The Major Component of Metin from Rabbit Skeletal and Bovine Cardiac Muscle*

Size: px

Start display at page:

Download "The Major Component of Metin from Rabbit Skeletal and Bovine Cardiac Muscle*"

Avis Woods
6 years ago
Views:

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 24, No. 1, October 1965 Printed in U.S.A. The Major Component of Metin from Rabbit Skeletal and Bovine Cardiac Muscle* NAOMI AzUMAt AND SHIZUO WATANABE~ From the Cardiovascular Reseawh Institute and the Department of Biochemistry, University of California School of Medicine, San FTancisco, California 941ZV2 (Received for publication, April 19, 1965) It has been generally accepted that myosin B is actomyosin, a complex of actin and myosin (or myosin A) which can be separately extracted from muscle (1). It was sometimes noticed that myosin B behaves differently than actomyosin in the light scattering effect, for example, on the addition of adenosine triphosphate (2-4). But the concept has remained unshaken until recently when two groups of investigators have called attention to a difference between myosin B and (reconstituted) actomyosin. Several investigators had noted that a calcium-chelator, 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane (ethylene glycol bis(p-aminoethyl ether)-n,n -tetraacetic acid) consistently inhibits the contraction phenomenon called superprecipitation of myosin B (in the presence of physiological concentrations of magnesium and adenosine triphosphate), but that it often fails to inhibit superprecipitation of actomyosin. Then, Ebashi and Ebashi (5, 6) found a new protein with which superprecipitation of actomyosin becomes consistently inhibited by EGTA. On the other hand, Szent-Gydrgyi and Kaminer (7) found that myosin B (in suspension) is, but actomyosin is not always, stained metachromatically by toluidine blue. Therefore, they suggested that myosin B is a tertiary complex of a metachromatic component, actin, and myosin. They actually reported three different methods for preparing the component and named it metin on the basis of its metachromatic property. We have undertaken a study to see if metin has an effect similar to that of Ebashi protein on the superprecipitation of actomyosin. We found that a major component of metin preparat,ions is tropomyosin, and that there is a difference between the major component of metin and Ebashi protein in their effect on the superprecipitation of actomyosin. Metin was prepared from both rabbit skeletal and bovine cardiac muscles; however, we will not discuss them separately because cardiac metin was exactly the same as skeletal metin in all of the aspects we studied. * Sunnorted by _ - grants from the American Heart Association (a special contribution of the San Francisco Heart Association), the National Science Foundation (G-19442), and the United States Public Health Service (HE-6285). Presented at the Sixteenth Fall Meeting of the American Physiological Society, t On leave from the Department of Chemistrv., Hokknido Ga- k&e; College, Asahikawa-shi, Japan. $ Established Investigator of the American Heart Association (196 to 1965). 1 The abbreviation used is: EGTA, 1,2-bis-@dicarboxymethyl- aminoethoxy)ethane (ethylene glycol his@-aminoethyl ether)- N,N -tetraacetic acid). EXPERIMENTAL IMaterials PROCEDURE and Methods Rabbit skeletal muscle was used in preparing actin, myosin A, and tropomyosin. G-Actin was prepared by the method of Straub and Feuer as described by Szent-Gyorgyi (8), except that the acetone treatment was carried out in the cold instead of at room temperature. G-Actin thus obtained was further purified according to Mommaerts (9). F-Actin was prepared by polymerizing G-actin in.3 M KC1 at room temperature and then centrifuging it at 1, X g for 3 hours. Myosin A (or myosin) was prepared according to Szent- Gyiirgyi (8). However, the first precipitate of myosin X collected by centrifugation was dissolved in.28 M KC1 by using.3 M KCl; if the first precipitate was once dissolved in.6 M KC1 and then the solution was diluted to M KCl, a slight contamination of myosin B in the final preparation of myosin A was hard to avoid. Such a slight contamination of myosin B was detected neither in an ultracentrifuge pattern (see Fig. 3a) nor in the light scattering change at relatively high KC1 concentrations upon addition of ATP. But it was detectable by the superprecipitation test at low KC1 concentrations2 (Fig. 1). From the turbidity method for superprecipitation (11, 12)) it was found that superprecipitation can be detected when only 3 parts of actin are added to 1 parts of myosin A (by weight). Fig. 2 shows the increase in turbidity on the addition of increasing concentrations of actin to a fixed concentration of myosin A (.142 mg per ml). An apparent saturation occurred at a weight ratio of approximately 1:4 for actin to myosin A. We therefore use the 1:4 actomyosin in the experiments reported in this paper. Actin, 1 part, was incubated with myosin A, 4 parts, at for 3 min in.5 M KCl; actomyosin thus formed was collected by centrifugation, and it was dissolved or suspended in appropriate concentrations of KCl. Preparation and crystallization of tropomyosin were carried out according to Bailey (13). Metin was prepared from rabbit skeletal or bovine cardiac muscle tissue by the isoelectric precipitation method of Szent- Gyorgyi and Kaminer (7), except that the first washing of muscle brei was done with 3 nli\i NaHCOe in place of distilled water. The use of NaHC3 was found to be essential to remove blood from heart muscle brei. Average yields of metin were ap- 2 Kessler and Spicer (1) described the superprecipitation test at.1 to.18 M KC1 as a sensitive test for actin contamination in myosin A preparations. However, in the presence of magnesium, KC1 concentrations lower than.1 M have to be used. 3847

3848 Major Component of Metin Vol. 24, No. 1.3r OA : B.<orc ATIP FIG. 1. Superprecipitation test for two different preparations of myosin A. Myosin A (dashed curues),.

2 3848 Major Component of Metin Vol. 24, No. 1.3r OA : B.<orc ATIP FIG. 1. Superprecipitation test for two different preparations of myosin A. Myosin A (dashed curues),.36 mg per ml, and myosin A (solid curves),.3 mg per ml, were used. X and + were obtained 5min after the addition of 1 mm ATP in the presence of 1 mm MgC12 and 16 mm Tris-maleate buffer (ph 7); 25. ration is therefore reasonably similar to Szent-Goyorgyi and Kaminer s preparation. The minor component of metin can be removed by salting out at about 5% saturation of neutral ammonium sulfate. The major component was then precipitated at 7% saturation of ammonium sulfate, dissolved in water, dialyzed against water overnight, and reprecipitated isoelectrically at ph 5. The precipitate was dissolved in water and the above procedures were repeated twice for a further purification. The final precipitate was dissolved in water, and insoluble materials were removed by centrifugation at 3, x g for 1 hour. The sedimentation pattern of the purified major component shows only one peak (Fig. 3~). The major component of metin thus isolated was identified as tropomyosin (13), based on the following properties. The sedimentation constant (2,.1 M KCl) of protein, obtained by extrapolating to zero concentration, is 2.5 X lo-13 for both tropomyosin and the major component of metin (Fig. 4). The intrinsic viscosity obtained from a relationship between [A CT I Nl (mg/l) FIG. 2. Effect of the actin concentration on the turbidity (optical density at 5 rnp) of myosin A suspension. Myosin A,.142 mg per ml, in.6 M KC1 buffered with 16 mm Tris-maleate (ph 7), 1 mm MgCL, and.1 mm ATP. proximately.15 and.9% of muscle, wet weight, for rabbit skeletal muscle and bovine heart muscle, respectively. The protein concentration was determined by the Kjeldahl method, with a factor of 6.25 g of protein per g of nitrogen, or by a biuret reaction (14), standardized by the Kjeldahl method. Analytical ultracentrifugation was performed in a Spinco model E ultracentrifuge, with an An-D rotor and 12-mm 4 sector cells with standard or wedge windows. RESULTS Characterization of Metin-Szent-Gyiirgyi and Kaminer (7) reported that their preparation of metin contains a major component with a sedimentation coefficient, szo,w = 2.85 x lo-13 (4.7 mg per ml,.1 M NaCl), and a minute quantity of a slightly faster sedimenting component (~2,~ = 3.6 X 1-13). Our metin preparation also showed two peaks in an ultracentrifuge pattern (Fig. 3b) ; for example, in.1 M KC1 at 2, a major component with a sedimentation coefficient of 2.7 x lo+ (3.4 mg per ml), and a minor component with a sedimentation coefficient of 3.7 X lo+ (3.4 mg per ml) are seen. Our prepa- FIG. 3. Sedimentation patterns of myosin A, crude metin, and a major component of metin. a, 1.6 mg per ml of myosin A in.6 M KC1 (standard window); 112 min after the maximum speed, 5,74 rpm, was reached. Diaphragm angle 7 ; b, 3.77 mg per ml of Szent-Gybrgyi and Kaminer metin in.6 M KC1 (wedge window) and 3.4 mg per ml of Szent-Gyorgyi and Kaminer metin in.1 M KC1 (standard window) ; 77 min after the maximum speed, 59,78 rpm, was reached. Diaphragm angle 6 ; 26. c, 5 mg per ml of Bailey tropomyosin (wedge window) and 4 mg per ml of major component of metin (standard window) in.2 M KCl; 143 min after the maximum speed, 59,78 rpm, was reached. Diaphragm angle 55 ; 2. ;; $3 t [PR T E i N] (g/looml) FIG. 4. Sedimentation constants of tropomyosin and of a major component of metin. The medium contained.21 M KC1 and no buffer; ph was adjusted to 6.8; 2. l, t.ropomyosin prepared according to Bailey (13) ;, a major component of metin (see text).

3 October 1965 N. Azuma and S. Watanabe 3849 reduced viscosity (v,,/c) and the protein concentration (C) is the same for both proteins (.5, Fig. 5). The drastic effect of KC1 concentration on viscosity, which is characteristic to tropomyosin (15), is also observed with the major component of metin (Fig. 6). The ultraviolet absorption spectrum is exactly the same for both proteins. The absorbance ratio, 278 to 26 rnp, is 2.1 for both proteins, indicating that neither protein contains nucleotides (16). The fluorescence spectra of the major component of metin and of tropomyosin show a fluorescence emission peak of tyrosine residues at 35 rnp upon activation at 29 mp. The tryptophancontaining proteins, myosin A and actin, show fluorescence emission peaks at 35 and 338 rnp, respectively, which are close to the fluorescence wave length of tryptophan residues (Fig. 7). In accordance with the fluorescence spectra, the tryptophan content estimated by the glyoxylic acid method (17) is almost nil. It is less than.1 mole per lo5 g of protein for both tropomyosin and the major component of metin. Fig. 8 shows that. both proteins have a metachromatic property. It should be mentioned, however, that metachromasis > P w : P :oo: *.o (PROTEIN : Cl (g/looml) FIG. 5. Reduced viscosity of Bailey tropomyosin and a major component of metin. Tropomyosin () or a major component of metin () in.2 M KC1 buffered with 2 mm phosphate (ph 7); 25. An Ubbelohde viscosimeter was used (flow time for distilled water = 96.1 see). [ K Cl J (M 1 FIG. 6. Effect of the KC1 concentration on the viscosities of Bailey tropomyosin and of a major component of metin. Tropomyosin (o), 3. mg per ml, and a major component of metin (), 3.2 mg per ml. The relative viscosity was measured at 25 in various concentrations of KC1 (no buffer, but the ph was adjusted to 6.8)) with a Cannon-Fenske viscosimeter (flow time for distilled water = 12.7 set). FIG. 7. Fluorescence spectra of Bailey tropomyosin, a major component of metin, and some other proteins. Protein,.1 mg per ml, in a medium consisting of.6 M KC1 and 5 mm phosphate buffer (ph 8.); room temperature. Activation at 29 rnp for all proteins. Coordinate, relative intensity of fluorescence emission (the scale is 5 times larger in the lower photo than in the upper photo). Abscissa, fluorescence wave length in millimicrons (peaks at 29 rnp are scattering peaks). Upper photo: a, actin (fluorescence peak at 338 rnp) ; b, myosin A (35 mm) ; c, Bailey tropomyosin (35 mr); d, medium. Louler photo: a, Ebashi protein (35 mp); b, Bailey tropomyosin (35 mp); c, major component of metin (35 mp); d, medium..8- > l- v,.6- z W n.4 - -I a E.2.O' WAVE LENGTH (m,u) FIG. 8. Toluidine blue metachromasis with metin and other substances. All of the samples were dialyzed against distilled water, and then 2 X 1-S M toluidine blue (aqueous) was added. Dashed curves, toluidine blue alone,.4 mg per ml of chondroitin sulfate, and.4 mg per ml of heparin. Solid curves,.375 mg per ml of Bailey s tropomyosin (lozuer curve) and.193 mg per ml of major component of metin (upper curve).

385 Major Component of Metin Vol. 24, No. 1 FIG. 9. Crystals of major component of metin. Crystallized in 16 g per liter of ammonium sulfate buffered with.1 M acetate (ph 5.4). Scale = 1 per 1 mm.

4 385 Major Component of Metin Vol. 24, No. 1 FIG. 9. Crystals of major component of metin. Crystallized in 16 g per liter of ammonium sulfate buffered with.1 M acetate (ph 5.4). Scale = 1 per 1 mm. Rabbit skeletal (I&) and bovine cardiac (right) metins..3 4 ( (NH&S4] ~~.so+uro+ion) FIG. 1. Salting out profiles of Bailey tropomyosin and a major component of metin. Major component of metin,.39 mg per ml, or tropomyosin,.73 mg per ml, in various concentrations of neutral ammonium sulfate were kept at 25 for 15 hours, then filtered through a filter paper (Whatman No. 31), and the absorbance at 278 rnp of the filtrate was measured. is greatly dependent on the salt concentration,3 ph, protein concentration, and dye concentration. Finally, applying Bailey s method (13) for crystallization of tropomyosin, the major component of metin is crystallized in the same form as tropomyosin (Fig. 9). Actually, ammonium sulfate salting out profiles (Fig. 1) indicate that the major component preparation is a bett er preparation of tropomyosin than Bailey s tropomyosin. E$ect of Metin on Superprecipitation-The effects of metin and tropomyosin on the superprecipitation of actomyosin were 8 In a previous abstract (18), we stated that tropomyosin does not show the metachromatic effect. However, the test was done at that time in the presence of KC1 and therefore the test was negative. 8 3 In s.2 E iii z w n -I.I a v i= g.o jb ta ATP T I M E -(minutes) FIG. 11. Effect of metin on the superprecipitation of actomyosin. Actomyosin,.2 mg per ml, in.45 M KC1 (A) or.9 M KC1 (B), buffered with 16 mix Tris-maleate (ph 7)) 1 mm MgC12, and.5 mm ATP. Szent-Gyorgyi and Kaminer metin: (a), 63 (b), 125 (c), and 25 pg per ml (d). studied at two different concentrations of KU,.45 M and.9 M. The crude metin preparation alone, which shows two peaks in the ultracentrifuge pattern, exhibits some activation effect on the superprecipitation in.45 M KCl, but it inhibits the superprecipitation in.9 M KCl. Both the activation effect and the inhibition effect increase with increasing concentrations of metin (Fig. 11). EGTA alone (without metin) inhibits only slightly, but EGTA plus metin greatly inhibits superprecipitation, although the inhibition by EGTA plus metin is less in.45 M KC1 than in.9 M KC1 (Fig. 12). Therefore, crude metin has an effect similar to that of Ebashi protein (5, 6) on superprecipitation. Tropomyosin and the major component of metin in the absence of EGTA behave somewhat like crude metin in the superprecipitation test. However, in the presence of EGTA,

5 October 1965 N. Azuma and S. Watanabe 3851 I.I fatp I 5 1c T I ME ( minutes) ;A-rP I I I FIG. 12. Effect of metin preparations on the superprecipitat,ion of actomyosin in the presence of EGTA. A,.152 mg per ml of actomvosin in.45 M KC1 buffered with 16 mm Tris-maleate (ph 7), 1 mm MgC12, and.5 mm ATP. a, no EGTA and no metin; b, 25 FM EGTA; c, 58 pg per ml of major component of metin; d, 25 PM of EGTA plus 58 rg per ml of major component of metin; e, 25 WM of EGTA plus 56 pg per ml of Szent-Gyorgyi and Kaminer metin; 26. B, mg per ml of actomyosin-in.9 M KC1 buffered with 16 rnm Tris-maleate (nh 7). 1 mm MaC12. and.5 mm ATP. a, no EGTA and no met&-; b, 25 PM EGTA; ci 75 rg per ml of major component of metin; cl, 25 PM of EGTA plus 75 pg per ml of major component of metin; e, 25 PM of EGTA plus 45 rg per ml of Szent-Gyorgyi and Kaminer metin; 25. both proteins have only a weak effect4 on superprecipitation in.9 hf KCl, and almost no effect on superprecipitation in.45 M KC1 (Fig. 12). Therefore, it may be suggested that the effect of the crude metin similar to that of Ebashi protein is not due to the major component of metin but is due to the minor component. DISCUSSION The major component of metin is tropomyosin, and tropomyosin in the absence or in the presence of low concentrations of KC1 exhibits a metachromatic effect with toluidine blue. Therefore, it may be said that metin is nothing but tropomyosin. However, upon increasing the KC1 concentration to approximately.5 M, the metachromatic effect of tropomyosin becomes undetectable while the crude metin preparation still retains its metachromatic property, suggesting that the minor component is stronger in producing the metachromasis than the major component. Besides, the minor component seems to have the same effect on the superprecipitation of actomyosin as has the new protein reported by Ebashi and Ebashi (5, 6). We therefore would like to reserve the name metin for the minor component of crude metin, a further study of which will be reported elsewhere (19). Ebashi and Ebashi (6) stated that tropomyosin prepared according to Bailey does not have the inhibition effect of their 4 It has been not,iced that the inhibitory effect of tropomyosin is stronger when tropomyosin is mixed with actomyosin solution in.6 M KC1 and then diluted to.9 M KC1 for the superprecipitation test, than when it is mixed with actomyosin suspension in.9 M KCI. new protein on the superprecipitation of actomyosin. On the other hand, Katz (2) has recently reported that tropomyosin does have an inhibitory effect on superprecipitation. This discrepancy seems to be due to the difference in the KC1 concentrations employed. Ebashi and Ebashi tested the effect at low concentrations of KC1 (.4 M), whereas Katz has employed relatively high concentrations of KC1 (.15 M). Our result obtained at.9 M KC1 seems to be in accord with Katz s report, and our result obtained at.45 M KC1 confirms Ebashi and Ebashi s statement. SUMMARY A major component of metin was isolated from metin prepared according to Szent-Gybrgyi and Kaminer (7), and it was identified as tropomyosin on the basis of its sedimentation constant, intrinsic viscosity, effect of KC1 concentration on viscosity, ultraviolet absorption spectrum, fluorescence emission spectrum, tryptophan content, metachromatic effect, and crystalline form. A major component of metin inhibited the superprecipitation of actomyosin not inhibited by the chelating agent, 1,2-bis-(2. dicarboxymethylaminoethoxy)ethane, alone, only when a high KC1 medium (.9 M KCl) was used. However, Szent-Gyorgyi and Kaminer s metin did so in a low KC1 medium (.45 M KCI) as well as in a high KC1 medium (.9 M KU). Ackno&edgment-We wish to thank Professor M. Morales for his interest and encouragement in this research. REFERENCES 1. SZENT-GY~RGYI, A., The chemistry of muscular contraction, Academic Press, Inc., New York, BLUM, J. J., Arch: Biochem. Biophys., 43, 176 (1953). 3. VON HIPPEL, P. H., SCHACHMAN, H. K., APPEL, P., AND Mo- RALES, M. F., Biochim. et Biophys. Acta, 28, 54 (1958). 4. GERGELY, J., J. Biol. Chem.,22, 917 (1956). 5. EBASHI. S.. Nature (1963). 6. EBASHI~ S:, AND ~BAS&, F., J. kochem. (Tokyo), 66, 64 (1964). 7. SZENT-GY~RGYI, A., AND KAMINER, B., Proc. Natl. Acad. Sci. U. S., 5, 133 (1963). 8. SZENT-GY~RGYI, A., The chemistry of muscular contraction, Ed. 2. Academic Press. Inc.. New York MOMMA~RTS, W. F. H. G., J. kol. Chem., 198, 445 (1952). 1. KESSLER, V., AND SPICER, S. S., Biochim. et Biophys. Acta, 8, 474 (1952). 11. EBASHI, S., J. Biochem. (Tokyo), 6, 236 (1961). 12. YASIJI, T., AND WATANABE, S, J. Biol. Chem., i4, 98 (1965). 13. BAILEY. K.. Biochem. J (1948). 14. GORNA~L, A. G., BARDA~IL;, C. k., AND DAVID, M. M.. J. Biol. &em., 177, 751 (1949): 15. TSAO. T.-C.. BAILEY. K.. AND ADAIR. G. S.. Biochem. J (1951). 16. SIMMONS, N. S., COHEN, C., SZENT-GY~RGYI, A. G., WET- LAUFER, D. B., AND BLOUT, E. R., J. Am. Chem. Sot., 83, 4766 (1961). 17. CARPENTER; D. C., Anal. Chem., 2, 536 (1948). 18. AZUMA. N.. AND WATANABE. S.. The Phusioloaist (1964). 19. AZUMA; N:, AND WATANABE, k., J. &ol. fihek., 24; 38k2 (1965). 2. KATZ, A. M., J. Biol. Chem., 239, 334 (1964)

Calcium Regulation in Squid Mantle and Scallop Adductor Muscles 1

. Biochem. 89, 581-589 (1981) Calcium Regulation in Squid Mantle and Scallop Adductor Muscles 1 Kunihiko KONNO,* Ken-ichi ARM,* Mikiharu YOSHIDA,** and Shizuo WATANABE*** Department of Food Science, Faculty