Heat resistance of MgATPase and contractility in muscle models

J. therm. Biol. Vol. 14, No. 4, pp. 183-186, 1989

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HEAT RESISTANCE OF MgATPase AND CONTRACTILITY IN MUSCLE MODELS N. S. SHELUD'KOand V. L. STADNIKOV Laboratory of Molecular Biophysics, Institute of Marine Biology, Far East Science Branch, Academy of Sciences of the U.S.S.R., Vladivostok 690022, U.S.S.R. (Received 24 September 1988; accepted in revisedform 25 February 1989)

AIw~'act--1. During the heating of a synthetic actomyosin suspension, the following sequence of events were observed. First, the rate of superprecipitation decreased; secondly the extent of superprecipitation decreased and finally the MgATPase activity was inhibited. At the same time the dissociating capability of actomyosin decreased in a solution of high ionic strength. 2. A similar lack of coincidence between the mechanical and the enzymatic activities of actomyosin was observed with an increasing proportion of inactivated myosin occurring in the reconstructed actomyosin complex. 3. The different heat resistance of contractility and MgATPase activity in muscle models may be caused by inactivated myosin bridges which form in the course of heat treatment so that the dissociating capacity of actomyosin in the presence of ATP is lost. Key Word Index--heat resistance of mucle models; actomyosin ATPase; actomyosin superprecipitation; myosin bridges.

INTRODUCTION

Contractile models of muscle have been widely used to study thermal injury. The models used include intact muscle, intact and glycerinated fibres, myofibrils and actomyosin. Depending on the model type, the effect of heat has been tested either by changes in contractility or in the ATPase activity. It is usually found that heat resistance of the MgATPase activity is generally higher than that of contractility. (Ushakov et al., 1971; Skholl, 1977a). Such an uncoupling between the levels of heat resistance was shown most convincingly by Skholl (1977b) on glycerinated fibres which allowed a simultaneous determination of mechanical and enzymatic activities. This may seem paradoxical, since contractility and ATPase activity are two different aspects of a single mechanochemical process, and therefore their changes must be correlated. The aim of the present work was the elucidation of the causes of this lack of coincidence between thermal sensitivity of the contractile and enzymatic activities. The simplest model, i.e. synthetic actomyosin, was used because it may allow the identification of site of heat action to be made. MATERIALS AND METHODS

Rabbit back and leg muscle was used for protein preparations. Myosin was prepared by the method of Offer et al. (1973) and actin by the method of Spudich and Watt (1971). Synthetic actomyosin (AM) was prepared by mixing myosin and actin (weight ratio 2:1) in 0.5M KCI solution. The mixture was then diluted until the suspension contained 0.75 mM KCI, 3 mM Tris-HCl pH 7.0, I mM ~-mercaptoethanol and 0.2-0.3 mg/ml protein. This low ionic strength

solution was used for heat treatment and measurement of light-scattering and ATPase activity. Heat treatment of the AM suspension was performed at 46 or 48°C for times up to 50 min. The samples (4-8 ml) were continuously stirred during heating and were immediately quenched on ice. In experiments where myosin and actin were heated separately, this procedure was carried out using solution of low ionic strength, but then, after heating, the ionic strength in samples was increased to 0.5 M KCI and AM-complexes were prepared as described above. In control experiments, one of proteins was not heated. Contractile activity of AM, i.e. superprecipitation (SPP) was evaluated by measuring changes in AM suspension light-scattering at an angle of 90 ° (Kropacheva et al., 1986) in response to the addition of 0.5 mM MgATP. We determined the extent of SPP = AI/I0, where AI is a change in light-scattering upon the addition of MgATP, Io is the initial light° scattering level; and SPP rate = 1/t, where t is the half-time (s) in light-scattering change. Kinetics of the ATPase reaction were recorded electrometrically by changes in pH of the medium (Kropacheva et al., 1986). The rate of activity was expressed as A = ApH/(t: - tl)(7, where t I (40 s) and t 2 (70 s) are the time after the onset of reaction corresponding to the beginning and the end of the interval within which the activity was measured, ApH is pH change during this time interval, and C is the concentration of myosin in the solution or suspension of actomyosin. The MgATPase and K+-EDTA ATPase activity were determined. In the first ease the reaction was run in conditions similar to those of SPP measurement, and in the second one, the ATPase activity was determined in a solution containing 0.5 M KCI, 3 mM Tris-HCl pH 7.0, 5 mM EDTA 183

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N, S, SI~LUD'KOand V, L. STADNIKOV

and 0.5 mM ATP. In both cases ATPase reaction was initiated by addition to suspension of 0.1 M MgATP (or ATP) solution, with pH chosen so that pH of suspension should change not higher than 0.005 as a result of addition. Dissociation ability of AM was determined by the change in the viscosity and light-scattering of an AM solution in 0.5 M KC1 upon addition of 0.5-1.0 mM MgATP. Viscosity was measured by means of capillary viscometer (Ubellode type). The extent of dissociation was calculated as the ratio of the changes in viscosity or light-scattering to their initial values.

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RESULTS Figure 1 shows the dependence of the rate and extent of SPP, MgATPase activity and dissociation of synthetic AM with the duration of preheating. As follows from this figure, heat treatment affects these features of AM differentially. With increased heating, the rate of SSP and dissociation ability decrease first, followed by a decrease in the extent of SPP, and finally there is a decrease in the MgATPase activity. A 50% decline in the SPP rate, SPP extent and MgATPase activity was observed after 4, 13 and 44 min, respectively. Thus the lack of coincidence between heat resistance curves of contractile and enzymatic activity may be reproduced on the simplest contractile model e.g. synthetic actomyosin. Hence, these mismatches are induced by changes in the major contractile proteins, i.e. myosin and actin. However, when actin and myosin are heated separately, the curves of dependence of SPP extent and MgATPase activity on the duration of heating decrease in parallel (Fig. 2). Hence, actomyosin bonds are essential for the manifestation of the effect discussed in heat treatment.

A

I

f,

I

1

2

3

Time of heat treatment, (mini

Fig. 2, The effect of heat treatment at 46°C upon the extent of SPP (0) and MgATPase activity (0) of synthetic actomyosin in separate heating of actin and myosin. Presented are the data of l out of 4 experiments. MgATPase activity is more heat-resistant than contractility. The activity of K+-EDTA ATPase, however, decreases even more rapidly than the extent of SPP (cf. Figs l and 3). The curve showing its dependence on the heat dose practically coincides with that of actomyosin dissociation (Fig. 3). A reduction in K+-EDTA ATPase activity, being in a strict correspondence with the reduction in actomyosin dissociation, su~l~'sts that heat treatment results in an increased number of inactivated molecules of myosin which have lost their ability to split the ATP and to break the complex with actin in the presence of ATP. To verify this hypothesis, we have prepared some actomyosin complexes with different myosin/actin ratios (from 0.1:1 to 2:1). These were heated (34 rain at 48°C) so as to suppress completely SPP and dissociation. The preparations

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Fig. 1. The effect of preheating on the extent of SPP (O), SPP rate (@), MgATPase activity (O) and the extent of dissociation (V) of synthetic actomyosin. Heat treatment was done at 48°C. Dissociation capacity was tested viscometrically in the presence of I mM MgATP. Presented are the data of 1 out of 9 experiments,

,o

6

6

I

,o

I

Time of heat treotrnent (mini

Fig. 3. The effect of preheating on MIATPa~ (O), K + EDTA ATPas¢ ~ t y (D) a ~ ~ ~ t of dissociation CV) of s y n ~ a c t o m y ~ Heat treatmmtt was ~ e d at 48°(3. ~ a t i o n ~ty wU ~ by changes in light-scattering. Presented are the data of I out of 7 experiments.

Heat resistance of MgATPase and contractility

150

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80

60

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Content of native myosin (%)

Fig. 4. Extent of SPP (Q), SPP rate (~), and MgATPase activity (O) of synthetic actomyosin with different proportion of inactivated myosin. Presented are the data of I out of 3 experiments. were then dissolved in 0.5 M KCI, added native myosin to obtain the equal (2:1) myosin/actin ratio and transferred again to a low-ionic-strength solution. Figure 4 shows the results of measurements of SPP and MgATPase activity in these preparations; as the proportion of inactivated myosin in actomyosin was increased, the SPP rate, the SPP extent and MgATPase activity showed the same pattern as described for actomyosin inactivation with duration of heat treatment (Fig. 1). DISCUSSION

When the AM system undergoes inactivating treatment, two types of alteration may be envisaged: either a gradual change in myosin structure accompanied by gradual changes in its properties, or a stepwise transition from the native state to an inactivated one. The latter may be of stochastic nature and hence occurs at different times for all myosin molecules in the system. The results in Fig. 4 show that when AM is heat-treated, a spasmodic change in myosin structure does occur resulting in the loss of the dissociation of actomyosin in the presence of ATP. The decreasing extent of AM dissociation induced by heating, is shown by two independent techniques, supplementing each other. Changes in AM viscosity upon the addition of MgATP unambiguously indicate dissociation of the AM complex, though the dependence between the extent of this change and the number of broken bonds is not linear (Abe and Maruyama, 1972). The interpretation of the results of optical measurements is more complex but if one is sure that light-scattering changes are indeed due to those in AM dissociation extent, we can speak of a linear dependence between these parameters (White, 1982). Hence an almost complete coincidence of dependence curves of K+-EDTA ATPase activity and the AM dissociation extent, according to light-scattering data, is remarkable. In our opinion, the emergence of non-dissociating myosin bridges in the course of heat treatment is also

185

the cause of the differences between the SPP and the MgATPase activity curves for the AM. These bridges provide a 'lacing' between thick and thin filaments of the contractile apparatus which impede the relative slide of filaments. As the number of 'laces' increases, they first affect the SPP rate and then the SPP extent which can be inhibited completely with a sufficient number of 'laces'. The native portion of myosin molecules retains its ability to interact normally with the ATP and actin and so maintain the ATPase activity when SPP is lacking completely. Thus at the level of elementary mechano-chemical transformers (myosin bridges) the lack of coincidence between the mechanical and the enzymatic phenomena is absent. A possibility of the lack of coincidence emerges at the level of contractile apparatus and is connected with the fact that contraction requires a coordinated operation of many bridges, whereas the ATPase activity is determined only by the number of operating bridges. Both in the case of AM heating (Fig. 1) and that of artificially changing the ratio between the native and inactivated myosin (Fig. 4), the MgATPase activity curves have a feature in common, i.e. the increase in the ATPase activity level at the initial stages of heat treatment or at a small content of inactivated myosin (compared with the control). This phenomenon, termed thermopotentiation, was also found in natural AM (Giambalvo and Dreizen, 1978). Within the framework of the hypothesis of 'lacing' between thick and thin filaments which appears during thermal treatment, we advance the following explanations for effect of thermopotentiation: (1) An increase in ATPase activity is due to SPP deceleration. It is known that, in the course of SPP the MgATPasc activity decreases significantly, which seems to be connected with the rearrangement of the AM structure (Strzelecka-Golaszewska et al., 1979). The decrease in the rate of SPP as a result of heating of AM prolongates the stage of high initial ATPase activity and result in increase in the mean value of activity. (2) A rise in ATPase activity is determined by changes in thin filament structure by the agency of non-dissociating myosin bridges as it occurs in the case of rigor bonds at low concentration of MgATP (Bremel and Weber, 1972). (3) Non-dissociating bridges--'laces'--heip retain the AM structure in a state sterically favourable for myosin/actin interaction. It is difficult to decide now which of the versions above is realistic. Our results allow for some considerations apropos the methods of determination of heat resistance of muscle models. Analysis of literature (Alexandrov, 1975) shows that by and large, the levels of heat resistance correlate in contractile and enzymatic reactions. For example, when these parameters are determined in parallel, in case of isolated myofibrils of adductor muscles of Crassostrea gigas and Spisula sachalinensis, it was shown that a 50% drop in the extent of contraction of myofibrils was observed at 43 and 34°C, and the same reduction in MgATPase activity, at 52 and 42°C, respectively (Yasnetsky and

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N.S. SrmLUD'KOand V. L. STAD~IKOV

Shelud'ko, not published). That is, the difference between heat resistance of myofibrils of two species of animals, measured by different ways, was practically the same. At the same time, however, there are examples of non-coordinated and even differently oriented changes in contractile and enzymatic activity when muscle models are affected by some agents of physical or chemical nature (Jones, 1971; Ikeuchi et al., 1980; Vorob'ev and Ovsyanko, 1972; Yamashita and Horigome, 1977; Kropacheva et al., 1986). What is more, there are described preparations of myofibrils from two muscles of the same animal, having a high ATPase activity with complete inability to contraction of one of the preparations (Sung et al., 1981). That is why we think it is better to test heat sensitivity of muscle models by measuring changes in ATPase activity, since contractile capacity changes seem to be always secondary to those in ATPase activity. It is quite possible that the most correct is determination of K + - E D T A ATPase activity of actomyosin, myofibrils or muscle homogenates, as it depends monotonously on the duration of heat treatment and lacks thermopotentiation, inherent in the MgATPase activity (Fig. 3). REFERENCES

Abe S. and Maruyama K. (1972) Interaction of myosin and F-actin in solution. J. Biochem. 71, 169-171. Aiexandrov V. Ya. (1975) Cells, Macromolecules and Temperature. Nauka, Leningrad. Bremel R. and Weber A. (1972) Cooperation within actin filament in vertebrate skeletal muscle. Nat. new Biol. 238, 97-101. Giamhalvo A. and Dreizen P. (1978) A thermally potentiated state for actomyosin ATPase of rabbit skeletal muscle. Biochim. biophys. Res. Com. 84, 208-214. Ikeuchi Y., Ito T. and Fukazawa T. (1980) Change of regulatory activity of tropomyosin and troponin on actoheavy-meromyosin ATPase during postmortem storage of muscle. J. fd Sci. 45, 13-16.

Jones J. M. (1971) Studies on chicken actomyosin. Biochem. J. 122, 61. Kropacheva I. V., Korchagin V. P. and Shelud'ko N. S. (1986) Changes in the mechano-chemical properties of actomyosin caused by desensitisation. Biokhimiya 51, 834-839. Offer G., Moos C. and Start R. J. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. J. molec. Biol. 74, 653-676. Skholl E. D. (1977a) The heat resistance of glycerinated muscles and actomyosin in the line of field mice obtained by selection of individuals with the greatest muscle heat resistance. Tsitologiya 19, 375-381. Skholl E. D. (19771))The heat resistance of ATP-ase activity and contractility of glycirinated muscle fibres in field mice. In Molecular Basis of Structure and Function of Cell, pp. 175-178. Nauka, Leningrad. Spudich J. A. and Watt S. (1971) The regulation of rabbit skeletal muscle contraction. J. biol. Chem. 246, 4866-4871. Strzelecka-Golaszewska H., Kiimaszewska U. and Dydynska M. (1979) Polyphasic character of A l P hydrolysis in actomyosin system. Eur. J. Biochem. 101, 523-530. Sung S., Ito T. and Izumi K. (1981) Myosin ATPase and acto-heavy meromyosin ATPase in normal and in pale soft and exudative (PSE) porcine muscle. Agric. biol. Chem. 45, 953-957. Ushakov V. B., Vasil'eva V. V. and Nikolaeva E. N. (1971) After-effect of heating of phasic muscle fibers of the frog upon soluble proteins, actomyosin adenosine triphosphatase and contractile properties of fibrils. Tsitologiya 13, 311-318. Vorob'ev V. I. and Ovsyanko E. P. (1972) Influence of exogeneous forces on contractivity and enzymatic splitting of ATP by glycerol muscle fibres. Tsitologiya 14, 981-989. White N. D. (1982) Special instrumentation and techniques for kinetic studies of contractile systems. In Methods in Enzymology, pp. 698-708. Academic Press, New York. Yamashita T. and Horigome T. (1977) The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. J. Biochem. 81, 933-939.