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The formation of filaments from barnacle myosin
Comp. Biochem. PhysioL, 1969, VoL 29, pp. 471 to 474. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION THE FORMATION OF FILAMENTS FROM BARNACLE MYOSIN R. J. BASKIN, W. C. SANFORD, P. D. M O R S E and M. L. B I G G S Department of Zoology, University of California, Davis, California (Received 26 August 1968)
Abstract--1. A method for the preparation of barnacle myosin has been developed. 2. The length of filaments formed from barnacle myosin has been measured and contrasted with rabbit myosin filaments. 3. Under similar ionic conditions, the length of filaments formed from barnacle myosin is smaller than the length of filaments formed from rabbit myosin. This is in contrast to the fact that, in vivo, the myosin-containing filaments from the barnacle are much larger than those from vertebrate striated muscle. INTRODUCTION FOLLOWING the description of large-diameter single muscle fibers in Balanus nubilis by Hoyle & Smyth (1963), another size variation in this animal has been recognized. Baskin & Wiese (1964), along with Hoyle et al. (1965), have shown the resting sarcomere length is much longer than in vertebrates, being up to 9/~ long. These sarcomeres contain the same array of thin and thick filaments as vertebrate striated muscle. The barnacle muscle filaments can be isolated using the same procedure that Huxley (1963) has applied to rabbit muscle in which filaments are relaxed from myofibril preparations with ATP, leaving the filaments suspended in the supernatant. Figure 1 shows a critical point dried and platinum-shadowed natural barnacle thick filament of 6.0/z length. These natural thick filaments vary from 4 to 6/~ length. Upon close examination a periodic surface roughness can be found on the thick filaments Zobel et aL (1967). In the rabbit themyosin-containing thick filament has an average length of 1-65/z. In view of this difference in native thick filament length we have explored some of the aggregation characteristics of extracted myosin from rabbit and barnacle muscle. We have attempted to obtain evidence to indicate whether the myosin molecule itself may regulate the length of filament formed. If this were the case, then barnacle myosin would then be expected to form longer filaments than rabbit myosin under identical conditions. Huxley (1963), as well as Noda & Ebashi (1960), found that lowering the ionic strength of myosin solutions to 0.15 or 0.20 caused an aggregation of the myosin 471
472
R. J. BASKIN,W. C. SANFORD,P. D. Morns ANY M. L. Btc,os
into filaments. Lowering the ionic strength by dialysis gives longer filaments than by direct dilution of myosin solutions. Josephs & H a r r i n g t o n (1966) have shown that p H , ionic strength and the nature of the ions present can alter the reconstituted myosin filament length. MATERIALS AND M E T H O D S We have utilized, a modified version of, the technique of Woods et al. (1963) to extract myosin from the freshly excised scutal-tergal depressor muscle of Balanus aquilia. Barnacle myosin was extracted by grinding the muscle in barnacle Guba-Straub solution composed of 0"4 M KCI, 0"15 M potassium PO4 buffer, pH 7"3, in a chilled Waring blender for 1 min at high speed. This homogenate was held at 4°C for 24 hr with constant agitation to extract actomyosin. It was then centrifuged at 14,500 rev/min for 30 min at 0°C to to remove cellular debris and the supematant diluted to 0"1 M ionic strength with glass-distilled water to precipitate the actomyosin. This was then spun at 3000 rev/min for 10 min on an International refrigerated centrifuge and the resulting pellet resuspended in 1"9 M KCI, 0.1 M potassium POt buffer, pH 7"0. ATP and MgClz, pre-dissolved in 1"9 M KCI and adjusted to pH 7.3, were added to give an end concentration of 20 mM ATP and 5 mM MgCI2 which separates the actin from the myosin. This mixture was then spun at 40,000 rev/min for 180 min on a Spinco model L preparative ultracentrifuge producing an actincontaining pellet and leaving the myosin in the supernatant. Purity of the barnacle myosin was investigated using a Spinco model E analytical refrigerated ultracentrifuge with Schlieren optics at a concentration of 0"2% myosin, 50 ° bar angle and 59,740 rev/min. Figure 3 shows the results obtained. The small side-peaks may be due to contaminating actomyosin, myosin degradation products, or myosin aggregates as described on Tuna myosin by Chung et al. (1967). It was not possible to concentrate the myosin by precipitation at low ionic strength and redissolve it in 2"0 M KCI as is readily done with rabbit myosin to remove actomyosin and other contaminating proteins, since the barnacle myosin is irreversibly denatured by this treatment. We isolated rabbit myosin (Fig. 2) utilizing a modified Szent-Gyorgyi (1960) technique, and the purity of our extract was visualized as a hypersharp peak on the Spinco model E ultracentrifuge at a speed of 59,740 rev/min, 70 ° bar angle and 0"4% myosin. Protein concentrations were determined by absorption at 280 mp for rabbit myosin and by the Folin-Ciocalteau technique on barnacle myosin because of the strong absorption due to the ATP present. Filaments were formed in all cases by dilution of the stock myosin solution from 0"6 M ionic strength to an end concentration of 0.2 M with cold 0"1 M standard salt solution. Constant brisk agitation was employed during dilution to avoid localized regions of low ionic strength. RESULTS Filaments were f o r m e d f r o m the barnacle myosin both with A T P present and f r o m myosin solutions containing 10 q M E D T A to chelate the M g 2+. Both barnacle and rabbit myosin were found to f o r m filaments over an ionic strength range of 0.2-0.125. Rabbit myosin maintained its normal filament formation ability for at least 2 weeks when stored at 4°C. With storage the barnacle myosin gives a higher percentage of thinner, smooth filaments of smaller diameter, which are probably L M M filaments f r o m degraded myosin. I n all cases of filament formation barnacle myosin consistently gave m o r e denatured masses of protein and fewer filaments than in the rabbit preparations. Figure 4A shows typical UO4Ac~ negative- and positive-stained reconstituted
SHORT COMMUNICATION THE FORMATION OF FILAMENTS FROM BARNACLE MYOSIN R. J. BASKIN, W. C. SANFORD, P. D. M O R S E and M. L. B I G G S Department of Zoology, University of California, Davis, California (Received 26 August 1968)
Abstract--1. A method for the preparation of barnacle myosin has been developed. 2. The length of filaments formed from barnacle myosin has been measured and contrasted with rabbit myosin filaments. 3. Under similar ionic conditions, the length of filaments formed from barnacle myosin is smaller than the length of filaments formed from rabbit myosin. This is in contrast to the fact that, in vivo, the myosin-containing filaments from the barnacle are much larger than those from vertebrate striated muscle. INTRODUCTION FOLLOWING the description of large-diameter single muscle fibers in Balanus nubilis by Hoyle & Smyth (1963), another size variation in this animal has been recognized. Baskin & Wiese (1964), along with Hoyle et al. (1965), have shown the resting sarcomere length is much longer than in vertebrates, being up to 9/~ long. These sarcomeres contain the same array of thin and thick filaments as vertebrate striated muscle. The barnacle muscle filaments can be isolated using the same procedure that Huxley (1963) has applied to rabbit muscle in which filaments are relaxed from myofibril preparations with ATP, leaving the filaments suspended in the supernatant. Figure 1 shows a critical point dried and platinum-shadowed natural barnacle thick filament of 6.0/z length. These natural thick filaments vary from 4 to 6/~ length. Upon close examination a periodic surface roughness can be found on the thick filaments Zobel et aL (1967). In the rabbit themyosin-containing thick filament has an average length of 1-65/z. In view of this difference in native thick filament length we have explored some of the aggregation characteristics of extracted myosin from rabbit and barnacle muscle. We have attempted to obtain evidence to indicate whether the myosin molecule itself may regulate the length of filament formed. If this were the case, then barnacle myosin would then be expected to form longer filaments than rabbit myosin under identical conditions. Huxley (1963), as well as Noda & Ebashi (1960), found that lowering the ionic strength of myosin solutions to 0.15 or 0.20 caused an aggregation of the myosin 471
472
R. J. BASKIN,W. C. SANFORD,P. D. Morns ANY M. L. Btc,os
into filaments. Lowering the ionic strength by dialysis gives longer filaments than by direct dilution of myosin solutions. Josephs & H a r r i n g t o n (1966) have shown that p H , ionic strength and the nature of the ions present can alter the reconstituted myosin filament length. MATERIALS AND M E T H O D S We have utilized, a modified version of, the technique of Woods et al. (1963) to extract myosin from the freshly excised scutal-tergal depressor muscle of Balanus aquilia. Barnacle myosin was extracted by grinding the muscle in barnacle Guba-Straub solution composed of 0"4 M KCI, 0"15 M potassium PO4 buffer, pH 7"3, in a chilled Waring blender for 1 min at high speed. This homogenate was held at 4°C for 24 hr with constant agitation to extract actomyosin. It was then centrifuged at 14,500 rev/min for 30 min at 0°C to to remove cellular debris and the supematant diluted to 0"1 M ionic strength with glass-distilled water to precipitate the actomyosin. This was then spun at 3000 rev/min for 10 min on an International refrigerated centrifuge and the resulting pellet resuspended in 1"9 M KCI, 0.1 M potassium POt buffer, pH 7"0. ATP and MgClz, pre-dissolved in 1"9 M KCI and adjusted to pH 7.3, were added to give an end concentration of 20 mM ATP and 5 mM MgCI2 which separates the actin from the myosin. This mixture was then spun at 40,000 rev/min for 180 min on a Spinco model L preparative ultracentrifuge producing an actincontaining pellet and leaving the myosin in the supernatant. Purity of the barnacle myosin was investigated using a Spinco model E analytical refrigerated ultracentrifuge with Schlieren optics at a concentration of 0"2% myosin, 50 ° bar angle and 59,740 rev/min. Figure 3 shows the results obtained. The small side-peaks may be due to contaminating actomyosin, myosin degradation products, or myosin aggregates as described on Tuna myosin by Chung et al. (1967). It was not possible to concentrate the myosin by precipitation at low ionic strength and redissolve it in 2"0 M KCI as is readily done with rabbit myosin to remove actomyosin and other contaminating proteins, since the barnacle myosin is irreversibly denatured by this treatment. We isolated rabbit myosin (Fig. 2) utilizing a modified Szent-Gyorgyi (1960) technique, and the purity of our extract was visualized as a hypersharp peak on the Spinco model E ultracentrifuge at a speed of 59,740 rev/min, 70 ° bar angle and 0"4% myosin. Protein concentrations were determined by absorption at 280 mp for rabbit myosin and by the Folin-Ciocalteau technique on barnacle myosin because of the strong absorption due to the ATP present. Filaments were formed in all cases by dilution of the stock myosin solution from 0"6 M ionic strength to an end concentration of 0.2 M with cold 0"1 M standard salt solution. Constant brisk agitation was employed during dilution to avoid localized regions of low ionic strength. RESULTS Filaments were f o r m e d f r o m the barnacle myosin both with A T P present and f r o m myosin solutions containing 10 q M E D T A to chelate the M g 2+. Both barnacle and rabbit myosin were found to f o r m filaments over an ionic strength range of 0.2-0.125. Rabbit myosin maintained its normal filament formation ability for at least 2 weeks when stored at 4°C. With storage the barnacle myosin gives a higher percentage of thinner, smooth filaments of smaller diameter, which are probably L M M filaments f r o m degraded myosin. I n all cases of filament formation barnacle myosin consistently gave m o r e denatured masses of protein and fewer filaments than in the rabbit preparations. Figure 4A shows typical UO4Ac~ negative- and positive-stained reconstituted