Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

404

Bmchimtca et Btophvsica Acta 912 (1987) 404-407 Ilsevier

BBA 32826

E n h a n c e m e n t of the actin-activated A T P a s e activity of m y o s i n from canine cardiac ventricle by purealin Jiro Takito, Hideshi Nakamura,

Jun'ichi Kobayashi

and Yasushi Ohizumi Mitsubisht-Kasei Institute of l,ife Sciences, "Fokvo (Japan) (Received 2 December 1986)

Key words: Purealin: Myosin; ATPase, actin-activated: Actin-myosin interaction: (Canine cardiac ventricle)

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activit3' of natural actomyosin and the actin-activated ATPase activiD' of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and MgZ+-ATPase activities of myosin were inhibited by purealin, whereas the K +-EDTA-ATPase activity, was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activi D of myosin.

i ntroduction

OCH•

Purealin (Fig. 1) is a bromotyrosine derivative isolated from an Okinawa sea sponge, Psammaplysilla purea [1]. Earlier studies have indicated that purealin modulates the ATPase activities of myosin and subfragment 1 from rabbit skeletal muscle [2]. Furthermore, purealin has been shown to alter the ATPase activities of dephosphorylated myosin from chicken gizzard smooth muscle [3]. In the present paper we report that purealin is the first known substance to increase the actinactivated ATPase activity of myosin from canine cardiac ventricle. Purealin has proved to be useful as a chemical tool for helping to resolve the mechanism of actin-activation of myosin ATPase. Abbreviations: Mops, 4-morpholinepropanesulphonic acid; F.GTA, ethylene glycol bis(,R-aminoethyl ether)-N,N.N',N'-tetraacetic acid. Correspondence: J. Takito, Mitsubishi-Kasei Institute of Life Sciences, I 1 Minamiooya. Machida-shi, Tokyo 194, Japan. 0167-4838/87/$03.50

.v.

HO ' ' " ~ H

Br

_/

°W-"~,, ~ / ~

°~.

H 0

]

N

..

H

6

<.~7-' ': H

Fig. 1. Chemical structure of purealin.

Materials and Methods Purealin was isolated from the Okinawa sea sponge, P. purea. Details of the purification method have been published elsewhere [1]. Purealin was dissolved in dimethyl sulfoxide and less than a 0.5% volume of the dimethyl sulfoxide solution was added to the assay mixture. The same volume of dimethyl sulfoxide was added for the control experiment. Myosin was prepared from canine heart

1987 Elsevier Science Publishers B.V. (Biomedical Division)

405

ventricle according to the method of Tada et al. [41, with slight modifications as follows. Fresh ventricular muscle, 20-30 g, was freed of connective tissue and then minced, The mince was homogenized in a Waring blender in 5 vol. of 0.05 M KCI, 1 mM MgCI 2 and 1 mM NaHCO 3. The homogenate was spun for 5 min at 10000 × g and the supernatant was discarded. This wash was repeated until the supernatant appeared colorless. The resulting pellet was extracted with 3 vol. of 0.3 M KCI, 1 mM ATP, 1 mM EDTA, 1 mM dithiothreitol, 0.1 M KH2PO 4 and 0.05 M K 2 H P O 4 (pH 6.5) for 10 min. The suspension was centrifuged at 1 0 0 0 0 × g for 10 rain and then filtered through glass wool. The filtrate was diluted with 15 vol. of cold water and the resulting precipitate was allowed to settle for an hour. The precipitate was collected by centrifugation and dissolved in 0.6 M KCI, 10 mM MgC12, 10 mM ATP, 1 mM EGTA, 20 mM Tris-HC1 (pH 8.0) in a final volume of 50 ml. The suspension was centrifuged at 80 000 x g for 1 h. The supernatant was diluted with an equal volume of 1 mM N a H C O 3, and dialyzed overnight against 10 vol. of 10 mM Mops (pH 6.8). The following day, the precipitate was collected by centrifugation, and dissolved in 0.5 M KCI, 20 mM Tris-HCl (pH 7.4) in a final volume of 20 ml. The suspension was centrifuged at 80000 x g for 1 h. The supernatant was used as canine cardiac ventricle myosin. Experiments using this myosin were done within a week. Natural actomyosin was prepared from canine heart ventricle according to the procedure of Szent-Gyorgyi [5]. F-actin was obtained from acetone-treated powdered rabbit skeletal muscle by the method of Spudich and Watt [6]. The purity of proteins was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli [7], and the gels were stained with Coomassie Blue. Protein concentrations were measured using the biuret reaction [8]. ATPase activities were determined by measuring the amount of Pi liberated according to the method of Martin and Doty [9]. The ATPase assay was performed as follows unless otherwise stated. A reaction mixture (1 ml) containing proteins and purealin was preincubated for 5 rain at 30 o C. The

ATPase reaction was started by adding ATP, and stopped by adding 0.5 ml of 20% trichloroacetic acid. Assay conditions are given in figure legends. The light-scattering intensity at an angle of 90 o was recorded using an AMINCO SRF-500 TM at 29°C. The wavelength of excitation (5 nm slit width) and emission (10 nm slit width) was 500 rim.

Results Superprecipitation of ventricular natural actomyosin suspension was followed by measuring the turbidity. Purealin enhanced superprecipitation of natural actomyosin with an increase in the concentrations up to 10 #M (Fig. 2). The activating effect of purealin on superprecipitation of natural actomyosin was prominent in the presence of 0.5 mM EGTA. Purealin shortened the time required to attain a maximum level of superprecipitation, but had little effect on the extent of superprecipitation. These results agreed well with our previous findings using myosin from rabbit skeletal muscle

[21. The Mg 2 *-ATPase activity of ventricular natural actomyosin in 0.05 M KCI was measured in the presence of various concentrations of purealin (Fig. 3A). Purealin at 10 ~M increased the ATPase

[

.

I

,

.

.

.

i

.

I

i

.

.

.

i

.

i

,

.

.

.

i

•

.

.

I

,

,

L

07

O6 O5

<

03 02 01

0

0

,

,

,

i

,

i

5 Time

10 (

rain

,

i

,

15

)

Fig. 2. Effects of purealin on superprecipitation of natural actomyosin. The change in the absorbance of ventricular natural actomyosin suspension at 550 nm was monitored at rcx)m temperature after addition of 0.1 mM ATP. Assay conditions: 50 mM KCI, 1 mM MgCI 2, 0.1 mM CaCI 2 (a) or 0.5 mM EGTA (b-e), 20 mM Mops (pH 6.8) and 0.42 m g / m l ventricular natural actomyosin. The concentrations of purealin were 10 ~M (b), 3 ~M (c), 1 p.M (d) and 0 (a, e), respectively.

406

250

~, . . . . . .

, ,

....

[

] ' ' ' ' I ....

I

100

B

~e 2O0

~50 E E

.~ 150

B E c

~,0

t, 50

3O

g

.E

I00 20

=,i

50 CE

,¢

1#1

,

01 1 , 6

5

- LOg { pureahn ]

z,

(M)

,

,

,

I

. . . .

10

Pureahn

I,

All

20

,

,

J

,

5 ATP

t

i

,

I

,

10 ( rnM )

i

,

"~1~_(

15

30

( ~JM )

Fig. 3. Effects of purealin on the ATPase activities of myosin, natural actomyosin (A) and the reconstituted actomyosin (B). Assay conditions: (A) 0.5 M KCI, 20 mM Mops (pH 6.8), 1 mM ATP and 10 mM CaC12 (Ca2*-ATPase, I), or 10 mM EDTA (K+-EDTA-ATPase, [21), or 2 mM MgCI z (Mg 2+ATPase, O). Actomyosin ATPase (e); 50 mM KCI, 2 mM MgCI 2, 20 mM Mops (pH 6.8), 1 mM ATP and 0.46 m g / m l ventricular natural actomyosin. (B) 50 mM KCI, 1 mM MgCI 2, 0.5 mM EGTA, 20 mM Mops (pH 6.8), 0.2 mM ATP, 0.1 m g / m l ventricular myosin and 0.05 m g / m l rabbit skeletal actin. Each symbol on the graph represents a percent of the control activity. Control values for the (7a2--, K ~-EDTA-, Mg 2 "- and actomyosin ATPase activities were 180, 110, 2.7 and 24 nmol P,/min per mg, respectively.

activity to 150% of the control value. Effects of purealin were also examined on the ATPase activity of actomyosin reconstituted from ventricular myosin and rabbit skeletal muscle actin, which, being devoid of troponin and tropomyosin, is advantageous for studies on the direct action of drug (Fig. 3B). The actin-activated ATPase activity of myosin increased with an increase in the purealin concentration, up to 20 #M, and then decreased when the concentration was further raised. The activity was increased to 650% of the control value by 20/.tM purealin. Effects of purealin on the Ca 2+-, K ~-EDTA-, and Mg2+-ATPase activities of ventricular myosin were examined and the results are shown in Fig. 3A. Purealin inhibited the Ca 2--, and Mg 2+ATPase activities of myosin in a concentration-dependent manner. The concentrations of purealin producing 50% inhibition of the Ca z÷-, and Mg2+-ATPase activities were 2 and 4 p,M, respectively. In contrast, the K ~-EDTA-ATPase activity

Fig. 4. Effects of purealin on the ATP-induced decrease in light-scattering of myosin. The relative intensity of lightscattering was defined by the relationship: [(1 - Ira.. ) / ( 1o -/ram)I× 1130 (I,> the light-scattering after addition of 0.1 mM ATP" 1. the minimal intensity of light-scattering after addition of ATP: Imin, the light-scattering after addition of 16.7 mM ATP in the presence of 0.4% dimethyl sulfoxide). Assay conditions: 0.15 M KC1, 10 mM MgC12. 1 mM EGTA, 40 mM Mops (pit 6.8), 0.3 m g / m l ventricular myosin and 40 ,ttM purealin (e) or 0.4c~ dimethyl sulfoxide (©) at 29 ° C.

of myosin was increased by 10 ~M purealin to 250% of the control value. These results clearly indicate that purealin acts directly on ventricular myosin to modulate its ATPase activities. Recently, it has been shown that purealin stabilizes thick filaments of dephosphorylated myosin from chicken gizzard smooth muscle against the disassembling action of ATP in 0.15 M KCI [3]. Therefore, effects of purealin on thick filaments of ventricular myosin were examined by light-scattering measurements. Fig. 4 shows the effect of purealin on the ATP-induced decrease in light-scattering of ventricular myosin in 0.15 M KCI. More than 10 mM ATP was required for a complete decrease in light-scattering of ventricular myosin. This value was in good agreement with that obtained using myosin from rabbit skeletal muscle [10]. The degree of the ATP-induced decrease in light-scattering was slightly reduced by 40/~M purealin. Discussion

Purealin decreases the Ca 2 ~- and Mg 2 +-ATPase activities of cardiac ventricular myosin, whereas it

407 increases the K+-EDTA-ATPase activity (Fig. 3A). These characteristics of purealin-induced modulation of the ATPase activities of ventricular myosin are in good agreement with observations obtained using myosin from rabbit skeletal muscle [2]. On the other hand, it has been reported that purealin increases the Ca 2+- and Mg2~-ATPase activities of dephosphorylated myosin from chicken gizzard smooth muscle, but inhibits its K - - E D T A - A T P a s e activity [3]. These observations suggest that cardiac muscle myosin is very similar to skeletal muscle myosin with regard to the structure of the purealin-binding site, which may be close to the active site of ATPase in the tertiary structure of the protein. It is of interest that purealin discriminates between skeletal or cardiac and smooth muscle myosin. Specific blocking of the highly reactive thiol group inhibits the K ' - E D T A - A T P a s e activity of myosin and activates the Ca2+-ATPase activity [11]. Sulfhydryl-protecting reagents cancel these modulations of the myosin ATPases, but purealin is effective in the presence of dithiothreitol [2,3]. Purealin enhances the ATPase activity of natural (Fig. 3A) and reconstituted actomyosin (Fig. 3B), and accelerates superprecipitation of natural actomyosin (Fig. 2). Taken together with the observations discussed above, it is possible that purealin binds to the myosin molecule and changes the actin-myosin interaction, resulting in enhancement of the actin-activated ATPase activity of myosin. With respect to the effects of purealin on other myosins, purealin is found to enhance the actin-activated ATPase activity of myosin, heavy meromyosin and subfragment 1 from rabbit skeletal muscle by increasing their kinetic affinities for actin, while having no effect on the actinactivated ATPase activity of dephosphorylated and phosphorylated myosin from chicken gizzard smooth muscle (unpublished data). Stoichiometric amounts of ATP readily disassemble thick filaments of dephosphorylated smooth muscle myosin into 10 S monomers [ 10,12,13]. Electron microscopic observations have shown that the thick filaments are preserved by

purealin even after addition of ATP [3]. This stabilizing effect of purealin on thick filaments against the disassembling action of ATP is also observed with ventricular myosin by light-scattering measurments (Fig. 4), although the apparent potency of the effect of purealin is considerably weaker when compared with dephosphorylated gizzard myosin [3]. It is of particular importance to elucidate the relation between the actions of purealin on the stability of thick filaments and the various ATPase activities of myosin described above. Acknowledgments We are grateful to Professor Y. Hirata University for his encouragement and Nonomura of Tokyo University for his advice. We also thank M. Nakai of our for preparing the manuscript.

of Meijo Prof. Y. valuable institute

References 1 Nakamura, H., Wu, H., Kobayashi, J., Nakamura, Y., Ohizumi, Y. and Hirata, Y. (1985) Tetrahedron Left. 26, 4517-4520 2 Nakamura, Y., Nakamura, H., Wu, H., Kobayashi, J. and Ohizumi, Y. (1985) Jap. J. Pharmacol. 39, p. 330 3 Takito, J., Nakamura, H., Kobayashi, J., Ohizumi, Y., Ebisawa, K. and Nonomura, Y. (1986) J. Biol. Chem. 261, 13861-13865 4 Tada, M., Bailin, G., Barany. K. and Barany, M. (1969) Biochemistry 8, 4842-4850 5 Szent-Gyorgyi,A. (1951) Chemistry of Mu~ular Contraction, 2nd Edn., p. 151, Academic Press, New York 6 Spudich, J.A. and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871 7 Laemmli, U.K. (1970) Nature 227, 680-685 8 Gronall, A., Bardawill, C.J. and David, M.M. (1949) J. Biol. Chem. 177, 751-766 9 Martin, M. and Doty, D.M. (1949) Anal. Chem. 21,965-967 10 Suzuki, H., Onishi, H., Takahashi, K. and Watanabe, S. (1978) J. Biochem. 84, 1529-1542 11 Sekine, T. and Kielley, W.W. (1964) Biochim. Biophys. Acta 81. 336-345 12 Scholey, J.M., Taylor, K.A. and Kendrick-Jones, J. (1980) Nature 287, 233-235 13 Trybus, K.M.. Huiatt, T.W. and Lowey, S. (1982) Prec. Nat. Acad. Sci. USA 79, 6151-6155