-Gyorgyi (1961) with slight modifications. The concentration was determined by a biuret method (Gornall, Bardawill & David, 1949). ATP was purchased from Sigma Chemical Co. and other chemicals from Katayama Chemical Co. (Osaka). (d) Preparations of solutions When F-a&in, tropomyosin and troponin were mixed at room temperature, the complex showed remarkable thixotropio properties and very intense forward light scattering, indicating the presence of very large aggregates of the order of several microns in diameter. These aggregates, which were observed also by viscosity (Ebashi & Kodama, 1966) and flow birefringence (Maruyama & Ebaahi, 1970) measurements, rotation viscometry and light scattering, gave disturbances of sutllcient magnitude to allow the study of the physicochemical properties of the complex by optical methods. However, once the solution of the complex was heated at 45°C for 10 mm after mixing, thixotropic properties as well ss intense forward light scattering were considerably diminished; that is, the dispersed state of the complex was obtained. Physiological activity of the “annealed” complex was confirmed by observing the effect of calcium ions on the superprecipitation of the suspension composed of myosin A and the annealed complex (Table 1). (Details of this experiment will be published elsewhere in the near future.) Therefore we used the solutions annealed at 45°C for 10 min throughout the present work. TABLE
1
Fuactimal assay of tropmyosin/troponin
system
Relaxing index (Y/X) Control (AM) AM+TM+TN
AM + TM + TN (46°C for 10 min) AM + TM + TN (20°C for 13 hr)
1.5 8 5 17
Abbreviations used: AM, actomyosin; (myosin, 160 &ml. 8nd F-ectin, 60 &ml.); TM, tropomyosin, 26 &ml.; TN, troponin, 26 &ml. Solvent, 3Omx-KCl, 201m4-Tris . IICI (pH 8*3), 0.6 mna-MgCl,, and 480 @r-GaGI, or 240 an-EGTA was further added. ATP was added at 0.6 mn. For a measure of relaxing aotivity, the ratio y/z, was used, where z and y are, reqeotively, the half times of the inorease of ebsorbance 8t 660 nm after adding ATP in the presence and absenoe of oaloium ions (Ebashi et al.. 1968). Since we did not take any special precaution to remove contamination by calcium ions during the preparation of muscle proteins, free calcium ions of about 30 F were usually present iu the solution of the F-actin/tropomyosin/troponin complex except when EGTA (ethylene glycol bis @-amino ethylether)N,N’-tetraacetic acid) or a Ca/EGTA buffer wae added.
(e) Apparatus Flow birefringence was determined by a Rae-type home-made apparatus. A Zeiss spectrophotometer was used to measure ultraviolet absorption and turbidity at 360 and 660 nm. The apparatus used in the quasi-elastic scattering experiment was essentially the same as that used previously (Fujime, 1970a,b; Fujime & Ishiwata, 1971). A 6328 A light beam from a He-Ne laser (N 15 mW) was passed through the solution. The soattered light was received by a photomultiplier tube and was analysed with a home-made apparatus of the
614
S. ISHIWATA
AND
S. FUJIME
self-beat (or homodyne) type, the bandwidth of which was 3 Hz. All measurements were made at room temperature (23°C). Spectral densities of scattered light were satisfactorily andysed with a single Lorentzian :
SK W)OCWW~+ PI,
(1)
end the half width at half height is r = 2DKa
+ l/1 (in Hz units),
(2)
where w is the frequency difference (in Hz units) between incident and scattered light, D and 7 are, respectively, the translational diffusion coefficient and the average relaxation time of bending motion of s, long polymer such as F-actin, and K is the scattering vector, the absolute value of which is defined by K = (4?r/Qsin(#2), (where h is the wavelength of incident light in a medium and 4 the scattering angle). Spectral densities were measured at various scattering angles between 40 and GO”, and Ka-dependence of r was obtained. As shown in Figure 1, the slope and the intercept with the ordinate of the straight line (equation (2)) give, respectively, the values of 20 and l/7. The decrease of I/T corresponds to the increase of the chain flexibility. For details, see Fujime (1970a,b) and Fujime & Ishiwata (1971).
3. Results In previous work (Ishiwata t Fujime, 1971b; Fujime & Ishiwata, 1971), we studied the change in flexibility of the F-actin/tropomyosin complex at various weight ratios of tropomyosin to F-a&in and at various salt concentrations. It was concluded that there are two types of binding of tropomyosin to F-ectin: i.e. tropomyosin can bind directly to F-actin up to a weight ratio of 1:6 and in addition up to 1:6 indirectly, in the sense that only directly bound tropomyosin molecules can make F-a&in stiffer. In this present work, we measured at first the spectral densities of scattered light from solutions of the F-actin/tropomyosin/troponin system at various weight ratios of tropomyosin to F-a&in. A weight ratio of 2 of tropomyosin to troponin was maintained throughout. Figure 1 shows the results of the analysis of the spectral densities. From this Figure, two effects of the tropomyosin/troponin complex on F-actin are clearly seen. One is that the presence of troponin caused the decrease in D; such a decrease in D was not observed in the F-actin/tropomyosin complex without troponin. The second effect was that by adding tropomyosin and troponin, the intercept of the r versus K2 line with the ordinate (i.e. I/T) gradually shifted to the higher frequency side and the extent of this shift was slightly larger than in the case of the F-actin/tropomyosin complex without troponin (Ishiwata & Fujime, 1971b; Fujime & Ishiwata, 1971). To sum up, the effects of troponin on the F-actin/tropomyosin complex (in the presence of free calcium ions of about 30 pM) are the apparent decreases of both D and of the flexibility. To investigate the effect of calcium ions qualitatively, EGTA was added to a solution of F-actin, tropomyosin and troponin at a weight ratio of 6 : 1: 0.5. This ratio was chosen on the basis of the fact that tropomyosin can bind directly to I”-actin up to a weight ratio of tropomyosin to F-ectin of 1:6 (Ishiwata & Fujime, 1971b; Fujime & Ishiwata, 1971), which is nearly equal to the composition of the thin filament in a living muscle (Ebashi et al., 1969; Hartshorne & Pyun, 1971). As shown in Figure 2, the addition of EGTA in sufficient quantity to remove free calcium ions increased the slope and the intercept of the r versus K2 line. Then, the addition of
DYNAMIC
STUDY
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515
FIG. 1. Half width at half height (r) wersu(I K2 at various weight ratios of tropomyosin to Factin. Weight rmtio of tropomyosin to troponin w&s kept 2. F-s&n, 3 mg/ml. Solvent, 100 mM-KCl, 5 rnM-Tris . HCl (pH 8.3) and 0.5 mM-ATP. (0) 0; (0) 1:30;(A) 1:lO; (A) 1:6; (0) 1:3.
150 1 (al
L.$
0
~ I
4 K’x 10-‘0cm-2
+
Fm. 2. Half width at half height (r) uer%ue Ka for a weight ratio of 1: 6. (a) (0) As prepared; (0) 200 FM-EUTA added; (0) further addition of 300 pM-cac1,. (b) (0) Aa prepared; (0) 60 q-EGTA added; (0) further addition of CmClp less than 50 PM; (m) further addition of 60 PM-EGTA. Other conditions, the same ES in the legend to Fig. 1. Arabic numermla attached to arrows indicate the order of measurements.
516
S. ISHIWATA
AND
8. FUJIME
excess amount of calcium ions to cancel the effect of added EGTA decreased the and the intercept to the initial values (Fig. 2(a)). When calcium ions nearly equivalent to added EGTA were added, however, the intercept of the r versus K2 line decreased to the initial value but the slope did not change (Fig. 2(b)). The addition of EGTA to this solution increased the intercept again. Thus, there are at least three states of the complex of F-actin, tropomyosin and troponin, depending on the concentration of free calcium ions ; at high concentrations of calcium ions, the complex has low D and l/r values ; in the absence of calcium ions, D and l/r are high; and at an intermediate concentration of calcium ions, D is high and l/r is low. All changes in D and l/7 were reversible. No appreciable change of the total intensity of scattered light was observed. At a low tropomyosin and troponin content, the complex was so well dispersed that the annealing treatment was not necessary. A complex of F-actin, tropomyosin and troponin at a weight ratio of 30 : 1: 0.5 was also examined to see the effect of annealing treatment. The result (Fig. 3) was similar to that shown in Figure 2. The annealing treatment did not have any effect on the change of flexibility of the complex by calcium ions. an
slope
FIQ. 3. Hmlf width at helf height (P) vema K2 for a weight ratio of 1: 30. Conditions and symbols am the same as in the legend to Fig. 2(e). In this cam, there was no annealing treatment.
FIQ. 4. Half width at half height (r) VW.TUSKa for control experiments. (0) F-a&in only; (O,.) F-actin/troponin; (A, A) F-actin/tropomyosin. Addition of 200 @-CsClp (open symbols) or 200 PM-EGTA (slled symbols) did not affeot the results. F-a&in, 3 mg/ml.; tropomyosin, 0.5 mg/ml.; troponin, @26 mg/ml. Other conditions, the same .ss in the legend to Fig. 1.
DYNAMIC
STUDY
OF F.ACTIN
617
Weight ratio of tropamyosin to F-win
FIG. 5. Summary of the qualitative results. l/7 (Hz) and the slope proportional to D (arbitrary unit) uemua weight ratios of tropomvosin to F-a&in. ( x ) F-a&in only (or F-a&in/troponin); (0) F-c&in/tropomyosin; (0) F-aatin/tropomyosin/troponin (a~ prepared); (0) F-aatin/tropomyoein/troponin &a 200 PM-EQTA. Conditions are the same aa in the legends to Figs 1, 2 and 3.
Figure 4 shows results of control experiments, in which it was confirmed that, in the case of F-a&in only, F-a&in and tropomyosin or F-a&in and troponin, the presence or the absence of calcium ions did not affect the P versu.s K2 relation. Only in the case of the three component complex of F-actin, tropomyosin and troponin, did calcium ions have the above-mentioned effect. In Figure 6, the qualitative aspect of the effect of troponin on the F-actin/tropomyosin complex described above is clearly shown. II(I-
i; I
lo( >-
-t: - 9(I-
8()-
’ Concentration
F-e&in, Solvent.
of EGTA
’
140
’
’
160
( pM 1
FIO. 6. 11~ ( 0) and the slope (0) uererBzu) added EGTA. 3 me/ml.; tropomyosin, 0.6 m&L; troponin, 0.26 nag/ml. 100 mar-KCl, 6 mM-Tris . HCl (pH 8.3), O-5 mm-ATP and 100 PM-C&l,.
S. ISHIWATA
618
AND
S. FUJIME
The order of the flexibility of the complexes w&s as follows: > F-actin/tropomyosin N (F-actin/tropomyosin/tro F-actin N F-actin/troponin ca2+. ponin),,a + > (F-actin/tropomyosin/troponin),,
-
I;;t100 0“O------l
90
I.0 80
-++k
s
-il!;1J05i P
05:
v)
i $ ! 0 20 40 Concentrotlon of EGTA
Conditions
68 (pM)
FIQ. 7. l/r (0) and the slope (0) vereua added EGTA. are the same as in the legend to Fig. 6, except that no CaCl, was added.
A solution of F-actin, tropomyosin and troponin at a weight ratio of 6 : 1: 0.5 was prepared with 100 EM-CaCl, at pH 8.3. EGTA was added to this solution step by step and the frequency spectra of scattered light measured at each step. Since the association constant between EGTA and calcium ions at pH 8.3 is very large (~10~ M-l), the concentration of free calcium ions decreased in proportion to the EGTA added, so long as the concentration of calcium ions was higher than that of EGTA. After the
I 8
I 7
I 6
I 5
I, 4,+
FIU. 8. l/r (open symbols) and the slope (filled symbols) ver8u8 pCe. The numerical Figs 6 and 7 are rewritten in terms of pCa. For details, sea text.
values in
DYNAMIC
STUDY
OF
F-ACTIM
519
concentration of EGTA exceeded that of calcium ions, the concentration of free calcium ions fell to 10-’ M or lower. As shown in Figure 6, at 120 PM-EGTA, the trenslational diffusion coefficient D suddenly increased. At about 145 PM-EGTA, the value of l/r suddenly increased. As described previously, there are two effects of the presence of calcium ions on the complex of F-actin, tropomyosin and troponin. The results of an experiment in which no calcium ions were ad&d beforehand are given in Figure 7 and are essentially similar to those shown in Figure 6. In the experiments shown in Figures 6 and 7 it was difficult to determine precisely the concentration of free calcium ions, because the concentration of contaminated calcium ions in the original sample solutions was not known. However, an attempt was made to translate the abscissa of these Figures into approximate values of the concentration of free calcium ions (Fig. 8). If this can be done, data of Figures 6 and 7 must coincide with each other. To obtain such a coincidence, the value of the concentration of contaminated calcium ions was adjusted by iteration, on the assumption that the binding constant between EGTA and calcium ions at pH 8.3 is about
PC0
Fm. 9. l/s (O), the slope (0) and the mean amplitude of bending motion (A) ver8218 pCa. (a) The concentration of Caa+ ions was controlled by a Ca/EGTA buffer solution. F-act& 3 mg/ml.; tropomyosin, @6 mg/ml.; troponin, 0.25 mg/ml. Solvent, 100 mrd-KCI, 10 mna-Tris * maleate (pH 6.8) and 0.6 mrtx-ATP. (b) The mean amplitude (6) of bending motion of an F-actiu/tropomyosiu/troponin complex of 1 II in lenath (L) under the same conditions ae above (a), w&s estimated accordk~ to the equation SAL3 -, ‘1 p = 3n’ 2‘n (Fujime & Ishiwata, 1971; Fujime, 1971), where the value of the (?a + 11214 parame&r A WA det&kned by using the relaxation time (7) shown in (8). 34
520
S. ISHIWATA
AND
S. FUJIME
1.6 x lo0 ~-l. Then it was found that the concentration of contaminated calcium ions was about 37 PM and that the concentration of free calcium ions when a change in l/~ was observed was about O-7 pM. The concentration when a change in D was observed is found to be about 20 PM. Finally, to determine quantitatively the calcium ion concentration when I/T changed, the concentration of free calcium ions was controlled by using a Ca/EGTA buffer to allow one to ignore the contamination of calcium ions. 10 mM-Tris * maleate, pH 6.8, instead of 5 mM-Tris * HCl, pH 8.3, was used as a pH buffer because it was convenient to prepare the Ca buffer. The binding constant between EGTA and calcium ions was assumed to be 2 x lo6 M-l at pH 6.8 (Schwarzenbach, Senn & Anderegg, 1957). As shown in Figure 9, the change of l/r occurred at around 1 PMCa2+ . This was not influenced by the addition of 2 mM-Mg2 + . The mean amplitude of bending motion of an F-actin/tropomyosin/troponin complex, the contour length of which is 1 pm, was also estimated (Fig. 9) according to the equations derived in previous papers (Fujime & Ishiwata, 1971; Fujime, 1971). The difference between the concentrations of free calcium ions where l/r changed at pH 8.3 and 6.8 was reasonable, if the pH dependence of the binding constant of troponin with calcium ions was taken into account (Fuoks, Reddy & Briggs, 1971).
4. Discussion (a) Trandationul
diffu.sion
coeficient
In the absence of free calcium ions, the translational diffusion coefficient D is nearly the same as that of F-actin only. This value of D can be interpreted as that of a single F-a&in filament of a few microns in length (Fujime, 1970a). Therefore, it is reasonable to consider that, under this condition, 7 gives the relaxation time of spontaneous bending motion of the single filament. Similarly, the increase of 7 found at about 1 p*M-Ca2+means the increase of the flexibility of the single filament, because the value of D did not change. At a concentration of calcium ions greater than 20 pM, the value of D decreased considerably, whereas the value of l/r changed only a little. This decrease in D is probably due to the interaction between filaments. However, this does not indicate the formation of large aggregates, because the turbidity at 350 run did not change. The observed decrease in D can be explained by loose coupling among a few filaments, which would have no appreciable effect on the intrafilament movement. At first sight, the change in D at about 20 pM4-Ca2+seems to have no connection with the contraction mechanism in the well-organized striated muscle. However, this phenomenon means that the concentration of calcium ions reversibly controls a “sol (a dispersed state of filaments) + gel (an aggregated state)” transformation. It is expected that such a sol-gel transformation would have some connection with the control of the motility of lower organisms such as Myxomycete plasmodia (for example, Hatano, 1970). (b) Binding
between troponin
and calcium ions
According to Ebashi et al. (1968), troponin contains about 4 moles of calcium ions per lo5 g protein. In the presence of 4 mM-MgCl, at pH 6.8, the binding constant of half of the sites is 1.3 x lo6 ~-l and that of the remaining half 5 x lo4 M-l. Fucks $ Briggs (1968) also reported that the binding constants are 2.1 x lo6 iwe (class 1)
DYNAMIC
STUDY
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F-ACTIN
621
and 3 x lo4 r.r-l (olass 2) in the presence of 2 rn&MgCl, at pH 7-O. On the other hand, apparent binding constants deduoed from Figures 8 and 9 are about 1 x lo8 at-’ and 5 x lo4 M-I. These values are in good agreement with those of Ebashi et al. (1968) and Fucks $ Briggs (1968), if the differenoes in solvent conditions are taken into account. Comparing these results, it can be concluded that troponin has et least two different effects corresponding to the degree of occupation of the binding sites by calcium ions, i.e. one half of the binding sites (lo6 M-‘) are related to the regulation of the flexibility chsnge of an F-actin/tropomyosin/troponin complex, and the other half ( lo4 M-I) to the change in the D vslue of the complex. (0) Supplementa y diwu88ion When calcium ions bind to troponin in a micromolar range of calcium ion concentration, a conformational change is expected to occur in troponin (Wakabayashi & Ebashi, 1968). This conformational change is considered to have an effect on the state of F-e&in through tropomyosin (Tonomura et al., 1969). A similar conclusion was reached from the present investigation: when troponin binds caloium ions, 8 transitional change of the flexibility of F-actin occurs only in the presence of tropomyosin. This is a typical example of “sequential control” in the biological system ; “ . . . --f membrane depolarization + calcium ion release + troponin + tropomyosin + actin 3 (acto)myosin + . . .“. The present results not only prove that calcium ions bound to troponin can regulate the state of F-actin in the presence of tropomyosin, but also suggest that the thin filament is not merely a rigid rod supporting tension developed in muscle, and that the dynamic nature of F-actin may have an important role in muscular contraction. What role can be given to the over-all flexibility change of the thin filament? One possibility is that, since there is a certain distance between the thin filament and the thick filament in muscle, the increase of the amplitude of spontaneous bending motion of the former may be required for the interaction between the two filaments (cf. Fig. 9). Even if the cross-bridge from myosin had a flexible hinge at the end, its interaction with actin might be made possible only by the help of flexibility of the thin filament,. Tropomyosin molecules are believed to bind to F-a&in along grooves between two strands of F-actin (Ebashi et aZ., 1969). Troponin binds not directly to F-actin but to tropomyosin, and is distributed periodically along the thin filament (Endo, Nonomum, Maseki, Ohtsuki t Ebeshi, 1966; Ohtsuki, Mesaki, Nonomura $ Ebashi, 1967). Each tropomyosin molecule covers more than half a pitch of the helix of F-a&in. When a conformational change of troponin induced by calcium ions is transmitted to tropomyosin, influences may extend to plural actin monomers in F-actin. Thus, troponin having no calcium ion, when bound to tropomyosin, makes the complex most rigid probably by strengthening the parallel binding between F-actin and tropomyosin. This effect is eliminated when troponin binds calcium ions. A recent preliminary study by the same method (Ishiwata $ Fujime, 1971c) showed that the increase in flexibility of F-actin by heavy meromyosin (Fujime & Ishiwata, 1971) can be regulated by the tropomyosin/troponin system, depending on the presence of free calcium ions at about micromolar concentrations, In the presence of calcium ions, the flexibility of the complex of F-a&in, tropomyosin and troponin is very much increased by binding of heavy meromyosin at a certain range of the molar ratio. In the absence of calcium ions, however, the flexibility of the complex is not changed by heavy meromyosin. It is well known that, in the presence of calcium ions,
822
S. ISHIWATA
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S. FUJIME
the complex of F-actin, tropomyosin and troponin activates the Mg-ATPase of (heavy mero)myosin, whereas in the absence of calcium ions, activation does not occur (e.g. Kominz, 1966; Eisenberg & Kielley, 1970). The ATPase activation may be a result of the local interaction of the filament with myosin (Ebashi $ Endo, 1968). The apparent parallelism between results of light scattering and of the ATPase measurements strongly suggests that the F-actin flexibility observed here is a direct reflection of rather local interactions, although light scattering indeed measures average properties of the whole filament. We intend to extend further this kind of study on the interaction between thin filaments and myosin in the presence of ATP, bearing in mind the plausible hypothesis that the dynamic nature of the thin filament may have an essential role in muscular contraction and in other cellular movements (e.g. Oosawa, Asakura & Ooi, 1961). We thank Professor Fumio Oosawa for his guidance and encouragement throughout the present work. We are indebted to Professor Setsuro Ebashi and Dr Fumiko Ebashi of the University of Tokyo for valuable discussions and advice on the preparation of troponin. We also wish t,o thank all the members of our laboratory for stimulating discussions. REFERENCES Ebashi, S. & Ebashi, F. (1965). J. Biochem., Tokyo, 58, 7. Ebashi, S., Ebashi, F. & Maruyama, K. (1964). Nature, 203, 645. Ebashi, S. & Endo, M. (1968). In Progress in Biophysics a,nd Molecular Biology, vol. 18, p. 123. Oxford: Pergamon Press, Ltd. Ebashi, S., Endo, M. & Ohtsuki, I. (1969). Quart. Rev. Biophys. 2, 351. Ebashi, S. & Kodama, A. (1965). J. Biochem., Tokyo, 58, 107. Ebashi, S. & Kodama, A. (1966). J. Biochem.., Tokyo, 59, 425. Ebashi, S., Kodama, A. & Ebashi, F. (1968). J. Biochem., Tokyo, 64, 465. Ebashi, S., Wakabayashi, T. & Ebashi, F. (1971). J. Biochem., Tokyo, 69, 441. Eisenberg, E. & Kielley, W. W. (1970). Biochem. Biophys. Rea. Comm. 40, 50. Endo, M., Nonomura, Y., Masaki, T., Ohtsuki, I. & Ebashi, 6. (1966). J. Biochem., Tokyo, 60, 605. Fucks, F. & Briggs, F. N. (1968). J. Gen. PhysioZ. 51, 655. Fucks, F., Reddy, Y. & Briggs, F. N. (1971). Biochim,. biophys. Acttr, 221, 407. Fujime, S. (1970a). J. Phys. Sot. Japan, 29, 751. Fujime, S. (19706). J. Phys. Sot. Japan, 29, 416. Fujime, S. (1971). J. Phys. Sot. Japan, 31, 1805. Fujime, S. & Ishiwata, S. (1971). J. Mol. Biol. 62, 251. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949). J. Biol. Chem. 177, 751. Hartshorne, I). J. & Pyun, H. Y. (1971). Biochim. biophy.s. Acta, 229, 698. Hatano, S. (1970). Exp. CeZE Res. 61, 199. Ishiwata, S. & Fujime, S. (1971a). J. Phys. Sot. Japan, 30, 303. Ishiwata, S. & Fujime, S. (1971b). J. Phys. Sot. Jwpan, 30, 302. Ishiwata, S. & Fujime, S. (1971c). J. Phys. Sot. Japan, 31, 1601. Kasai, M., Kawashima, H. & Oosawa, F. (1960). J. Polymer Sci. 44, 51. Kominz, D. R. (1966). Arch. Biochem. Biophys. 115, 583. Maruyama, K. & Ebashi, 8. (1970). Sci. Papers CoZ. Gen. Educ., Univ. Tokyo, 20, 171. Ohtsuki, I., Masaki, T., Nonomura, Y. & Ebashi, S. (1967). J. Biochem., Tokyo, 61, 817. Ooi, T. (1967). Biochemistry, 6, 2433. Oosawa, F., Asakura, S. & Ooi, T. (1961). Progr. Theoret. Phys., Kyoto, Suppl. 17, 14. Schwarzenbach, G., Senn, H. & Anderegg, G. (1957). HeZv. chim. Acta, 40, 1886. Straub, F. B. (1943). Studies Inst. Med. Chem.. Univ. Szeged. 3, 23. Szent-GyGrgyi, A. (1951). Chemistry of MzLscu.Zar Contraction, 2nd edn., p. 146. London: Academic Press Inc. Tonomura, Y., Watanabe, S. & Morales, M. F. (1969). Biochemistry, 8, 217 1. Wakabayashi, T. & Ebashi, S. (1968). J. Biochem., Tokyo, 64, 731.