Introduction

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The force of muscle contraction is produced by the interaction between actin and myosin molecules in a process that involves cross-bridge cycling coupled with the hydrolysis of ATP (dos Remedios and Moens, 1995; Holmes, 1996; Cooke, 1997). Actomyosin is a precise machine for the transduction of chemical energy in ATPase molecules into mechanical work (Sugi, 1993). Myosin is an ATPase whose activity is stimulated by the interaction with actin. Although the binding sites involved in the interaction between myosin and actin molecules have been determined (Rayment et al., 1993a,b), the role of conformational changes and the interactions of these proteins in muscle contraction remain to be elucidated. Furthermore, the regulatory protein system, the troponin/tropomyosin complex, plays an important role in the regulatory process of the skeletal muscle contraction (Holmes, 1995; Cooke, 1997; Squire and Morris, 1998). Therefore, novel tools that provide information of interactions of contractile proteins will be useful (Ohizumi, 1997). The superprecipitation of actomyosin is generally accepted to be basically the same phenomenon in vitro as a contraction in skeletal muscle cells (Szent-Györgyi, 1951). Circular dichroism (CD) has been extensively applied in the study of conformational changes of protein (William et al., 1982).

In the course of our survey on biologically active substances from marine resources, much attention has been given to compounds affecting the contractile apparatus, because these compounds are very useful as tools for elucidating the relationship between structure and function in the contractile and regulatory proteins (Ohizumi, 1997). Recently, we isolated several natural products that affect myosin and actin functions, such as purealin, which modulates myosin ATPase activity (Takito et al., 1986; Nakamura et al., 1987), and xestoquinone, which modulates the specific sulfhydryl groups of myosin (Kobayashi et al., 1991a,b; Sakamoto et al., 1995). In further research, goniodomin A (Fig. 1) was first obtained as an antibiotic substance from a marine dinoflagellate, Goniodoma pseudogoniaulax (Murakami et al., 1988). Goniodomin A has been shown to dramatically modify the ATPase activity of actomyosin reconstituted from actin and myosin (Furukawa et al., 1993). However, the detailed property of the potent stimulatory effect of this compound on the skeletal muscle contractile protein system has not been investigated. Here, we present the first report indicating that the marked enhancement of the skeletal muscle actomyosin ATPase activity by goniodomin A is highly sensitive to a regulatory protein system, the troponin/tropomyosin complex.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Chemical structure of goniodomin A.

	Experimental Procedures

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Materials. Goniodomin A was isolated from dinoflagellate G. pseudogoniaulax as previously reported (Murakami et al., 1988). Briefly, the dinoflagellate was cultivated, collected, and extracted with ethanol/dichloromethane (1:1). The extract was partitioned between water and ether. The organic layer was subjected to column chromatography on a silica gel, followed by reversed phase HPLC to yield pure goniodomin A, which was identified by the spectroscopic data of NMR, mass, UV, and infrared spectra (Murakami et al., 1988). Goniodomin A was dissolved in dimethyl sulfoxide, and a final concentration of dimethyl sulfoxide did not exceed 1%. Less than 1% dimethyl sulfoxide had little effect on the ATPase activities. Myofibrils, natural actomyosin, troponin, actin, myosin, and tropomyosin were prepared from rabbit skeletal muscle as described by Perry and Corsi (1958), Szent-Györgyi (1951), Ebashi et al. (1968), Spudich and Watt (1971), Weeds and Taylor (1975), and Kohama (1979), respectively. Natural actomyosin contains the principal protein components such as actin, myosin, and troponin/tropomyosin for the muscle contractile system. The animals used in this study were treated in accordance with the principles and guidelines of Tohoku University Council on Animal Care.

Measurement of ATPase Activity. The ATPase activities were examined as previously reported (Ohizumi et al., 1998). The reaction mixture for each ATPase consisted of 0.1 mg/ml myofibrils, 2 mM ATP, 1 mM EGTA, 2 mM MgCl₂, 0.76 mM CaCl₂, 50 mM KCl, and 20 mM Tris · HCl (pH 6.8) for myofibril ATPase; 0.3 mg/ml natural actomyosin, 2 mM ATP, 1 mM EGTA, 2 mM MgCl₂, 0.76 mM CaCl₂, 50 mM KCl, and 20 mM Tris · HCl (pH 6.8) for natural actomyosin ATPase; 0.1 mg/ml actin, 0.1 mg/ml myosin, 1 mM ATP, 0.76 mM CaCl₂, 2 mM EGTA, 50 mM KCl, 2 mM MgCl₂, and 20 mM Tris · HCl (pH 6.8) for the ATPase activity of actomyosin reconstituted from actin and myosin; 0.15 mg/ml myosin, 2 mM ATP, 10 mM CaCl₂, 500 mM KCl, and 50 mM Tris · HCl (pH 7.4) for the Ca²⁺-ATPase activity of myosin; 0.015 mg/ml myosin, 2 mM ATP, 5 mM EDTA-Tris, 500 mM KCl, and 50 mM Tris · HCl for the K⁺-EDTA-ATPase activity of myosin; and 1.5 mg/ml myosin, 2 mM ATP, 5 mM MgCl₂, 500 mM KCl, and 50 mM Tris · HCl (pH 7.4) for the Mg²⁺-ATPase activity of myosin. The mixture was preincubated in the absence of goniodomin A and ATP at 30°C for 5 min, followed by the addition of goniodomin A and further preincubation. The reaction was started by the addition of ATP and stopped by the addition of an equal volume of cold 10% trichloroacetic acid. The amount of inorganic phosphate liberated during the 5-min incubation was determined according to the method of Martin and Doty (1949).

Measurement of Superprecipitation Activity. The superprecipitation activity was examined as described previously (Ohizumi et al., 1998). The superprecipitation was induced by the addition of 0.4 mM ATP in 0.3 mg/ml natural actomyosin, 0.8 mM CaCl₂, 2 mM MgCl₂, 50 mM KCl, 1 mM EGTA, and 20 mM Tris · HCl at pH 6.8 and 25°C, and the change in the absorbance at 660 nm was followed.

Measurement of Contractile Response of Skinned Fibers. Psoas muscles of male guinea pig were excised and washed rapidly with a Ringer's solution containing 150 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 5.5 mM glucose, and 5 mM HEPES (pH 7.4) and were immediately transferred into relaxing solution containing 74.7 mM K-methanesulfonic acid, 5.4 mM Mg-methanesulfonic acid₂, 4 mM ATP, 10 mM EGTA, and 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid)-KOH (pH 7.0). A small muscle bundle of four or five fibers (~0.1 mm in diameter and ~3 mm in length) was dissected from the psoas muscle. One end of the fiber was secured to the tissue holder by a ligature and the other end was connected to a force-displacement transducer (Acers AE801; Horten, Norway; the compliance of tension measurement system was ~0.5 mm/g) for measurement of isometric contraction of the fiber at 20-23°C. Fibers were treated with the relaxing solution containing 50 µg/ml saponin for 30 min and then with a 0.5% Triton X-100 solution for 15 min (Endo and Iino, 1980; Horiuti, 1986). Various solutions for skinned fiber experiments were prepared as described elsewhere (Kobayashi et al., 1991a). A criterion for discarding skinned preparations was the total decline in a maximum contractile response to Ca²⁺, and the survival time of the preparation was at least 5 h.

CD Measurements. CD spectrum was measured as previously reported (Sakamoto et al., 1995) with a slight modification. F-actin (0.1 mg/ml) was incubated with various concentrations of goniodomin A in the medium containing 50 mM KCl, 1 mM MgCl₂, and 25 mM Tris · HCl (pH 6.8) at 37°C for 5 min. The reaction mixture was dialyzed into a buffer (50 mM KCl, 1 mM MgCl₂, 25 mM Tris · HCl, pH 6.8) at 4°C for 2 h to remove dimethyl sulfoxide used as a dissolving solvent of goniodomin A. Far-UV CD spectra of F-actin were measured in the 200 - to 300-nm region in the medium consisting of 50 mM KCl, 1 mM MgCl₂, and 25 mM Tris · HCl (pH 6.8) with a spectropolarimeter (J-720; Japan Spectroscopic Corporation, Inc.) at 25°C.

Statistical Analysis. The data are expressed as mean ± S.E. Statistical comparisons were made with the use of Student's t test. P < .05 was considered significant.

	Results

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Effects of Goniodomin A on Reconstituted Actomyosin ATPase Activity. Goniodomin A enhanced the ATPase activity of actomyosin reconstituted from actin and myosin in the range of 10⁸ to 10⁷ M in a concentration-dependent manner, but the ATPase activity was decreased by further increasing goniodomin A concentration (Fig. 2). The troponin-tropomyosin complex decreased goniodomin A-induced elevation of the actomyosin ATPase activity of the actin-myosin reconstituted system in a concentration-dependent manner (Fig. 2). Goniodomin A (10⁸ to 3 × 10⁶ M) caused a concentration-dependent inhibition of the ATPase activity of actomyosin reconstituted from 0.1 mg/ml actin and 0.1 mg/ml myosin in the presence of 0.8 mg/ml troponin and 0.8 mg/ml tropomyosin (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of goniodomin A on the ATPase activity of reconstituted actomyosin. , 0.1 mg/ml actin and 0.1 mg/ml myosin. , 0.1 mg/ml actin, 0.1 mg/ml myosin, 0.2 mg/ml troponin, and 0.2 mg/ml tropomyosin. , 0.1 mg/ml actin, 0.1 mg/ml myosin, 0.4 mg/ml troponin, and 0.4 mg/ml tropomyosin. , 0.1 mg/ml actin, 0.1 mg/ml myosin, 0.8 mg/ml troponin, and 0.8 mg/ml tropomyosin. The ATPase activity is expressed as a percentage against the control activity of actomyosin reconstituted from 0.1 mg/ml actin, 0.1 mg/ml myosin, 0.8 mg/ml troponin, and 0.8 mg/ml tropomyosin in the absence of goniodomin A at a Ca2+ concentration of 4 × 106 M. Values are mean ± S.E. (n = 3). *P < .05; **P  .01.

Effects of Goniodomin A on Natural Actomyosin ATP- ase Activity. Natural actomyosin was used for investigation of the effect of goniodomin A because it contained more principal protein components for the contractile system than actomyosin reconstituted from actin and myosin. As shown in Fig. 3A, goniodomin A caused a concentration-dependent increase in the natural actomyosin ATPase activity in the range of 3 × 10⁸ to 3 × 10⁷ M but its ATPase activity was decreased by further increasing the goniodomin A concentration. This effect was not affected by the SH group protecting reagent dithiothreitol (10³ M, data not shown). The troponin-tropomyosin complex decreased the goniodomin A-induced elevation of the natural actomyosin ATPase activity in a concentration-dependent manner (Fig. 3A). Goniodomin A caused a concentration-dependent decrease in the ATPase activity of 0.3 mg/ml natural actomyosin in the presence of 0.2 mg/ml troponin and 0.2 mg/ml tropomyosin (Fig. 3A). The goniodomin A-induced modulations of the ATPase activities of the actin-myosin reconstituted system (Fig. 2) and natural actomyosin (Fig. 3A) are closely correlated. The concentration-response curve of the ATPase activity for Ca²⁺ was shifted to the upper direction by goniodomin A (10⁷ M; Fig. 3B).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effects of goniodomin A on the ATPase activity of rabbit skeletal muscle natural actomyosin. A, the log concentration-response curve of the ATPase activity of natural actomyosin for goniodomin A. , 0.3 mg/ml troponin/tropomyosin-free natural actomyosin. , 0.3 mg/ml natural actomyosin. , 0.3 mg/ml natural actomyosin, 0.1 mg/ml troponin, and 0.1 mg/ml tropomyosin. , 0.3 mg/ml natural actomyosin, 0.2 mg/ml troponin, and 0.2 mg/ml tropomyosin. The ATPase activity is expressed as a percentage against the control activity of natural actomyosin with 0.2 mg/ml troponin and 0.2 mg/ml tropomyosin in the absence of goniodomin A at a Ca2+ concentration of 4 × 106 M. Values are mean ± S.E. (n = 3). **P  .01. B, the log concentration-response curve of the ATPase activity of natural actomyosin for Ca2+ in the absence () or presence () of 107 M goniodomin A. Relative ATPase activity is expressed as a percentage against a maximum activity in the absence of goniodomin A at a Ca2+ concentration of 105 M. Values are mean ± S.E. (n = 3). **P  .01.

Effects of Goniodomin A on Troponin/Tropomyosin-Free Natural Actomyosin ATPase Activity. The ATPase activity was enhanced with an increase in goniodomin A concentration and reached a peak at 10⁷ M (Fig. 3A). The peak value was 240% higher than that of the natural actomyosin ATPase activity. Further increase in the goniodomin A concentration up to 3 × 10⁷ to 3 × 10⁶ M decreased the ATPase activity.

Effects of Goniodomin A on Myofibril ATPase Activity. Goniodomin A decreased the ATPase activity of myofibrils in a concentration-dependent manner (>10⁸ M; Fig. 4A). This profile was similar to that found for the goniodomin A-induced inhibition of the ATPase activity of natural actomyosin (Fig. 3A) and actomyosin reconstituted from actin and myosin (Fig. 2) in the presence of a high concentration of the troponin/tropomyosin complex. The concentration-response curve of the myofibril ATPase activity for Ca²⁺ was decreased by goniodomin A at 10⁶ M to 10⁵ M Ca²⁺ (Fig. 4B).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of goniodomin A on the ATPase activity of rabbit skeletal muscle myofibrils. A, the log concentration-response curve of myofibril ATPase activity for goniodomin A. The ATPase activity is expressed as a percentage against the control activity in the absence of goniodomin A at a Ca2+ concentration of 4 × 106 M. Values are mean ± S.E. (n = 3). B, the log concentration-response curve of myofibril ATPase activity for Ca2+ in the absence () or presence () of 105 M goniodomin A. Relative ATPase activity is expressed as a percentage against the maximum ATPase activity at a Ca2+ concentration of 105 M. Values are mean ± S.E. (n = 3). **P  .01.

Effects of goniodomin A on Superprecipitation Activity of Natural Actomyosin. The effect of goniodomin A was examined on the superprecipitation of skeletal muscle natural actomyosin, an in vitro model reaction of muscle protein contraction, by monitoring the turbidity change. After the addition of ATP, the turbidity increased for 3 min. Figure 5A shows the typical recording traces of the superprecipitation activity of natural actomyosin in the presence of various concentrations of goniodomin A. As shown in Fig. 5B, goniodomin A (10⁸ to 3 × 10⁸ M) enhanced the superprecipitation activity in a concentration-dependent manner, but further increase in the goniodomin A concentration decreased it. This profile was similar to that found in the goniodomin A-induced modulation of the ATPase activity of natural actomyosin (Fig. 3A). The concentration-response curve of the superprecipitation activity of natural actomyosin for Ca²⁺ was shifted to the upper direction by goniodomin A (2 × 10⁸ M; Fig. 5C).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effects of goniodomin A on the superprecipitation activity of rabbit skeletal muscle natural actomyosin. A, typical recording traces in the presence of different concentrations of goniodomin A. B, the log concentration-activity curve of the superprecipitation of natural actomyosin for goniodomin A at a Ca2+ concentration of 4 × 106 M. Values are mean ± S.E. (n = 3). C, the log concentration-activity curve of the superprecipitation of natural actomyosin for Ca2+ in the absence () or presence () of 2 × 108 M goniodomin A. Relative initial velocity is expressed as a percentage against a maximum activity in the absence of goniodomin A at a Ca2+ concentration of 104 M. Values are mean ± S.E. (n = 3). **P  .01.

Effects of Goniodomin A on Contractile Response of Chemically Skinned Fibers. To measure the contractile force of chemically skinned fibers under the direct influence of Ca²⁺ concentration, the fibers were prepared from guinea pig skeletal muscles by sufficient treatment with detergents to destroy the function of both the cell membrane and sarcoplasmic reticulum membrane. As shown in Fig. 6A, goniodomin A (>10⁶ M) inhibited the contraction of skinned fibers in a concentration-dependent manner. The concentration-response curve of the contractile response of skinned fibers for Ca²⁺ was shifted to the lower direction by goniodomin A (10⁵ M; Fig. 6B).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of goniodomin A on the contraction of skinned fibers. A, the log concentration-response curve of the contraction for goniodomin A. The contraction is expressed as a percentage against the control tension in the absence of goniodomin A at a Ca2+ concentration of 4 × 107 M. Values are mean ± S.E. (n = 3). B, the log concentration-response curve of the contraction for Ca2+ in the absence () or presence () of 105 M goniodomin A. Relative contraction is expressed as a percentage against each maximum tension at a Ca2+ concentration of 106 M. Values are mean ± S.E. (n = 3). **P  .01.

Effects of Goniodomin A on CD of Actin. Figure 7A shows the far-UV CD spectra of actin treated with various concentrations of goniodomin A. Goniodomin A increased the negative ellipticity at 220 nm in a concentration-dependent manner (Fig. 7B).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effects of goniodomin A on the CD spectra of actin. A, typical CD spectra of actin in the presence or absence of goniodomin A. B, the change in the optical rotation at 220 nm by goniodomin A. Values are mean ± S.E. (n = 3). *P < .05; **P  .01.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Troponin and tropomyosin, the muscle regulatory proteins, in concert play a physiologically significant role in the regulatory process of the actomyosin ATPase activity and the striated muscle contraction (Holmes, 1995; Cooke, 1997; Squire and Morris, 1998). Goniodomin A (>10⁸ M, <10⁷ M) induced a profound enhancement of the ATPase activities of actomyosin reconstituted from actin and myosin, troponin/tropomyosin-free natural actomyosin, and natural actomyosin. At higher concentrations (>3 × 10⁷ M), goniodomin A caused an inhibition of the ATPase activity. It is well known that superprecipitation of skeletal natural actomyosin is an in vitro model reaction of muscle protein contraction (Szent-Györgyi, 1951). The superprecipitation activity of natural actomyosin was increased by goniodomin A at lower concentrations but was inhibited by it at higher concentrations. It has been previously reported that the conformational change of actin molecules, resulting from stoichiometric binding of goniodomin A to actin monomers in filaments, modifies the interaction between myosin and actin (Furukawa et al., 1993). Ca²⁺-, Mg²⁺-, or K⁺-EDTA-ATPase activity of myosin was not affected by goniodomin A, suggesting an elimination of the possible involvement of direct stimulation of myosin ATPase in the mechanism of actomyosin ATPase modulation. These results suggest that at lower concentrations, goniodomin A directly enhances the interaction of actin and myosin to activate the ATPase activity and thus increases the superprecipitation of natural actomyosin, but at higher concentrations, goniodomin A inhibits it, resulting in the decrease in the ATPase activity, and thus reduces the superprecipitation activity.

An interesting observation is that in the actin-myosin reconstituted system and natural actomyosin, the troponin/tropomyosin complex (regulatory proteins of muscle contraction) significantly reduced the goniodomin A-induced enhancement of the ATPase activities. In the presence of a sufficient amount of the troponin/tropomyosin complex, goniodomin A at any concentration used did not activate the actomyosin ATPase activity but rather inhibited it. The enhancement of the ATPase activity of troponin/tropomyosin-free natural actomyosin was greater than that obtained from natural actomyosin. Goniodomin A at any concentration used did not activate myofibril ATPase activity and the tension development of skinned fibers but rather decreased both the functions. A probable explanation for these findings is that goniodomin A fails to potentiate the actomyosin ATPase activity in the presence of a sufficient amount of the troponin/tropomyosin complex. Furthermore, it is probable that goniodomin A acts at two separate sites on actin: a stimulatory site and an inhibitory site. These results suggest that goniodomin A affects the stimulatory or the inhibitory site on actin to increase or decrease the actin-myosin interaction. It is also suggested that the troponin/tropomyosin complex causes a marked inhibition of the goniodomin A-induced activation of actomyosin ATPase activity through the stimulatory site on actin.

It is generally accepted that in skeletal muscles, Ca²⁺ first interacts with troponin-C, resulting in a shift of the position of tropomyosin on skeletal thin filament and leading to the physiological contraction of muscle fibers. Goniodomin A at a low concentration enhanced the response to Ca²⁺ in the superprecipitation and ATPase activity of natural actomyosin, but at a high concentration, goniodomin A decreased it in the ATPase activity of myofibrils and tension development of skinned fibers. These results suggest that goniodomin A can modify the function of actomyosin ATPase and contractile response at a wide concentration range of Ca²⁺.

Various physiological techniques, including electron spin resonance, fluorometry, and absorption spectrophotometry, have provided useful information about the conformational changes of physiologically important proteins. We previously reported that at higher concentrations (>10⁷ M), goniodomin A caused a concentration-dependent increase in the fluorescence intensity in tryptophan residues of actin (Furukawa et al., 1993). In the present study, the ATPase activity of actin-myosin reconstituted system and natural actomyosin increased with an increase in the goniodomin A concentration and reached a peak at 10⁷ M. Further increase in the goniodomin A concentration caused a concentration-dependent decrease in the ATPase activity. The profile of a concentration-response curve of the goniodomin A-induced decrease in the ATPase activity was similar to that of the goniodomin A-induced increase in the fluorescence intensity. It is possible that there is a close correlation between the decrease in the ATPase activity and the increase in the fluorescence intensity induced by goniodomin A. (A detailed study on both the relationship is under way.) On the other hand, the relationship between the conformation of the actin molecule and its function has been extensively studied by the analysis of the CD spectra (Murphy, 1971; De La Cruz and Pollard, 1995). The far-UV CD spectra of actin shows two negative ellipticities at 208 and 220 nm, and negative optical rotation around 220 nm indicates the -helical content (Feinberg et al., 1996). Goniodomin A increased the negative ellipticity at 220 nm in the CD spectrum of actin in a concentration-dependent manner, suggesting binding to a site that changes the conformation of actin into -helical. These results suggest that goniodomin A shifts the dynamic equilibrium of actin between a helical conformation and a more random structure toward a helical. It is also suggested that the increase in -helical content of actin by goniodomin A at a concentration of 10⁷ M enhances actomyosin ATPase activity but at a concentration of 3 × 10⁷ M inhibits it.

Goniodomin A may provide a selective modulator of actin for the study of not only the relationship between structure and function of actin but also the molecular mechanism of the activation of actomyosin ATPase that is regulated by the troponin/tropomyosin complex.