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Journal of Physiology (1991), 437, pp. 409-430 With 5 figures Printed in Great Britain

EFFECTS OF SULPHYDRYL MODIFICATION ON SKINNED RAT SKELETAL MUSCLE FIBRES USING 5,5'-DITHIOBIS(2-NITROBENZOIC ACID)

BY GREGORY J. WILSON*, CRISTOBAL G. DOS REMEDIOSt, D. GEORGE STEPHENSONt AND DAVID A. WILLIAMS§ From the tDepartment of Zoology, La Trobe University, Bundoora, Melbourne, Victoria 3083, Australia, the tMuscle Research Unit, Department of Anatomy F13, University of Sydney, Sydney, New South Wales 2006, Australia, and the §Department of Physiology, University of Melbourne, Parkville, Victoria 3052, Australia

(Received 5 March 1990) SUMMARY

1. The sulphydryl groups of skinned skeletal muscle fibres have been reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in order to determine whether the effects of modifications to the contractile proteins are reflected in changes in the physiological properties of the contractile apparatus and Ca2+-regulatory system. 2. Results obtained from fast-twitch and slow-twitch rat fibres which were treated with DTNB (10 mm, pH 8-6, 5 C) for various periods of time under relaxing conditions showed that a major effect of the modification was to reduce the level of maximally Ca2+-activated force and fibre stiffness. Force and fibre stiffness were found to decline in proportion. Treatment with DTNB under these conditions did not cause a rise in force or fibre stiffness in relaxed fibres of either type. 3. The effects induced by DTNB under relaxing conditions were substantially reversed by exposure to the reducing agent dithiothreitol (DTT) (10 mM, pH 741, 23 °C). Force abolished by 30-35 s treatment with DTNB recovered after subsequent DTT treatment to 67+3 % (mean + S.E.M., n = 4) in fast-twitch fibres and to 91 + 2 % (n = 7) in slow-twitch fibres. These results were significantly different (t test, P < 0f001) indicating that the level of force recovery depended upon the fibre type. 4. DTNB was found to affect not only the maximal Ca2+-activated force, but also the force-pCa (pCa = -logl0[Ca2+]) relationships of the fibres in a complex, fibretype specific way. DTT treatment partially reversed these DTNB effects. 5. The skinned fibre preparations reacted differently with DTNB under rigor conditions than under relaxing conditions, indicating that rigor modifies the reactivity of the functional sulphydryl groups to the thiol-targeted agents. 6. When superprecipitation assays (an in vitro analogue of fibre contraction) were * Present address: Cardiovascular Research Institute and Department of Biochemistry and Biophysics, Box 0524, University of California San Francisco, San Francisco, CA 94143, USA. I To whom correspondence and reprint requests should be addressed.

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carried out with recombined mvofibrillar proteins which had been previously reacted with DTNB it was found that modification of myosin, but not modification of thin filament proteins, led to changes in the superprecipitation reaction. 7. Both the skinned fibre results and the superprecipitation results indicate that the effects of DTNB upon the fibre characteristics are primarily due to modifications of the sulphydryl groups of myosin. Therefore, these results show that myosin is not only involved in determining the ability of the contractile apparatus to develop force but also in determining the Ca2+-regulatory characteristics of the muscle fibre. INTRODUCTION

The thick and thin filaments of the contractile apparatus of vertebrate skeletal muscle contain many sulphydryl groups which can be covalently modified with oxidizing agents (Reisler, 1982; Wtells & Yount, 1982; Williams & Swenson, 1982; Titus, Ashiba & Szent-Gyorgyi, 1989). Such modifications have been widely used to attach a variety of fluorescent and spin label probes to different components of the contractile and regulatory system in muscle in order to report changes in the conformation and position of specific structures during contractile activation (e.g. Potter, Seidel, Leavis, Lehrer & Gergely, 1976; dos Remedios, Miki & Barden, 1987; Thomas, 1987). There are, however, relatively few studies concerned with changes in the physiological properties of a structurally intact contractile and regulatory system following sulphydryl modifications (e.g. Moss, Giulian & Greaser, 1982; Crowder & Cooke, 1984; Chaen, Shimada & Sugi, 1985; Ishiwata, Kinosita, Yoshimura & Ikegami, 1987). Since several reactive sulphydryls occur at functionally important positions on myofibrils, the use of sulphydryl-directed oxidizing agents with mechanically skinned fibre preparations could yield interesting results regarding the mechanisms of muscle contraction and its regulation by Ca2 . In this study we have exposed mechanically skinned muscle fibres to the sulphydryl-directed oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) which has often been utilized to modify the sulphydryl groups on myosin in solution (Reisler, 1982; Wells & Yount, 1982; dos Remedios et al. 1987) but not so in preparations with an intact contractile and regulatory system (Moss et al. 1982). DTNB is convenient to use because of the reversibility of the oxidizing reaction in the presence of a reducing agent. DTNB can react with sulphydryl groups in two ways. It can oxidize two appropriately spaced sulphydryl groups forming a disulphide bridge between them, creating cystine (Wells & Yount, 1982) and two 5thio-2-nitrobenzoic acid (TNB) molecules, the cleavage products of the reaction with DTNB. Alternatively DTNB can create a mixed disulphide between TNB and cysteine (Reisler, 1982; Wells & Yount, 1982). Reducing agents such as dithiothreitol (DTT) (Seidel, 1969; Pemrick, 1977; Mocz, Biro & Balint, 1982; Grabarek, Grabarek, Leavis & Gergely, 1983) reduce disulphide bridges, both those which join cysteine with TNB and those of cystine, forming the cysteinyl sulphydryl groups. DTT was therefore used in this study to reverse some of the effects of DTNB upon the contractile apparatus. Since the number and properties of the sulphydryls on different isotypes of the contractile proteins are known to vary (Cummins & Perry, 1974; Carraro, Catani, Dalla Libera, Vascon & Zanella, 1981; Smillie, 1982; Wagner, 1982) it was of interest

MYOFIBRILLAR SLULPHYDR YL MODIFICATION 411 to compare the effects of DTNB upon different muscle fibre types. The rat soleus and the rat extensor digitorum longus (EDL) muscles were therefore used to obtain slowtwitch and fast-twitch skeletal muscle fibres respectively (Stephenson & Williams, 1981). There is a great deal of complexity within the contractile apparatus and considering the number of sulphydryls which occur in these systems it was important to locate and characterize the sites of modification by DTNB and DTT. DTNB and DTT were therefore also used in studies of the modification of reconstituted actomyosin preparations which were investigated using superprecipitation assays. Superprecipitation is a complex phenomenon in which actomyosin undergoes a reaction which is considered to be an in vitro analogue of muscle fibre contraction (Ebashi, 1963). This phenomenon is observed when myosin and Factin (or Ca2+-regulated F-actin) are combined in appropriate proportions, similar to the molar ratios found in skeletal muscle, and low concentrations of MgATP (or MgATP and Ca2+ for Ca2+-regulated F-actin) are added to initiate the reaction. The results show that modifications to the myofibrillar sulphydryls markedly affect the Ca2+-activated force characteristics in both fast- and slow-twitch muscle fibres. The effects of DTNB, some of which are fibre-type specific, help to show better not only the importance of the positions of myofibrillar sulphydryl groups for muscle function but also that the mechanism of force generation and its regulation by Ca2+ are affected by modification of these groups. Since we have largely localized the functional effects of DTNB on the myosin molecule, this implicates myosin in Ca2+-regulation or its modulation. Some of these results have been presented in preliminary form (Wilson, dos Remedios, Stephenson & Williams, 1988). METHODS

Skinned fibres Single fast-twitch and slow-twitch mechanically skinned muscle fibres were obtained from the extenisor digitorum longus (EDL) and soleus muscles of adult rats. The rats were first killed by deep anaesthesia using diethyl ether. Fibres were then removed from the ablated muscles by microdissection and were mechanically skinned under paraffin oil. The fibres were then attached using brai(lecd surgical silk (I)eknatel, size 10) between a pair of jewellers forceps. which were fixed to a micromanipulator, and a steel hook, which was fixed to a piezoresistive force transducer (Aksjeselskapet AE 875). The average sarcomere lengths of the fibres were measured from the diffraction pattern produced by the beam of a He-Ne laser (Spectra Physics) passed across the fibre and were adjusted to between 2-65 and 2-75 ,um using the micromanipulator (Stephenson, Stewart & XVilson, 1989). Force production was displayed on a chart recorder. These procedures have been described in more detail elsewhere (Stephenson & Williams, 1981 Fink, Stephenson & WVilliams, 1986). For relative fibre stiffness measurements, fibres were mounted using surgical silk between a force transducer (AME-802) and the stainless-steel beam of a vibrating motor which was driven by a power oscillator as previously described (Rees & Stephenson, 1987; Stephenson et al. 1989). The resonant frequency of the transducer with the fibre attached was 3-2 kHz. Relative fibre stiffness was determined from the amplitude of force oscillations generated by sinusoidal length oscillations (20 kHlz) of fixed amplitude ( 15 min (Pr = 709 + 5 7 %). C, results from single slowtwitch fibre (sarcomere length = 269 ium). S (1), fresh fibre; 0 (2), after DTNB treatment for 8 s (Pr = 23 % of PO); X (3), after DTT treatment for 23 8 (Pr = 74 % of Po); * (4), after further DTT treatment for 9 min (Pr = 74% of P1). D, Hill curves fitted through pooled paired results from three slow-twitch fibres (all sarcomere length= 2-69 gm). * (1), fresh fibres; 0 (2), after DTNB treatment for 6-5-9-5 s (Pr = 37-6+12%); X (3), after DTT treatment for 22-42 s (Pr = 838 ± 39%); * (4), after DTT treatment for 6-5-10-5 min (Pr = 82-6+2-9%).

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within the contractile apparatus in a complex way. After treatment with DTT for 30 s P, increased to 48 % of Po while the force-pCa curve moved markedly to the left (pCa50 = 6W03) without a change in shape. This showed that the fibre had become more sensitive to Ca2" by a factor of 155 than it had been prior to DTNB treatment (pCa50 = 5 84). After further treatment with DTT for 60 min P1 increased to 68 % of I'0 while the force-pCa curve became steeper, rising from 10 to 90 % of Po over a pCa range of 0-54 pCa units. However, the mid-point activation did not change

(p(a50 = 6*03). The paired results from this and five other fast-twitch fibres in which force was not abolished after DTNB treatment and which were then treated with DTT are pooled in Fig. 4B. The force-pCa relationships in these fibres became less steep after DTNB treatment for 5-10 s and in all except one case, the 50 % Po point was shifted to the right. The decrease in the steepness of the force-pCa relationship was statistically significant (P < 0 05) but the shift to the right of the 50 % P0 point was not. After DTT treatment for 30-70 s the curves shifted to the left without markedly changing in shape. After further treatment with DTT for > 15 min the curves became steeper. These results showed that with short DTNB and short subsequent DTT treatment the fibres could be made more sensitive to Ca2" than they had been prior to DTNB treatment. They also showed that the effects of DTNB upon Ca2" sensitivity and cooperativity in fast-twitch fibres were substantially altered by long subsequent DTT treatment. Slow-twitch fibres The results in Fig. 4C are the force-pCa data from a single slow-twitch fibre which was treated with DTNB and then with DTT. The data obtained from the fresh fibre were fitted with a Hill curve generated using a value for n of 2-6 and a pCa50 of 5 89. The changes in the force-pCa data after treatment with DTNB and then DTT were qualitatively similar to those which occurred in fast-twitch fibres. After DTNB and DTT treatments the curves remained symmetrical by the criterion mentioned above (Wilson & Stephenson, 1990) and data were therefore fitted using the Hill equation. After DTNB treatment for 8 s the curve became very shallow (n < 1t0), again indicating complex changes in the Ca2+-regulatory units, as was observed for fasttwitch fibres. Concomitantly Pr declined to 23% of P0 and the Ca2+ sensitivity declined by a factor of 1P66 (pCa50 = 5 67). After DTT treatment for 23 s P. recovered to 74% of Po. The force-pCa curve became only marginally steeper (n 1-2) and was shifted a long way to the left (pCa50 = 6 27), indicating that the Ca2+ sensitivity increased by a factor of 2-4 compared with that before DTNB treatment (pCa50 = 5 89). With further DTT treatment for a total of 9 min Pr remained the same (73 % of P0). The force-pCa curve became steeper (n = 2-2) and very similar in shape to that representing the fresh fibre and the fibre was only slightly more sensitive to Ca2+ by a factor of 1 15 (pCa50 = 5 95). The force-pCa results from this and two other slow-twitch fibres which received similar treatment were pooled in Fig. 4D. After treatment with DTNB for 6 5-9-5 seconds the curves became more shallow (n = 14) and the Ca2+ sensitivity became variable although the curves retained their symmetry. They were then always shifted to the left after short (22-42 s) subsequent DTT treatment without change in shape -

MYOFIBRILLAR SUJLPHYDR YL MODIFICATION4423 and after longer ineubations with DTT (6 5-10 5 min) the curves became steeper and returned to positions and shapes similar to those representing the fresh fibres.

DTNBVB modifications in relaxed fibres under conditions different from standard The DTNB incubation conditions of the relaxed fibres were different from standard (pH 86, temperature 50) in several experiments: pH 75, temperature 5-8 °C for five fast-twitch EDL fibres; pH 7410, 23-25 °C for three fast-twitch EDL fibres, one slow-twitch soleus fibre; pH 8 0, 23-25 °C for two fast-twitch EDL and two slow-twitch soleus fibres. In all these experiments, the changes observed in the ability to develop Ca2+-activated force, force-pCa relation and force recovery after DTT treatment were similar to those reported above, except that the reduction in force was generally slower at lower pH and lower temperature. In other experiments three fast-twitch EDL fibres and one slow-twitch soleus fibre were treated for 120, 140, 300 and 240 s respectively in a 10 mm-DTNB relaxing solution (pH 8 0, 23 °C). Force was abolished under these circumstances (see Fig. 2A and D). After extensive treatment with DTT (10 mM) for between 40 and 60 min, force recovered to 16, 14 and 7 4 % of Po in the fast-twitch fibres and to 53 % of Po in the slow-twitch fibre. The fibres were then exposed to protein extracts obtained after Triton-treated, fast-twitch fibre bundles had been incubated for several hours in a DTNB relaxing solution (see Methods). The extracts were dialysed extensively in the presence of DTT (1 mM) and concentrated. Force partially recovered to 19, 19 5 and 27-5 % of Po respectively in all three fast-twitch fibres after extensive (longer than 40 min) treatment with the extract but deteriorated to 43 % of Po in the slowtwitch fibre. The amount of force recovery in the three fast-twitch fibres represents 19, 39 and 270 % of the respective force level before incubation in the extract.

Superprecipitation The results shown in Fig. 5 are from superprecipitation assays performed by reacting myosin with either F-actin or Ca2+-regulated actin in low (50-200 /iM) MgATP concentrations. In the control experiment illustrated in Fig. 5A, unmodified, unregulated F-actin was gently homogenized with unmodified myosin in the molar ratio 5: 1 corresponding to G-actin: myosin. Upon addition of ATP there was a rapid increase in light scattering at 660 nm which corresponds to the formation of actomyosin complexes and which reaches a maximum in under 60 s. In this reaction DTT (10 mM) was added to the reaction medium before the addition of ATP so that the result could be compared with the result from the following experiment, in which the DTT was added after the addition of ATP to reverse the DTNB modification. The upper trace in Fig. 5A is the time course of light scattering of an actomyosin (5:1) complex in which the F-actin was first reacted with a 100-fold molar excess (10 mM) of DTNB for 10 min. This experiment demonstrates that DTNB modification of F-actin has practically no effect on its ability to interact with myosin. A similar result was obtained when F-actin was reacted with DTNB for 30 s. Thus, it appears that the results of the DTNB modification of muscle fibres cannot be attributed to modification of F-actin with DTNB. In the second series of superprecipitation experiments (Fig. 5B), myosin was

G. J. WILSON AND OTHERS

424

Maximally DTNBlabelled actin

A

DTT

Control

t I

DTT ATP

Maximally DTNBlabelled

Ca2"-regulated

B

actin

Ca2"-regulated

control

A

A As/> 9 0) as indicated at the arrows and then by the addition of CaCl2, in aliquots, when indicated by

425 MYOFIBRILLAR SULPHYDRYL MODIFICATION combined with Ca2`-regulated F-actin. This complex conferred a Ca2+-dependent regulation upon actomyosin, it being no longer able to superprecipitate unless the free Ca2+ concentration exceeded a certain critical value. In Fig. 5B the control Ca2+regulated F-actin was combined with untreated myosin (in the molar ratio 5: 1 corresponding to G-actin: myosin). The result demonstrates the so-called 'clearing' effect when excess (0-2 mM) MgATP is added. This state is analogous to relaxed fibres in the presence of MgATP but where pCa is high (> 9) due to the presence of EGTA (20 mM). Successive, cumulative additions of Ca2+ (0-5 mm changes in total Ca concentration) progressively raised the level of free Ca2+ to pCa < 5-0, where the inhibition by tropomyosin-troponin of the actomyosin interaction was removed and superprecipitation was able to proceed. A similar result was obtained using Ca2+regulated actin which had been reacted with DTNB for 28 min (superimposed) combined with unmodified myosin. These experiments demonstrate that the reaction of excess DTNB for long periods of time with Ca2+-regulated F-actin does not inhibit its ability to interact with myosin. Also, although there is a small change in the time course of the reaction, there being a slight increase in the level of light scattering produced by the interaction of modified Ca2+-regulated F-actin with myosin, the Ca2+ sensitivity is not appreciable changed. Practically identical results were obtained when Ca2+-regulated actin was first reacted with DTNB (10 mm) for 4-5 h and then reacted with unmodified myosin. Finally, we reacted myosin with DTNB for approximately 30 s. It was then combined with unmodified, unregulated F-actin. This briefly reacted, DTNBmodified myosin (partially labelled myosin) produced a small but reproducible (approximately 10% of maximum light scattering) decrease in both the rate and extent of the superprecipitation reaction which was not apparently reversed by the addition of DTT (Fig. 5C). The control experiment using unreacted myosin has already been described above except that the stepwise decrease in optical density upon addition of DTT (final concentration 10 mM) is simply due to dilution of the reaction mixture. The effects of DTNB modification can be seen more dramatically when the myosin was modified using DTNB under the same conditions for 10 min (Fig. 5C). Addition of ATP (50 /M) produced no detectable actomyosin interaction. Addition of DTT (10 mM) produced a delayed and rather small (less than 20 % of the final change in the control light scattering) and slow reversal of the inhibition of the superprecipitation by DTNB modification. This result strongly suggests that the superprecipitation is inhibited mainly by the reaction of myosin with DTNB and that the effect is substantially irreversible. the arrow-heads, which increase the total [Ca2+] by 0.5 mm each time (final pCa < 5 0). A, superprecipitation of normal myosin with F-actin which had been maximally reacted with DTNB (10 mM) for 10 min (as described in the text and as determined by absorption at 325 nm) compared with control in which normal myosin and F-actin were used. B, reaction between normal myosin and maximally DTNB-treated Ca2+-regulated actin. The solution cleared upon addition of ATP and superprecipitation then proceeded gradually as the [Ca2+] increased. C, reactions between normal F-actin and normal myosin (control), myosin partially labelled using DTNB and myosin fully labelled using DTNB. The reduction in maximum light scattering in A and C after addition of DTT (10 mm final) was due to dilution of the sample. The ordinate of all the curves is indicated by the marker arrow absorbance at 660 nm. The time axis was constant as indicated by the 60 s bar.

426V

G. J. WILSON AND OTHERS DISCUSSION

The results in this paper have shown that modification of the myofibrillar sulphydryl groups in intact myofibrils of skinned mammalian skeletal muscle fibres had pronounced effects on the contractile properties of the fibres, including the level of maximum Ca2+-activated force and fibre stiffness, and the force-pCa relationships. There were also quantitative differences in the effects of DTNB upon slow-twitch and fast-twitch fibres. DTNB treatment did not cause a rise in force or stiffness of relaxed fibres which would be expected if DTNB had modified the interactions between the actin and myosin filaments in the absence of Ca2 . Many of the effects of DTNB upon the fibres were found to be substantially reversed following treatment of the fibres with DTT. The reversible component of the loss of ability to develop Ca2+-activated force after short exposure of the relaxed skinned fibre preparation to DTNB is due to the oxidation of the sulphydryl groups on myosin. This is clearly demonstrated by the superprecipitation results which show that only myosin was functionally modified by the reaction with DTNB. Furthermore, the rigor results indicate that the binding of myosin to actin profoundly modifies the reactivity of DTNB with the sulphydryl groups which are located at important positions for muscle contractility. If these groups were on the thin filament rather than on the myosin molecules, then one could not have expected that the rigor state would have prevented the action of DTNB on either the vast excess of actin monomers which were not complexed with myosin heads or on the similarly exposed troponin-tropomyosin system. This is further supported by (i) recent results which show convincingly that the five thiol groups on actin are not accessible to thiol-targeted reagents if actin exists in the filamentous form as would be found in skinned fibres (Liu, Wang & Stracher, 1990), (ii) by the observations of Duke, Takashi, Ue & Morales (1976) which show that the sulphydryl groups on tropomyosin and troponin T were equally reactive in both the relaxed and in the rigor states and (iii) by several studies in which modifications to the sulphydryl groups on troponin C (e.g. Potter et al. 1976; Grabarek et al. 1983) or tropomyosin Ca2+-regulated by troponin (Williams & Swenson, 1982) do not affect their functional properties. Other indirect evidence is consistent with the conclusion that the observed DTNB effects on muscle contractility are not due to modifications of the tropomyosin molecules. There are two main isoforms of tropomyosin in mammalian skeletal muscle. Fast-twitch fibres contain predominantly a-tropomyosin while slow-twitch fibres contain more fl-tropomyosin (Cummins & Perry, 1974; Carraro et al. 1981; Smillie, 1982). /f-Tropomyosin contains a higher proportion of cysteine (Cummins & Perry, 1974) and could therefore be more likely to be functionally affected by DTNB. However, the fact that slow-twitch fibres are affected by DTNB to a lesser extent than fast-twitch fibres despite their higher tropomyosin sulphydryl content provides further support for the conclusion that modification of tropomyosin by DTNB is not responsible for the effects we have observed. The most reactive of the sulphydryl groups on myosin are the SHR and SH2 groups which reside on the myosin head (Reisler, 1982; Wells & Yount, 1982). These thiol groups are probably not essential for normal ATPase activity of myosin

MYOFIBRILLAR SULPHYDR YL MODIFICATION42 427 (Okamoto & Sekine, 1987) but the SH1-SH2 region of the myosin head may participate in constructing the functional nucleotide hydrolytic site (Mornet, Bonet, Audemard & Bonicel, 1989). It is known that DTNB promotes cross-linking between SH1 and SH2 which modifies the enzymatic properties of myosin by trapping MgADP -at the active site and abolishing ATPase activity (Wells & Yount, 1982). This is likely to happen in our experiments when the relaxed fibres are exposed to DTNB and would fully explain the reversible loss in ability of myosin to interact with actin and develop Ca2+-activated force in both fast- and slow-twitch fibre preparations. However, there is a marked difference in the reactivity of the skinned fibres to DTNB when the preparation was in the relaxed or in the rigor state. This implies that the sulphydryl groups on myosin in fibres in the rigor state undergo a different modification to that of myosin in the relaxed fibres. As the 8H1-8H2 region on myosin is thought to participate in the construction of the strong actin binding site (Mornet et al. 1989) it is likely that cross-linking between SH, and 1SH2 cannot take place when myosin is strongly attached to actin as it is in rigor. The development of a Ca2+-independent MgATPase in the DTNB-rigor-treated fibres suggests that some myosin heads were specifically modified at the SH1 group since it has been recently reported that such specific modification of the SH1 group elevates the actin-activated myosin MgATPase activity in the absence of Ca21 (Titus et al. 1989). It is important to note that neither the DTNB modification of myosin sulphydryl groups when the fibre was relaxed, nor that when the fibre was in rigor, lead to loss of Ca21 sensitivity of force production. This differs from the observations of Titus et al. (1989) that specific modifications to SH, by thiol reagents which do not promote 811-1SH2 crosslinking lead to the loss of Ca21 sensitivity of the MgATPase actomyosin system. However, our results may be reconciled with those of Titus et al. (1989) if force production becomes dissociated from the actin-activated SH1-modified myosin MgATPase activity in a Ca2+-dependent manner so that full dissociation between force and MgATPase occurs in the absence of Ca2+ while coupling between MgATPase and force occurs at higher [Ca2+]. If the observed effects are indeed due to modification of myosin SH, and SH2 then the reactivities of these groups in fibres must be greater than in isolated myosin as it only took 30 s of DTNB treatment under relaxed conditions to abolish force in both fast-twitch and slow-twitch fibres, whereas it took up to 10 min of similar DTNB treatment to modify isolated myosin such that superprecipitation could not occur. It is possible that the organization of the proteins in the myofilaments could render the sulphydryls more reactive since myofilament organization can influence protein properties such as cross-bridge characteristics (Goldman & Brenner, 1987). Also, since the presence of nucleotides increases the reactivity of the myosin sulphydryls (Duke et al. 1976; Reisler, 1982), the rate of abolition of superprecipitation and force production is probably influenced by the presence of MgATP. DTT is more successful in restoring the DTNB-dependent loss of force in slowtwitch than in fast-twitch fibres (Fig. 2 C and F). The component of force loss which cannot be restored by extensive DTT treatment is severalfold higher in fast-twitch than in slow-twitch fibres and it can be partly recovered following incubation of the fast-twitch (but not slow-twitch) skinned fibre in a crude DTNB muscle extract. This

0. J. WILSON AND OTHERS .428 strongly indicates that a myofibrillar component is gradually being lost from the fibres during prolonged exposure to DTNB. Since DTNB is known to remove specifically myosin light chain 2 (LC2) from native fast-twitch myosin (Weeds & Lowey, 1971; Wagner, 1982) but not from the native slow-twitch myosin (Weeds, 1976; Wagner, 1982) it is likely that the removal of LC2 in the presence of DTNB is responsible for this effect. It follows, therefore, that LC2 may be necessary for Ca2+_ activated force production. Indeed, it has been found (Pemrick, 1977; Schaub, Huber, Jauch & Brunner, 1988) that modification or removal of LC2 could affect both the direct interaction between actin and myosin and the regulation of this interaction by Ca2+ (Borovikov & Levitski, 1984; Moss et al. 1982). Results from this study also indicate that the Ca2+-activation characteristics (Fig. 4) are modified in a characteristic way by DTNB treatment in both fast- and in slowtwitch fibres. The similarity in the Ca2+ dependence of the superprecipitation results between controls and Ca2+-regulated actin maximally labelled with DTNB (Fig. 5C) indicates that the DTNB-induced changes in Ca2+-activation properties of skinned fibres are also mainly due to modifications on myosin. The similarities in the effects of DTNB on the force-pCa curves of both fast- and slow-twitch fibre preparations indicates that LC2 may not be involved in this process because slow LC2 contains no cysteine and cannot be readily extracted with DTNB (Weeds, 1976) while fast LC2 contains two cysteine residues and can be readily extracted with DTNB (Mocz et al. 1982). However, we cannot exclude the involvement in this effect of the so called essential myosin light chains which possess cysteinyl sulphydryl groups (Frank & Weeds, 1974; Mocz et al. 1982) and are known to react with thiol-modifying reagents (dos Remedios et al. 1987). The major DTNB effect after short treatment (8-10 s) on the isometric force-pCa curves in both fast- and slow-twitch fibres was a wider pCa range over which the force rose from 10 to 90 % with a concomitant reduction of maximum Ca2+-activated force by a factor of between 4 and 7 (Fig. 4). This effect can be interpreted as a loss of Ca21 co-operativity in activating the functional unit for Ca2+ regulation. We wish to point out that this apparent decrease in cooperativity is not related to the reduced level of force and smaller number of attached cross-bridges as suggested by the stiffness results because the shape of the force-pCa curve did not markedly change after short (22-70 s) DTT treatment yet maximum Caa2+-activated force and fibre stiffness rose by a factor greater than 3 in both the fast- and slow-twitch fibre as shown in Fig. 4A and C. Moreover, further extensive (>15 min) treatment with DTT produced a marked increase in force-pCa curve steepness but only a modest rise in maximum Ca2+-activated force (Fig. 4). This is an important observation because it clearly shows that the apparent Ca2+ cooperativity in force regulation (force-pCa curve steepness) is not dependent on either force or the number of active cross-bridges. This is contrary to the view that increased number of actin-myosin interactions affects the Ca2+ co-operativity of the regulatory system in striated muscle (Brandt, Cox, Kawai & Robinson, 1982). In conclusion, the results presented in this paper strongly suggest that modifications to the thiol groups on the myosin molecules affect not only the mechanism of force production but also the mechanism of force regulation by Ca2+, indicating that myosin may play an important modulatory role in Ca2+ regulation of isometric force. The results also show that attaching probes to the thiol groups on myosin may affect the contractile and regulatory properties of muscle.

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We would like to thank Dr Roger Cooke for critically reading this manuscript and Mrs Ruth Cafarella for invaluable technical assistance. This work was supported by the National Health and Medical Research Council and by the Australian Research Grants Committee. REFERENCES

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