J. Biochem. 86, 663-673 (1979)
Kiyoyoshi N1SH1TA,* Takao OJIMA,* and Shizuo WATANABE** •The Department of Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, and **the Department of Chemistry, Faculty of Science, Tokyo Institute of Technology, Ookayama, Meguro-lcu, Tokyo 152 Received for publication, March 19, 1979
Myosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant Cr!j)jW) and the intrinsic viscosity, [ij\ of Akazara myosin thus purified were estimated to be 6.6 S and 2.10 dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of lightchain component (17,000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206,000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1 : 1 : 1 . (2) The EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin. Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum.
Among shell-fish muscles, the anterior byssus retractor muscle of Mytilus has frequently been used in physiological studies of muscular contraction (see 1-3). However, shell-fish myosins of good purity, adequate for biochemical studies, have been obtained only from the adductor muscle of scallop (4, 5) and from the foot muscle of abalone (6). In both scallop and abalone muscles, Abbreviations: DTNB, 5,5'-ditnio bis(2-nitrobenzoic acid); EGTA, ethyleneglycol bis(^-aminoethylether> yV.W-tetraacetate; DTT, dithiothreitol. Vol. 86, No. 3, 1979
663
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Myosin from Striated Adductor Muscle of Chlamys nipponensis akazara
calcium sensitivity was shown to be associated with myosin rather than with actin (7-10). We have been able to obtain myosin in a pure form from striated adductor muscle of Chlamys nipponensis akazara, and have characterized it to some extent, MATERIALS AND METHODS
Preparation of Myofibrils from Smooth and Striated Muscles of Chlamys nipponensis akazara— Muscles were cut from living Akazara, soaked quickly in an ice-cold solution of 50% glycerol
664
K. NISHTTA, T. OJIMA, and S. WATANABE
by centrifugation at 80,000xg for 1 h. The supernatant was diluted with 10 volumes of icecold water, and the myosin precipitates thus formed were collected by centrifugation at 8,000 Xg for 15 min, and redissolved in 0.6M KC1 and 5 mM ATP. The myosin solution was centrifuged at 100,000X0 for 90 min, removing insoluble sediments. Only the upper half of the supernatant (Fig. 3) was collected, and it was then dialyzed against 0.6 M KC1, 50 mM Tris-maleate (pH 7.0), and 1 mM DTT. Actin was prepared from rabbit skeletal muscle by the method of Spudich and Watt (12). The ATPase reaction was carried out at 25°C in the presence of 1 mM ATP and 25 mM Trismaleate buffer (pH 7.0), and it was stopped by adding perchloric acid (5%). Pi liberated was determined by Gomori's method (75), using pmethylaminophenol (Elon) as the reducing reagent. The ATPase activity was expressed in /imol Pi per min per mg of myosin. The protein concentration was determined by the biuret method Preparation of Myosin from Striated Adductor (70. Muscle of Akazara—Akazara myosin was prepared Sodium dodecyl sulfate (SDS)-gel electrofrom fresh striated adductors essentially by the phoresis was performed according to Weber and method used by Barany and Barany for scallop Osborn (75) using 5, 8, or 10% polyacrylamide myosin (4). All the solutions used in the prepa- gel rods. Coomassie Brilliant Blue (0.2%) or ration procedures described below contained 1 mM 0.5% Fast Green FCF was employed to stain the DTT (or 2mM 2-mercaptoethanol). Striated gel rods (8). Urea-gel electrophoresis was conadductor muscle (100 g) was cut from living ducted according to the original method of Perrie Akazara shell-fish, and homogenized in a Waring et al. (76). A recording densitometer (Shimadzu blender for 15 s with a washing medium (1 liter) CS-900 dual-wavelength scanner) was used to containing 40 mM KC1 and 10 mM potassium measure the density of Coomassie Brilliant Blue phosphate (pH 7.0). The homogenate was cen- or Fast Green FCF staining. trifuged to remove the supernatant, yielding A Spinco L5-65 ultracentrifuge was used to washed muscle residues. The washing was obtain schlieren patterns of myosin preparations, repeated twice more, and washed residues were and a Shimadzu recording spectrophotometer then extracted at 0°C for 15 min by gently stirring (UV-210A) was used to determine UV absorption with 1 liter of 0.6 M KC1 containing 10 mM potas- spectra of proteins. sium phosphate (pH 6.4), 1 mM ATP, and 2 mM 2-mercaptoethanol. The extract was obtained by RESULTS AND DISCUSSION centrifugation at 8,000 x g for 15 min, and diluted with ice-cold water (2 liters). Myosin precipitates 1. Myofibrils—Figure 1 shows the morthus formed were collected by centrifugation at phology of Akazara adductor myofibrils under a 8,000xg for 15 min, and redissolved in 0.6M phase-contrast microscope (Nikon SUR-Ke). KC1, 10 mM potassium phosphate (pH 7.0) and Smooth adductor myofibrils appeared swollen in 1 mM DTT. 0.1 M MgCl2 was added to the the medium we used here: 0 . 1 M KC1, 25 mM myosin solution, and the solution was stirred for Tris-HCl, pH 7.5. 15 min (see ref. 4). The precipitate formed (actin Myofibrils were dissolved in 1 % SDS and and a small amount of paramyosin) was removed 2 mM 2-mercaptoethanol. They were kept at J. Biochem.
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buffered with 40 mM sodium phosphate (pH 7.2), kept at 0°C for about 12 h, and then stored at — 15°C in the same glycerol solution for about a month. Myofibrils were prepared from the stored glycerinated fibers by applying the method of Perry and Grey (77). Glycerinated muscle fibers (10 g) were rinsed once with 0 . 1 M KC1 buffered with 25 mM Tris-HCl (pH 7.5) and homogenized with the same solution (50 ml) in a Waring blender for 15 s; homogenization was repeated eight times. The homogenate was then diluted with 0.1 M KC1 (50 ml), mixed thoroughly, and centrifuged at 600 xg for 15min, removing the supernatant. The washing with 0.1 M KC1 was repeated twice more. Muscle residues thus washed were again homogenized four times (15 s each) with 0.1 M KC1 (50 ml), and centrifuged at 400 x g for 3 min. This time, an upper layer of gluey fluid was obtained by decantation into a new centrifuge tube, and it was centrifuged again at 600x g for 15 min; the supernatant was removed, leaving the sedimented myofibrils.
665
MYOS1N FROM Chlamys nipponensis akazara
20°C for 1 h, and then analyzed by SDS-gel electrophoresis (Fig. 2). From the staining density measured at 600 nm, the relative contents of myosin (heavy chain plus light chains), paramyosin, actin, and tropomyosin were estimated, and are listed in Table I. The density of the total staining per gel rod was taken as 100%. Myofibrils applied to each gel rod (0.5x8 cm) contained 75 fig of protein. A control run with rabbit skeletal myofibrils was conducted in exactly the same way as the runs with Akazara adductor myofibrils. 2. Myosin—Judging from the SDS-gel electrophoretic patterns, Akazara myosin prepared as described in " MATERIALS AND METHODS" was usually in a highly purified form. However, we sometimes found it necessary to use DEAESephadex A50 in a batch method to remove minor impurities. DTT at 1 or 2 mM was present in all the solutions employed in the following procedures for the batch method. Akazara myosin was dissolved in 0.06 M sodium pyrophosphate buffer (pH 7.5). DEAE-Sephadex A50 was equilibrated in Vol. 86, No. 3, 1979
-Heavy chain (206,000) Paramyosin — (110,000)
-ActinU30OO) -Tropomyosin (35000) -A1 light chain(26,000) Light chain — (17,000)
-DTNB light chain (17,500) - A 2 light chain
(15500)
Fig. 2. SDS-polyacrylamide gel electrophoresis of Akazara and rabbit myofibrils. Eight percent gel rods (0.5x8 cm each) were used. Coomassie Brilliant Blue was employed to stain the gel rod. 1, Akazara striated adductor myofibrils (75 fig protein); 2, Akazara smooth adductor myofibrils (75 fig protein); 3, Rabbit skeletal myofibrils (75 fig protein).
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Fig. I. Myofibrils from striated (A) and smooth (B) adductor muscles of Chlamys nipponensis akazara. The myofibrils were suspended in 0.1 M K.CI, 0.25 M Tris-HCI (pH 7.5) and observed with a phase-contrast microscope (Nikon SUR-Ke type).
666
K. NISHITA, T. OJ1MA, and S. WATANABE
TABLE f. Protein composition of Akazara adductor myofibrils and rabbit skeletal myofibrils. Myofibrils were dissolved in sodium dodecyl sulfate, and analyzed by polyacrylamide gel electrophoresis (see Fig. 2). The content of each protein (myosin, i.e., heavy chain plus light chain, paramyosin, actin, and tropomyosin) was estimated simply from the staining density, and expressed as a percentage of the total staining density.
Myosin Paramyosin Actin Tropomyosin Others Investigators
Akazara striated 56 5.0 26
Akazara smooth
Rabbit skeletal
Rabbit skeletal
28
57
60
30
0
0
29
13
10
5.6
5.8
9
7.6
7.0
16
Present authors
Vertebrate smooth
4.5 15.5 Ebashi and Nonomura (17)
7. 1 ±0. 6 12.2+1.0 3. 3±0.2
Cohen and Murphy (18)
Heavy chain — ( 206.000 ) Paramyosin—r ( 110.000) Actin — 143,000)
Light chain — ( 17,000)
Fig. 3. SDS-polyacrylamide gel electrophoresis of the myosin and actin preparations used in the experiments reported in this paper. Akazara myosin before treatment with DEAE-Sephadex (batch method); 1, 25 fig and 2, 50 fig. AJcazara myosin after the treatment; 3, 25 fig and 4, 50 fig. Rabbit skeletal actin; 5, 25 fig and 6, 50 fig. Ten percent gel rods (0.5x8 cm each) were used.
Fig. 4. Sedimentation patterns of Akazara and scallop myosins. Ultracentrifugation was performed in 0.6 M KCI, 1 mM DTT, and 10 mM potassium phosphate, pH 7.0 at 10±0.1°C. Bar angle 72°; rotor speed 60,000 rpm. The pictures were taken 72 min after the rotor had reached maximum speed. (A) Akazara myosin, 4.0 mg/ml; (B) scallop myosin, 4.0 mg/ml.
As shown in Fig. 3, DEAE-Sephadex A50 in the batch method was useful to improve the purity of Akazara myosin preparations. Akazara myosin thus purified contained a single light-chain component of 17,000 daltons as determined by SDSadvance with the same buffer, and filtered through gel electrophoresis. Figure 4 shows the sedimentation pattern of a filter paper. DEAE-Sephadex on the filter paper (approximately 30 ml) was mixed with Akazara myosin compared with that of scallop Akazara myosin solution (70-100 mg per 10- myosin prepared from striated adductor muscle 20 ml), stirred for 30 min (0°C), and then filtered of scallop by the procedures described for the through a filter paper. Using the same procedures, preparation of Akazara myosin. Preparations of DEAE-Sephadex on the filter paper was washed Akazara myosin gave a single sharp peak, having first with 0.06 M KCI-0.06 M sodium pyrophos- a sedimentation coefficient (s^ w) of 6.6S. On phate, pH7.5, and then with 0.15 M KCI-0.06 M the other hand, preparations of scallop myosin sodium pyrophosphate, pH 7.5. DEAE-Sephadex gave two peaks: one major peak and one minor thus washed was extracted with 3—4 ml of 0.5 M peak which is probably an aggregated form of KCI buffered with 0.06 M sodium pyrophosphate, myosin (cf. ref. 4 and 8). The intrinsic viscosity, pH 7.5, for a few hours with occasional stirring. [jj], of Akazara myosin was 2.10 dl/g at 10°C in a /. Biochem.
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Protein
MYOSIN FROM Chlamys nipponensis akazara
667
0.6 E c o
0.1.
cw o
c o
CO
oil
0.2-
(J O o
o 20
40 60 Tube Number
80
Fig. 5. Ion-exchange chromatography of Akazara myosin on DEAE-Sephadex A50. Eighty mg of the myosin in 0.06 M sodium pyrophosphate, pH 7.5, was applied to a 1.9 x 15 cm column and eluted with a linear gradient of KCI in 0.06 M sodium pyrophosphate, pH 7.5. The flow rate was lOml/h. Fraction size was 10 ml. All the solutions used here contained 1 mM DTT. (O) Absorbance at 280 nm; ( x ) absorbance at 260 nm; ( A ) Ca-ATPase activity. medium containing 0.6 M KCI, 10 mM potassium phosphate buffer (pH 7.0), and 2 mM 2-mercaptoethanol. These values of and [rj] are in good agreement with those of scallop myosin reported by Szent-Gyorgyi et al. (8). The UV absorption spectrum of Akazara myosin was that of a typical protein, and the extinction coefficient measured at 280 nm in 0.6 M KCI buffered with 10 mM potassium phosphate, pH 7.0 was 7.92 for 10 mg/ml Akazara myosin. The sulfhydryl content measured in 7 M urea by Ellman's method {19) was 38 mol per 500,000 g of Akazara myosin. Column chromatography can be used in place of the batch method for improving the purity of Akazara myosin preparations. Figure 5 shows a chromatogram of a preparation of Akazara myosin. A major component was eluted with 0.07-0.12 M KCI in 0.06 M sodium pyrophosphate, pH 7.5. In this particular case, the Ca-ATPase activity of Akazara myosin (/imol per min per mg of myosin) was 2.8 for the eluted fraction as compared with 2.6 for the preparation before the chromatography. The activity was measured at Vol. 86, No. 3, 1979
25°C in 5 mM CaCl,, 1 mM ATP, 0.06 M KCI, and 25 mM Tris-maleate, pH 7.0. 3. A TPase Activity of Myosin—The ATPase activity of Akazara myosin in the steady state was measured, and the results obtained are summarized in Table II. The Ca-ATPase activity of Akazara myosin was high, approximately 2-4 times those of scallop adductor and rabbit skeletal myosins. The EDTA-ATPase activity is higher (2-3 times) than that of scallop myosin but lower (approximately one-half) than that of rabbit skeletal myosin. The Mg-ATPase activity of Akazara myosin was higher than those of scallop myosin and rabbit skeletal myosin. 4. Calcium Sensitivity—According to Kendrick-Jones et al. (7), calcium regulation of scallop adductor actomyosin is myosin-linked, and it is lost when a light-chain component of scallop myosin is removed by washing with EDTA. Our preparations of Akazara myosin, when combined with skeletal pure actin (free from troponin and tropomyosin), were found to be sensitive to calcium ions, thus indicating that a calcium regulatory system is associated with Akazara myosin. Figure 6
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UD
K. NISHITA, T. OJIMA, and S. WATANABE
668
TABLE II. ATPase activity of myosins. The reaction was carried out at 25°C in a medium containing 1 ITIM ATP, 25 ITIM Tris-maleate, pH 7.0, KCI at the concentration indicated in the left-hand column, and one of the additions indicated in the upper line.
0.012 0.06 0.6
a
Akazara
5 HIM CaCI,
1 iriM MgCU
0.030
4.40
0.53
1 mM EDTA —
Scallop
0.036
1.08
0.13
—
Akazara
0.068
3.50
0.45
—
Scallop
0.065
1.45
0.13
Akazara
0.24
2.10
0. 14
0.68
Scallop
0.21
0.96
0.06
0.24
—
0.6
Scallop'
—
0.26
0.09
0.07
0.6
Gizzard b
—
0.13
0.003
0.35
0.6
Skeletal"
—
0.27
0.004-0. 025
1.3
Barany and Barany (4).
_
None
b
Onishi el al. (20).
1.25
100-
1.00-
+EGTA
0.75 Q_
zo0-25-
0
4 8 12 Incubation Time (min) Fig. 7. Effect of adding EGTA on the actin-activated 0 05 1.0 ATPase reaction in the presence of calcium ions. Pi Actin/Myosin (w/w) liberation was measured in a medium containing Fig. 6. Actin activation of Akazara myosin in the 30 mM KCI, 20 mM Tris-maleate (pH 6.8), 2 mM MgCI,, presence of calcium ions. ATPase activity was 0.67 mM ATP, 0.04 mg/ml Akazara myosin, 0.013 mg/ml measured in a medium containing 12 mM KCI, 25 mM rabbit skeletal actin, and 0.1 mM CaCI, ((},•) or Tris-maleate, pH 7.0, 1 mM MgCI,, I mM ATP, and 0.5 ITIM EGTA (A, A ) . 0.17 mM EGTA was added 0.1 mM CaCI, or 0.5 mM EGTA at 25°C. ( • ) In the 7 min after the addition of ATP (arrows marked presence of CaCI,; (O) in the presence of EGTA. + EGTA). Preincubation was carried out in 0.18 M KCI, 25 mM Tris-maleate, pH 7.5, 5 mM MgCI,, 1.44 shows that Mg-ATPase of Akazara myosin was mg/ml Akazara myosin, 7 mM ATP, 0.7 mM CaCI, or activated by skeletal actin in the presence of Ca l+ EGTA (total volume 5.0 ml). After prcincubation for 1 h at 20°C, the myosin was precipitated by dilution and not in its absence. Moreover, as shown in Fig. 7, the actin-activated ATPase reaction in the with 10 volumes of IOITIM Tris-maleate, 2 mM MgCI,, presence of Ca1+ was suppressed by subsequent pH 7.0. Tlie myosin was collected by centrifugation at 20,000 Xff for 15 min, and then dissolved in 0.6 M addition of EGTA (to remove Ca1+). This was KCI, 0.05 M Tris-maleate, pH 7.0. true regardless of whether Akazara myosin was J. Biochem.
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Addition Omol Pi/min/mg myosin)
KCI (M)
669
MYOSFN FROM Chlamys nipponensis akazara
slightly decreased, thus reducing the calcium sensitivity. Figure 9A shows that the staining density of the 17,000 dalton light-chain band in SDS-gel electrophoresis was gradually decreased as the EDTA washing was repeated. The density ratio of the light-chain band to the heavy-chain band was estimated, and is shown in Fig. 8. In the particular series of experiments shown in Fig. 8, the density ratio reached a minimum value of approximately 0.08 after 6 or 7 washings, and complete loss of calcium sensitivity (O, • ) occurred concurrently. Therefore, it appears that the calcium sensitivity of Akazara muscle is
Heavy chain (206,000) y (11O000)
•
0.2
1 2
-Light chain (17,000) (Chain weight)
5 6
0.1
.. 0
o &
2 4 6 Number of EDTA Washings
Fig. 8. Desensitizing effect of EDTA washing on the Mg-ATPase activity of rabbit acto-Akazara myosin. Akazara myosin was washed with a solution containing 10 min EDTA, 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT. The myosin concentration during EDTA washing was maintained at about 4 mg/ml. SDS-gel electrophoresis was carried out (see Fig. 9) to determine the ratio of staining density ( x ) of the light chain component (17,000 daltons) to the heavy chain component (206,000 daltons), and (A) of the EDTA-light chain component to the SH-light chain component (17,000 daltons). The ATPase activity was measured at 25°C in reaction medium containing rabbit skeletal actin (0.1 mg/ml), Akazara adductor myosin (0.2 mg/ml), 30 mM KCI, 2 mM MgCI,, 20 mM Trismaleate of pH 6.8, 0.5 mM ATP, and 0.1 mM CaCIi ( # ) or 0.5 mM EGTA (O).
Vol. 86, No. 3, 1979
-EDTA-light chain -SH-light chain
1 2
3
L
Fig. 9. Removal of EDTA-light chains from Akazara myosin. (A) SDS-gel electrophoresis: Eight percent polyacrylamide gel rods (0.5x8 cm) were used. I, Unwashed myosin; 2, washed once; 3, washed twice; 4, washed four times; 5, washed seven times; 6, washed fifteen times. Forty-five fig of myosin was applied to each gel. (B) Urea-gel electrophoresis: Polyacrylamide (1.5%) gel rods (0.5x8 cm each) containing 8 M urea were used. Samples 1-6 are the same as those indicated in (A). One hundred ng of myosin was applied to each gel.
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preincubated with ATP in the presence or absence of Ca ! + . Therefore, the actin activation does not seem to involve phosphorylation of light-chain components, as reported for gizzard myosin (Ikebe et a/. (21)). 5. Desensitization of Myosin—Akazara myosin was washed essentially in the same way as described by Szent-Gyorgyi et al. (8, 9) for scallop myosin: myosin was suspended in a solution containing 10 mM EDTA, 40 mM NaCl, 5 mM potassium phosphate, pH 7.0 and 0.5 mM DTT, stirred for lOmin at 0-5°C, and then centrifuged to obtain precipitates of EDTA-washed myosin. Combining the EDTA-washed myosin of Akazara adductor with rabbit skeletal actin (in a weight ratio of 2 : 1 ) , the Mg-ATPase activity was measured in the presence of 0.1 mM CaCI2 or 0.5 mM EGTA. Figure 8 shows that as the number of EDTA washes increased, the MgATPase activity of rabbit acto-Akazara myosin in the absence of Ca ! + (i.e. in the presence of EGTA) increased, whereas that in the presence of Ca 5+
K. NISHITA, T. OJIMA, and S. WATANABE
670
TABLE III. Desensitization of Akazara myosin by washing with EDTA in the presence and absence of DTT. Akazara adductor myosin was washed at 0-5°C with 10 mM EDTA in the presence of 0.5 mM DTT or in its absence. The washed myosin was then combined with rabbit skeletal actin in a weight ratio of 2 : I, and the Mg-ATPase activity of the combined system was measured in the presence of 0.1 mM CaCl, or 0.5 mM EGTA. The calcium sensitivity was expressed in terms of (I —activity in EGTA/activity in CaCl,) x 100 (%). Number of EDTA washes 0 1 2 4 7
Ca sensitivity (%) Washed without DTT
Washed in 0.5 mM DTT
62.9 54.4 39.2 18.9 12.6
64.0 50.4 46.9 23.1 12.9
100 g of Akazara striated muscle, and suspended in 2 liters of an extracting solution containing 10 mM EDTA, 40 mM NaCl, and 5 mM potassium phosphate, pH 7.0. The suspension was stirred at 4°C for 30 min, and then centrifuged at 8,000 XQ for 15 min. The supernatant was collected, and the residue was extracted again in a similar manner. The supernatant thus obtained was combined and 0.25 mM DTT was added. DEAESephadex A50 (approximately 40 ml), which had been pre-equilibrated with 25 mM potassium phosphate, pH 7.0, and 0.25 mM DTT and allowed to settle in a cylinder, was added. The mixture was stirred for 1 h at 4°C, so that EDTA-light chains would be adsorbed on the DEAE-Sephadex. The mixture was then filtered through a filter paper, and the Sephadex on the filter paper was washed twice with 50 ml of 0 . 1 5 M potassium phosphate, pH 7.0. The washed Sephadex was then extracted with 20 ml of 0.5 M potassium phosphate, pH 7.0, and the extract was made up to 4 5 % saturation with saturated ammonium sulfate solution (containing 0.5 mM DTT). The precipitates thus formed (mainly of actin, myosin, and paramyosin) was removed by centrifugation, and additional ammonium sulfate was added to give 75% saturation. The precipitate formed was sedimented by centrifugation, dissolved in 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT, and then dialyzed against the same solution. The yield of this preparation of Akazara EDTA-light chains was approx. 20-30 mg/lOOg myofibrils. Gel electrophoretic patterns of this preparation of Akazara EDTA-light chain are shown in Fig. 11 A, 2 and 4. SH-light chain was also prepared from Akazara myosin. Dissociation of SH-light chains from the myosin with 6 M urea and the removal of myosin heavy chain by the addition of ethanol and centrifugation were performed by the method of Kendrick-Jones et al. (9). The supernatant thus obtained was diluted with 10 volumes of cold distilled water, followed by DEAE-Sephadex A50 chromatography and ammonium sulfate fractionaxion as described for the preparation of EDTA-light chains. Finally, the SH-light chains were subjected to urea-gel electrophoresis in order to eliminate contaminating EDTA-light chains. Gel-electrophoretic patterns of this preparation of SH-light chain are shown in Figs. 11 A, 3 and 5.
/ . Biochem.
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also myosin-linked and that the EDTA-light chain component is responsible for the sensitivity. Moreover, as shown in Fig. 9B, the 17,000 dalton light chain was separated into two bands in ureagel electrophoresis, and only one of the two bands was removed by EDTA washing, causing a complete loss of calcium sensitivity (Fig. 8). These properties of Akazara myosin are practically identical with those reported for scallop myosin (9). However, we found one important difference from scallop myosin: not half the amount of EDTA-light chains but the total amount had to be removed to achieve complete loss of calcium sensitivity (7 washes; Fig. 8, see also Fig. 9B-5). In the experiments shown in Figs. 8 and 9, EDTA washing was conducted in the presence of 0.5 mM DTT. On the other hand, KendrickJones et al. (9) reported that DTNB treatment can facilitate the removal of EDTA-light chain from scallop myosin. Thus, EDTA washes were carried out in the absence of DTT, and the loss of calcium sensitivity was estimated. As shown in Table I IF, the absence of DTT did not enhance the loss of calcium sensitivity as long as the washing was conducted at low temperature. 6. Light chains—Isolation of the EDTAlight chain component from myofibrils was conducted essentially by the method of KendrickJones et al. (9). Myofibrils were prepared from
MYOSIN FROM Chlamys nipponcnsis akazara
671
(A)
myosin practically free from EDTA-light chains (see Fig. 9) and purified preparations of Akazara EDTA-light chains, we examined whether or not the resensitization is also incomplete with our preparations from Akazara adductors. Recombination of the isolated EDTA-light chains with EDTA-washed myosin was performed by the following procedure. One ml of "desensitized" (washed five times with EDTA) Akazara myosin solution (1.5-2.0 mg/ml in 0.6 M KC1, 4 mM MgCU, 0.05 M Tris-maleate, pH 7.0, and 0.5 mM DTT) and two ml of the EDTA-light chain solution (0.3-0.9 mg/ml in 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT) were mixed, and dialyzed overnight against 0.3 or 0.6 M KC1, 0.05 M Tris-maleate, pH 7.0, 2 mM MgCl,, and 0.5 mM DTT. The dialyzed solution was diluted with 14 volumes of ice-cold 2 mM MgCl, solution, and the precipitate formed was collected by centrifugation. It was redissolved in 0.6 M K.CI and 0.5 mM DTT and the solution was cleared by centrifugation. The cleared solution was diluted once more with 10 volumes of
(B)
(C)
0.8H 2-
2
0
•Ca*Eg 240
260
280
300
Wavelength (nm)
320
250
270 290 310 330 Wavelength (nm)
Fig. 10. (A) UV absorption spectra of Akazara light chains. Concentrations of EDTA-light chains and of SH-light chains were 3.8 mg/ml and 1.4 mg/ml, respectively. They were dissolved in 40 mM potassium phosphate, pH 7.0, and 1 mM DTT: ( ) EDTA-light chains; ( ) SH-light chains. (B) UV absorption difference spectra of Akazara light chains. Concentrations of EDTA-light chains and of SH-light chains were the same as in (A). 1 mM EGTA was present in the reference cell and 1 mM CaCI, in the sample cell: ( ) EDTA-light chain; ( ) SH-light chain; ( ) before addition of CaCI,. (C) Disc-gel electrophoretic pattern of EDTA-light chains: 7.5% polyacrylamide gel rods (0.5x8 cm each) and 50 mM Tris-glycine buffer, pH 8.3 were used. +Ca: In the presence of I mM CaCI,. +Eg: In the presence of 1 mM EGTA. Vol. 86, No. 3, 1979
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The UV absorption spectra of Akazara EDTAlight chains and SH-light chains (Fig. 10A) were very similar to those of the corresponding light chains from scallop adductors (see ref. 9). Nevertheless, we were able to obtain a calcium-induced difference spectrum of Akazara EDTA-light chains (Fig. 10B): this is in contrast to the report of Kendrick-Jones et al. (9) that the UV absorption spectrum of scallop EDTA-light chains was not changed on addition of calcium ions. Moreover, we found (Fig. IOC) that calcium ions cause changes in the disc electrophoretic patterns of Akazara EDTA-light chains, suggesting that at least a portion of Akazara EDTA-light chains binds with calcium ions (22). However, no clear evidence for Ca2+ binding to Akazara SH-light chains was obtained. 7. Resensitization—Kendrick-Jones et al. (9) reported that removal of more than half the total amount of EDTA-light chains from scallop myosin resulted in incomplete resensitization of desensitized myosin on re-adding EDTA-light chains. Since we obtained desensitized Akazara
672
ice-cold 2 mM MgCI,, and the precipitate formed was collected by centrifugation. As shown in Fig. 12 (see also Fig. 11B, 4) EDTA-light chains rccombincd with desensitized Akazara myosin, showing EDTA-light chain and SH-light chain bands in approximately equal amounts. Moreover, resensitization was nearly complete: approximately 80% of the lost calcium sensitivity was recovered on mixing EDTA-light chains and desensitized myosin in a molar ratio of 2 : 1. It can therefore be concluded that, unlike the case with scallop adductor myosin reported by Kendrick-Jones et a/. (9), EDTA-light chains can be completely removed from and fully reintegrated in Akazara adductor myosin. We should add that these studies with Akazara adductor myosin were motivated by similar findings with myosins from other sources: calcium ions induce a UV absorption difference spectrum of squid mantle EDTA-light chains (K. Konno, K. Arai, & S. Watanabe, unpublished data), and EDTA-light chains can be completely removed from and fully reintegrated in clam foot myosin (T. Asada, G. Ashiba, & S. Watanabe, 23).
0 0
1 2 5 10 EDTA-LC(odded)/Desensitized Myosin (Molar Ratio)
Fig. 12. Resensitization of desensitized Akazara myosin by isolated EDTA-light chain preparations. Desensitized Akazara myosin (4.0 mg in 0.6 M KCI, 0.05 M Tris-maleate, pH 7.0, 2 mM MgCI,, and 0.5 mM DTT) and various amounts of Akazara EDTA-light chains (0.96 mg/ml in 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT) were mixed, and rabbit skeletal actin (2.0 mg in 0.6 M KCI and 0.05 M Trismaleate, pH 7.0) was added to the mixture. The ATPase activity of the combined system was measured in a medium containing 30 mM KCI, 20 mM Tris-maleate, pH 6.8, 2 mM MgCU, 0.5 mM ATP, in the presence of 0.1 mM CaCI, or 0.5 mM EGTA. Ca sensitivity is as defined in Table III: it was 50% ( • ) for the unwashed myosin we employed. MW: Desensitized myosin, 450,000; EDTA-light chain, 17,000. ( • ) +Ca; (O) + EGTA; ( A ) unwashed myosin, +Ca; ( A ) unwashed myosin, +EGTA. Urea-gel electrophoretic patterns: ratio of EDTA-light chains/desensitized myosin, a = 0 , b = 2, c-»10, and d = unwashcd myosin.
The authors wish to thank Dr. K. Arai and Mr. K. Konno, Faculty of Fisheries, Hokkaido University for valuable advice and discussions. The authors also wish to thank Mr. H. Yokota for his technical assistance in preparing Akazara myosin. REFERENCES 1. Twarog, B.M. (1954) / . Cell. Comp. Physiol. 44, 141-163 2. Johnson, W.H., Kahn, J.S., & Szent-Gydrgyi, A.G. (1959) Science 130, 160-161 3. Twarog, B.M. (1967) J. Gen. Physiol. 50, 157-168 4. Barany, M. & Barany, K. (1966) Biochem. Zeir. 345, 37-56 J. Biochem.
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' -> 3 L 5 1 2 Fig. 11. Recombination of EDTA-light chains with desensitized Akazara myosin. (A) Preparations; 1. Desensitized Akazara myosin (45 fig); 2, Akazara EDTA-light chains (50 fig); 3, Akazara SH-light chains (80 fig); 4, Akazara EDTA-light chains (60 fig); 5. Akazara SH-light chains (50 fig). 1-3, SDS-gel electrophoresis (8% gel rods); 4-5, urea-gel electrophoresis (7-5% gel rods). (B) Recombination: 1, Unwashed myosin (control) (150 fig); 2, desensitized myosin (150 fig); 3, Akazara EDTA-light chains (30 fig); 4, recombined Akazara myosin with Akazara EDTA-light chains (100 fig). Urea-gel electrophoresis (7.5% gel rods).
K. NISHITA, T. OJIMA, and S. WATANABE
MYOSIN FROM Chlamys nipponensis akazara
Vol. 86, No. 3, 1979
16. Perrie, W.T., Smillie, L.B., & Perry, S.V. (1973) Biochem. J. 135, 151-164 17. Ebashi, S. & Nonomura, Y. (1973) in The Structure and Function of Muscle (Bourne, G.H., ed.) Vol. 3, pp. 286-362, Academic Press, New York 18. Cohen, D.M. & Murphy, R.A. (1977) Fed. Proc. 36,602 19. Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77 20. Onishi, H., Yamada, Y., Ikebe, M., Suzuki, H., & Watanabe, S. (1978) / . Biochem. 83, 129-135 21. Ikebe, M., Aiba, T., Onishi, H., & Watanabe, S. (1978) / . Biochem. 83, 1643-1655 22. Gaffin, S.L. & Oplatka, A. (1974) /. Biochem. 75, 277-281 23. Asada, T., Ashiba, G., & Watanabe, S. (1979) /. Biochem. 85, 1543-1546
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5. Szent-Gydrgyi, A.G., Cohen, C , & Kendrick-Jones, J. (1971) / . Mol. Biol. 56, 239-258 6. Azuma, N., Asakura, A., & Yagi, K. (1975) / . Biochem. 77, 973-981 7. Kendrick-Jones, J., Lehman, W., & Szent-Gyorgyi, A.G. (1970) / . Mol. Biol. 54, 313-326 8. Szent-Gy6rgyi, A.G., Szentkiralyi, E.M., & Kendrick-Jones, J. (1973) / . Mol. Biol. 74, 179-203 9. Kendrick-Jones, J., Szentkiralyi, E.M., & SzentGyorgyi, A.G. (1976) / . Mol. Biol. 104, 747-775 10. Azuma, N. (1976) / . Biochem. 80, 187-189 11. Perry, S.V. & Grey, T.C. (1956) Biochem. J. 64, 184-192 12. Spudich, J.A. & Watt, S. (1971) / . Biol. Chem. 246, 4866-^*871 13. Gomori, G. (1942) /. Lab. Clin. Med. 11, 955-960 14. Gornall, A.G., Bardawill, C.S., & David, M.M. (1949) / . Biol. Chem. 177, 751-766 15. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406-4412
673
Kiyoyoshi N1SH1TA,* Takao OJIMA,* and Shizuo WATANABE** •The Department of Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, and **the Department of Chemistry, Faculty of Science, Tokyo Institute of Technology, Ookayama, Meguro-lcu, Tokyo 152 Received for publication, March 19, 1979
Myosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant Cr!j)jW) and the intrinsic viscosity, [ij\ of Akazara myosin thus purified were estimated to be 6.6 S and 2.10 dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of lightchain component (17,000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206,000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1 : 1 : 1 . (2) The EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin. Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum.
Among shell-fish muscles, the anterior byssus retractor muscle of Mytilus has frequently been used in physiological studies of muscular contraction (see 1-3). However, shell-fish myosins of good purity, adequate for biochemical studies, have been obtained only from the adductor muscle of scallop (4, 5) and from the foot muscle of abalone (6). In both scallop and abalone muscles, Abbreviations: DTNB, 5,5'-ditnio bis(2-nitrobenzoic acid); EGTA, ethyleneglycol bis(^-aminoethylether> yV.W-tetraacetate; DTT, dithiothreitol. Vol. 86, No. 3, 1979
663
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Myosin from Striated Adductor Muscle of Chlamys nipponensis akazara
calcium sensitivity was shown to be associated with myosin rather than with actin (7-10). We have been able to obtain myosin in a pure form from striated adductor muscle of Chlamys nipponensis akazara, and have characterized it to some extent, MATERIALS AND METHODS
Preparation of Myofibrils from Smooth and Striated Muscles of Chlamys nipponensis akazara— Muscles were cut from living Akazara, soaked quickly in an ice-cold solution of 50% glycerol
664
K. NISHTTA, T. OJIMA, and S. WATANABE
by centrifugation at 80,000xg for 1 h. The supernatant was diluted with 10 volumes of icecold water, and the myosin precipitates thus formed were collected by centrifugation at 8,000 Xg for 15 min, and redissolved in 0.6M KC1 and 5 mM ATP. The myosin solution was centrifuged at 100,000X0 for 90 min, removing insoluble sediments. Only the upper half of the supernatant (Fig. 3) was collected, and it was then dialyzed against 0.6 M KC1, 50 mM Tris-maleate (pH 7.0), and 1 mM DTT. Actin was prepared from rabbit skeletal muscle by the method of Spudich and Watt (12). The ATPase reaction was carried out at 25°C in the presence of 1 mM ATP and 25 mM Trismaleate buffer (pH 7.0), and it was stopped by adding perchloric acid (5%). Pi liberated was determined by Gomori's method (75), using pmethylaminophenol (Elon) as the reducing reagent. The ATPase activity was expressed in /imol Pi per min per mg of myosin. The protein concentration was determined by the biuret method Preparation of Myosin from Striated Adductor (70. Muscle of Akazara—Akazara myosin was prepared Sodium dodecyl sulfate (SDS)-gel electrofrom fresh striated adductors essentially by the phoresis was performed according to Weber and method used by Barany and Barany for scallop Osborn (75) using 5, 8, or 10% polyacrylamide myosin (4). All the solutions used in the prepa- gel rods. Coomassie Brilliant Blue (0.2%) or ration procedures described below contained 1 mM 0.5% Fast Green FCF was employed to stain the DTT (or 2mM 2-mercaptoethanol). Striated gel rods (8). Urea-gel electrophoresis was conadductor muscle (100 g) was cut from living ducted according to the original method of Perrie Akazara shell-fish, and homogenized in a Waring et al. (76). A recording densitometer (Shimadzu blender for 15 s with a washing medium (1 liter) CS-900 dual-wavelength scanner) was used to containing 40 mM KC1 and 10 mM potassium measure the density of Coomassie Brilliant Blue phosphate (pH 7.0). The homogenate was cen- or Fast Green FCF staining. trifuged to remove the supernatant, yielding A Spinco L5-65 ultracentrifuge was used to washed muscle residues. The washing was obtain schlieren patterns of myosin preparations, repeated twice more, and washed residues were and a Shimadzu recording spectrophotometer then extracted at 0°C for 15 min by gently stirring (UV-210A) was used to determine UV absorption with 1 liter of 0.6 M KC1 containing 10 mM potas- spectra of proteins. sium phosphate (pH 6.4), 1 mM ATP, and 2 mM 2-mercaptoethanol. The extract was obtained by RESULTS AND DISCUSSION centrifugation at 8,000 x g for 15 min, and diluted with ice-cold water (2 liters). Myosin precipitates 1. Myofibrils—Figure 1 shows the morthus formed were collected by centrifugation at phology of Akazara adductor myofibrils under a 8,000xg for 15 min, and redissolved in 0.6M phase-contrast microscope (Nikon SUR-Ke). KC1, 10 mM potassium phosphate (pH 7.0) and Smooth adductor myofibrils appeared swollen in 1 mM DTT. 0.1 M MgCl2 was added to the the medium we used here: 0 . 1 M KC1, 25 mM myosin solution, and the solution was stirred for Tris-HCl, pH 7.5. 15 min (see ref. 4). The precipitate formed (actin Myofibrils were dissolved in 1 % SDS and and a small amount of paramyosin) was removed 2 mM 2-mercaptoethanol. They were kept at J. Biochem.
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buffered with 40 mM sodium phosphate (pH 7.2), kept at 0°C for about 12 h, and then stored at — 15°C in the same glycerol solution for about a month. Myofibrils were prepared from the stored glycerinated fibers by applying the method of Perry and Grey (77). Glycerinated muscle fibers (10 g) were rinsed once with 0 . 1 M KC1 buffered with 25 mM Tris-HCl (pH 7.5) and homogenized with the same solution (50 ml) in a Waring blender for 15 s; homogenization was repeated eight times. The homogenate was then diluted with 0.1 M KC1 (50 ml), mixed thoroughly, and centrifuged at 600 xg for 15min, removing the supernatant. The washing with 0.1 M KC1 was repeated twice more. Muscle residues thus washed were again homogenized four times (15 s each) with 0.1 M KC1 (50 ml), and centrifuged at 400 x g for 3 min. This time, an upper layer of gluey fluid was obtained by decantation into a new centrifuge tube, and it was centrifuged again at 600x g for 15 min; the supernatant was removed, leaving the sedimented myofibrils.
665
MYOS1N FROM Chlamys nipponensis akazara
20°C for 1 h, and then analyzed by SDS-gel electrophoresis (Fig. 2). From the staining density measured at 600 nm, the relative contents of myosin (heavy chain plus light chains), paramyosin, actin, and tropomyosin were estimated, and are listed in Table I. The density of the total staining per gel rod was taken as 100%. Myofibrils applied to each gel rod (0.5x8 cm) contained 75 fig of protein. A control run with rabbit skeletal myofibrils was conducted in exactly the same way as the runs with Akazara adductor myofibrils. 2. Myosin—Judging from the SDS-gel electrophoretic patterns, Akazara myosin prepared as described in " MATERIALS AND METHODS" was usually in a highly purified form. However, we sometimes found it necessary to use DEAESephadex A50 in a batch method to remove minor impurities. DTT at 1 or 2 mM was present in all the solutions employed in the following procedures for the batch method. Akazara myosin was dissolved in 0.06 M sodium pyrophosphate buffer (pH 7.5). DEAE-Sephadex A50 was equilibrated in Vol. 86, No. 3, 1979
-Heavy chain (206,000) Paramyosin — (110,000)
-ActinU30OO) -Tropomyosin (35000) -A1 light chain(26,000) Light chain — (17,000)
-DTNB light chain (17,500) - A 2 light chain
(15500)
Fig. 2. SDS-polyacrylamide gel electrophoresis of Akazara and rabbit myofibrils. Eight percent gel rods (0.5x8 cm each) were used. Coomassie Brilliant Blue was employed to stain the gel rod. 1, Akazara striated adductor myofibrils (75 fig protein); 2, Akazara smooth adductor myofibrils (75 fig protein); 3, Rabbit skeletal myofibrils (75 fig protein).
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Fig. I. Myofibrils from striated (A) and smooth (B) adductor muscles of Chlamys nipponensis akazara. The myofibrils were suspended in 0.1 M K.CI, 0.25 M Tris-HCI (pH 7.5) and observed with a phase-contrast microscope (Nikon SUR-Ke type).
666
K. NISHITA, T. OJ1MA, and S. WATANABE
TABLE f. Protein composition of Akazara adductor myofibrils and rabbit skeletal myofibrils. Myofibrils were dissolved in sodium dodecyl sulfate, and analyzed by polyacrylamide gel electrophoresis (see Fig. 2). The content of each protein (myosin, i.e., heavy chain plus light chain, paramyosin, actin, and tropomyosin) was estimated simply from the staining density, and expressed as a percentage of the total staining density.
Myosin Paramyosin Actin Tropomyosin Others Investigators
Akazara striated 56 5.0 26
Akazara smooth
Rabbit skeletal
Rabbit skeletal
28
57
60
30
0
0
29
13
10
5.6
5.8
9
7.6
7.0
16
Present authors
Vertebrate smooth
4.5 15.5 Ebashi and Nonomura (17)
7. 1 ±0. 6 12.2+1.0 3. 3±0.2
Cohen and Murphy (18)
Heavy chain — ( 206.000 ) Paramyosin—r ( 110.000) Actin — 143,000)
Light chain — ( 17,000)
Fig. 3. SDS-polyacrylamide gel electrophoresis of the myosin and actin preparations used in the experiments reported in this paper. Akazara myosin before treatment with DEAE-Sephadex (batch method); 1, 25 fig and 2, 50 fig. AJcazara myosin after the treatment; 3, 25 fig and 4, 50 fig. Rabbit skeletal actin; 5, 25 fig and 6, 50 fig. Ten percent gel rods (0.5x8 cm each) were used.
Fig. 4. Sedimentation patterns of Akazara and scallop myosins. Ultracentrifugation was performed in 0.6 M KCI, 1 mM DTT, and 10 mM potassium phosphate, pH 7.0 at 10±0.1°C. Bar angle 72°; rotor speed 60,000 rpm. The pictures were taken 72 min after the rotor had reached maximum speed. (A) Akazara myosin, 4.0 mg/ml; (B) scallop myosin, 4.0 mg/ml.
As shown in Fig. 3, DEAE-Sephadex A50 in the batch method was useful to improve the purity of Akazara myosin preparations. Akazara myosin thus purified contained a single light-chain component of 17,000 daltons as determined by SDSadvance with the same buffer, and filtered through gel electrophoresis. Figure 4 shows the sedimentation pattern of a filter paper. DEAE-Sephadex on the filter paper (approximately 30 ml) was mixed with Akazara myosin compared with that of scallop Akazara myosin solution (70-100 mg per 10- myosin prepared from striated adductor muscle 20 ml), stirred for 30 min (0°C), and then filtered of scallop by the procedures described for the through a filter paper. Using the same procedures, preparation of Akazara myosin. Preparations of DEAE-Sephadex on the filter paper was washed Akazara myosin gave a single sharp peak, having first with 0.06 M KCI-0.06 M sodium pyrophos- a sedimentation coefficient (s^ w) of 6.6S. On phate, pH7.5, and then with 0.15 M KCI-0.06 M the other hand, preparations of scallop myosin sodium pyrophosphate, pH 7.5. DEAE-Sephadex gave two peaks: one major peak and one minor thus washed was extracted with 3—4 ml of 0.5 M peak which is probably an aggregated form of KCI buffered with 0.06 M sodium pyrophosphate, myosin (cf. ref. 4 and 8). The intrinsic viscosity, pH 7.5, for a few hours with occasional stirring. [jj], of Akazara myosin was 2.10 dl/g at 10°C in a /. Biochem.
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Protein
MYOSIN FROM Chlamys nipponensis akazara
667
0.6 E c o
0.1.
cw o
c o
CO
oil
0.2-
(J O o
o 20
40 60 Tube Number
80
Fig. 5. Ion-exchange chromatography of Akazara myosin on DEAE-Sephadex A50. Eighty mg of the myosin in 0.06 M sodium pyrophosphate, pH 7.5, was applied to a 1.9 x 15 cm column and eluted with a linear gradient of KCI in 0.06 M sodium pyrophosphate, pH 7.5. The flow rate was lOml/h. Fraction size was 10 ml. All the solutions used here contained 1 mM DTT. (O) Absorbance at 280 nm; ( x ) absorbance at 260 nm; ( A ) Ca-ATPase activity. medium containing 0.6 M KCI, 10 mM potassium phosphate buffer (pH 7.0), and 2 mM 2-mercaptoethanol. These values of and [rj] are in good agreement with those of scallop myosin reported by Szent-Gyorgyi et al. (8). The UV absorption spectrum of Akazara myosin was that of a typical protein, and the extinction coefficient measured at 280 nm in 0.6 M KCI buffered with 10 mM potassium phosphate, pH 7.0 was 7.92 for 10 mg/ml Akazara myosin. The sulfhydryl content measured in 7 M urea by Ellman's method {19) was 38 mol per 500,000 g of Akazara myosin. Column chromatography can be used in place of the batch method for improving the purity of Akazara myosin preparations. Figure 5 shows a chromatogram of a preparation of Akazara myosin. A major component was eluted with 0.07-0.12 M KCI in 0.06 M sodium pyrophosphate, pH 7.5. In this particular case, the Ca-ATPase activity of Akazara myosin (/imol per min per mg of myosin) was 2.8 for the eluted fraction as compared with 2.6 for the preparation before the chromatography. The activity was measured at Vol. 86, No. 3, 1979
25°C in 5 mM CaCl,, 1 mM ATP, 0.06 M KCI, and 25 mM Tris-maleate, pH 7.0. 3. A TPase Activity of Myosin—The ATPase activity of Akazara myosin in the steady state was measured, and the results obtained are summarized in Table II. The Ca-ATPase activity of Akazara myosin was high, approximately 2-4 times those of scallop adductor and rabbit skeletal myosins. The EDTA-ATPase activity is higher (2-3 times) than that of scallop myosin but lower (approximately one-half) than that of rabbit skeletal myosin. The Mg-ATPase activity of Akazara myosin was higher than those of scallop myosin and rabbit skeletal myosin. 4. Calcium Sensitivity—According to Kendrick-Jones et al. (7), calcium regulation of scallop adductor actomyosin is myosin-linked, and it is lost when a light-chain component of scallop myosin is removed by washing with EDTA. Our preparations of Akazara myosin, when combined with skeletal pure actin (free from troponin and tropomyosin), were found to be sensitive to calcium ions, thus indicating that a calcium regulatory system is associated with Akazara myosin. Figure 6
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UD
K. NISHITA, T. OJIMA, and S. WATANABE
668
TABLE II. ATPase activity of myosins. The reaction was carried out at 25°C in a medium containing 1 ITIM ATP, 25 ITIM Tris-maleate, pH 7.0, KCI at the concentration indicated in the left-hand column, and one of the additions indicated in the upper line.
0.012 0.06 0.6
a
Akazara
5 HIM CaCI,
1 iriM MgCU
0.030
4.40
0.53
1 mM EDTA —
Scallop
0.036
1.08
0.13
—
Akazara
0.068
3.50
0.45
—
Scallop
0.065
1.45
0.13
Akazara
0.24
2.10
0. 14
0.68
Scallop
0.21
0.96
0.06
0.24
—
0.6
Scallop'
—
0.26
0.09
0.07
0.6
Gizzard b
—
0.13
0.003
0.35
0.6
Skeletal"
—
0.27
0.004-0. 025
1.3
Barany and Barany (4).
_
None
b
Onishi el al. (20).
1.25
100-
1.00-
+EGTA
0.75 Q_
zo0-25-
0
4 8 12 Incubation Time (min) Fig. 7. Effect of adding EGTA on the actin-activated 0 05 1.0 ATPase reaction in the presence of calcium ions. Pi Actin/Myosin (w/w) liberation was measured in a medium containing Fig. 6. Actin activation of Akazara myosin in the 30 mM KCI, 20 mM Tris-maleate (pH 6.8), 2 mM MgCI,, presence of calcium ions. ATPase activity was 0.67 mM ATP, 0.04 mg/ml Akazara myosin, 0.013 mg/ml measured in a medium containing 12 mM KCI, 25 mM rabbit skeletal actin, and 0.1 mM CaCI, ((},•) or Tris-maleate, pH 7.0, 1 mM MgCI,, I mM ATP, and 0.5 ITIM EGTA (A, A ) . 0.17 mM EGTA was added 0.1 mM CaCI, or 0.5 mM EGTA at 25°C. ( • ) In the 7 min after the addition of ATP (arrows marked presence of CaCI,; (O) in the presence of EGTA. + EGTA). Preincubation was carried out in 0.18 M KCI, 25 mM Tris-maleate, pH 7.5, 5 mM MgCI,, 1.44 shows that Mg-ATPase of Akazara myosin was mg/ml Akazara myosin, 7 mM ATP, 0.7 mM CaCI, or activated by skeletal actin in the presence of Ca l+ EGTA (total volume 5.0 ml). After prcincubation for 1 h at 20°C, the myosin was precipitated by dilution and not in its absence. Moreover, as shown in Fig. 7, the actin-activated ATPase reaction in the with 10 volumes of IOITIM Tris-maleate, 2 mM MgCI,, presence of Ca1+ was suppressed by subsequent pH 7.0. Tlie myosin was collected by centrifugation at 20,000 Xff for 15 min, and then dissolved in 0.6 M addition of EGTA (to remove Ca1+). This was KCI, 0.05 M Tris-maleate, pH 7.0. true regardless of whether Akazara myosin was J. Biochem.
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Addition Omol Pi/min/mg myosin)
KCI (M)
669
MYOSFN FROM Chlamys nipponensis akazara
slightly decreased, thus reducing the calcium sensitivity. Figure 9A shows that the staining density of the 17,000 dalton light-chain band in SDS-gel electrophoresis was gradually decreased as the EDTA washing was repeated. The density ratio of the light-chain band to the heavy-chain band was estimated, and is shown in Fig. 8. In the particular series of experiments shown in Fig. 8, the density ratio reached a minimum value of approximately 0.08 after 6 or 7 washings, and complete loss of calcium sensitivity (O, • ) occurred concurrently. Therefore, it appears that the calcium sensitivity of Akazara muscle is
Heavy chain (206,000) y (11O000)
•
0.2
1 2
-Light chain (17,000) (Chain weight)
5 6
0.1
.. 0
o &
2 4 6 Number of EDTA Washings
Fig. 8. Desensitizing effect of EDTA washing on the Mg-ATPase activity of rabbit acto-Akazara myosin. Akazara myosin was washed with a solution containing 10 min EDTA, 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT. The myosin concentration during EDTA washing was maintained at about 4 mg/ml. SDS-gel electrophoresis was carried out (see Fig. 9) to determine the ratio of staining density ( x ) of the light chain component (17,000 daltons) to the heavy chain component (206,000 daltons), and (A) of the EDTA-light chain component to the SH-light chain component (17,000 daltons). The ATPase activity was measured at 25°C in reaction medium containing rabbit skeletal actin (0.1 mg/ml), Akazara adductor myosin (0.2 mg/ml), 30 mM KCI, 2 mM MgCI,, 20 mM Trismaleate of pH 6.8, 0.5 mM ATP, and 0.1 mM CaCIi ( # ) or 0.5 mM EGTA (O).
Vol. 86, No. 3, 1979
-EDTA-light chain -SH-light chain
1 2
3
L
Fig. 9. Removal of EDTA-light chains from Akazara myosin. (A) SDS-gel electrophoresis: Eight percent polyacrylamide gel rods (0.5x8 cm) were used. I, Unwashed myosin; 2, washed once; 3, washed twice; 4, washed four times; 5, washed seven times; 6, washed fifteen times. Forty-five fig of myosin was applied to each gel. (B) Urea-gel electrophoresis: Polyacrylamide (1.5%) gel rods (0.5x8 cm each) containing 8 M urea were used. Samples 1-6 are the same as those indicated in (A). One hundred ng of myosin was applied to each gel.
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preincubated with ATP in the presence or absence of Ca ! + . Therefore, the actin activation does not seem to involve phosphorylation of light-chain components, as reported for gizzard myosin (Ikebe et a/. (21)). 5. Desensitization of Myosin—Akazara myosin was washed essentially in the same way as described by Szent-Gyorgyi et al. (8, 9) for scallop myosin: myosin was suspended in a solution containing 10 mM EDTA, 40 mM NaCl, 5 mM potassium phosphate, pH 7.0 and 0.5 mM DTT, stirred for lOmin at 0-5°C, and then centrifuged to obtain precipitates of EDTA-washed myosin. Combining the EDTA-washed myosin of Akazara adductor with rabbit skeletal actin (in a weight ratio of 2 : 1 ) , the Mg-ATPase activity was measured in the presence of 0.1 mM CaCI2 or 0.5 mM EGTA. Figure 8 shows that as the number of EDTA washes increased, the MgATPase activity of rabbit acto-Akazara myosin in the absence of Ca ! + (i.e. in the presence of EGTA) increased, whereas that in the presence of Ca 5+
K. NISHITA, T. OJIMA, and S. WATANABE
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TABLE III. Desensitization of Akazara myosin by washing with EDTA in the presence and absence of DTT. Akazara adductor myosin was washed at 0-5°C with 10 mM EDTA in the presence of 0.5 mM DTT or in its absence. The washed myosin was then combined with rabbit skeletal actin in a weight ratio of 2 : I, and the Mg-ATPase activity of the combined system was measured in the presence of 0.1 mM CaCl, or 0.5 mM EGTA. The calcium sensitivity was expressed in terms of (I —activity in EGTA/activity in CaCl,) x 100 (%). Number of EDTA washes 0 1 2 4 7
Ca sensitivity (%) Washed without DTT
Washed in 0.5 mM DTT
62.9 54.4 39.2 18.9 12.6
64.0 50.4 46.9 23.1 12.9
100 g of Akazara striated muscle, and suspended in 2 liters of an extracting solution containing 10 mM EDTA, 40 mM NaCl, and 5 mM potassium phosphate, pH 7.0. The suspension was stirred at 4°C for 30 min, and then centrifuged at 8,000 XQ for 15 min. The supernatant was collected, and the residue was extracted again in a similar manner. The supernatant thus obtained was combined and 0.25 mM DTT was added. DEAESephadex A50 (approximately 40 ml), which had been pre-equilibrated with 25 mM potassium phosphate, pH 7.0, and 0.25 mM DTT and allowed to settle in a cylinder, was added. The mixture was stirred for 1 h at 4°C, so that EDTA-light chains would be adsorbed on the DEAE-Sephadex. The mixture was then filtered through a filter paper, and the Sephadex on the filter paper was washed twice with 50 ml of 0 . 1 5 M potassium phosphate, pH 7.0. The washed Sephadex was then extracted with 20 ml of 0.5 M potassium phosphate, pH 7.0, and the extract was made up to 4 5 % saturation with saturated ammonium sulfate solution (containing 0.5 mM DTT). The precipitates thus formed (mainly of actin, myosin, and paramyosin) was removed by centrifugation, and additional ammonium sulfate was added to give 75% saturation. The precipitate formed was sedimented by centrifugation, dissolved in 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT, and then dialyzed against the same solution. The yield of this preparation of Akazara EDTA-light chains was approx. 20-30 mg/lOOg myofibrils. Gel electrophoretic patterns of this preparation of Akazara EDTA-light chain are shown in Fig. 11 A, 2 and 4. SH-light chain was also prepared from Akazara myosin. Dissociation of SH-light chains from the myosin with 6 M urea and the removal of myosin heavy chain by the addition of ethanol and centrifugation were performed by the method of Kendrick-Jones et al. (9). The supernatant thus obtained was diluted with 10 volumes of cold distilled water, followed by DEAE-Sephadex A50 chromatography and ammonium sulfate fractionaxion as described for the preparation of EDTA-light chains. Finally, the SH-light chains were subjected to urea-gel electrophoresis in order to eliminate contaminating EDTA-light chains. Gel-electrophoretic patterns of this preparation of SH-light chain are shown in Figs. 11 A, 3 and 5.
/ . Biochem.
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also myosin-linked and that the EDTA-light chain component is responsible for the sensitivity. Moreover, as shown in Fig. 9B, the 17,000 dalton light chain was separated into two bands in ureagel electrophoresis, and only one of the two bands was removed by EDTA washing, causing a complete loss of calcium sensitivity (Fig. 8). These properties of Akazara myosin are practically identical with those reported for scallop myosin (9). However, we found one important difference from scallop myosin: not half the amount of EDTA-light chains but the total amount had to be removed to achieve complete loss of calcium sensitivity (7 washes; Fig. 8, see also Fig. 9B-5). In the experiments shown in Figs. 8 and 9, EDTA washing was conducted in the presence of 0.5 mM DTT. On the other hand, KendrickJones et al. (9) reported that DTNB treatment can facilitate the removal of EDTA-light chain from scallop myosin. Thus, EDTA washes were carried out in the absence of DTT, and the loss of calcium sensitivity was estimated. As shown in Table I IF, the absence of DTT did not enhance the loss of calcium sensitivity as long as the washing was conducted at low temperature. 6. Light chains—Isolation of the EDTAlight chain component from myofibrils was conducted essentially by the method of KendrickJones et al. (9). Myofibrils were prepared from
MYOSIN FROM Chlamys nipponcnsis akazara
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(A)
myosin practically free from EDTA-light chains (see Fig. 9) and purified preparations of Akazara EDTA-light chains, we examined whether or not the resensitization is also incomplete with our preparations from Akazara adductors. Recombination of the isolated EDTA-light chains with EDTA-washed myosin was performed by the following procedure. One ml of "desensitized" (washed five times with EDTA) Akazara myosin solution (1.5-2.0 mg/ml in 0.6 M KC1, 4 mM MgCU, 0.05 M Tris-maleate, pH 7.0, and 0.5 mM DTT) and two ml of the EDTA-light chain solution (0.3-0.9 mg/ml in 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT) were mixed, and dialyzed overnight against 0.3 or 0.6 M KC1, 0.05 M Tris-maleate, pH 7.0, 2 mM MgCl,, and 0.5 mM DTT. The dialyzed solution was diluted with 14 volumes of ice-cold 2 mM MgCl, solution, and the precipitate formed was collected by centrifugation. It was redissolved in 0.6 M K.CI and 0.5 mM DTT and the solution was cleared by centrifugation. The cleared solution was diluted once more with 10 volumes of
(B)
(C)
0.8H 2-
2
0
•Ca*Eg 240
260
280
300
Wavelength (nm)
320
250
270 290 310 330 Wavelength (nm)
Fig. 10. (A) UV absorption spectra of Akazara light chains. Concentrations of EDTA-light chains and of SH-light chains were 3.8 mg/ml and 1.4 mg/ml, respectively. They were dissolved in 40 mM potassium phosphate, pH 7.0, and 1 mM DTT: ( ) EDTA-light chains; ( ) SH-light chains. (B) UV absorption difference spectra of Akazara light chains. Concentrations of EDTA-light chains and of SH-light chains were the same as in (A). 1 mM EGTA was present in the reference cell and 1 mM CaCI, in the sample cell: ( ) EDTA-light chain; ( ) SH-light chain; ( ) before addition of CaCI,. (C) Disc-gel electrophoretic pattern of EDTA-light chains: 7.5% polyacrylamide gel rods (0.5x8 cm each) and 50 mM Tris-glycine buffer, pH 8.3 were used. +Ca: In the presence of I mM CaCI,. +Eg: In the presence of 1 mM EGTA. Vol. 86, No. 3, 1979
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The UV absorption spectra of Akazara EDTAlight chains and SH-light chains (Fig. 10A) were very similar to those of the corresponding light chains from scallop adductors (see ref. 9). Nevertheless, we were able to obtain a calcium-induced difference spectrum of Akazara EDTA-light chains (Fig. 10B): this is in contrast to the report of Kendrick-Jones et al. (9) that the UV absorption spectrum of scallop EDTA-light chains was not changed on addition of calcium ions. Moreover, we found (Fig. IOC) that calcium ions cause changes in the disc electrophoretic patterns of Akazara EDTA-light chains, suggesting that at least a portion of Akazara EDTA-light chains binds with calcium ions (22). However, no clear evidence for Ca2+ binding to Akazara SH-light chains was obtained. 7. Resensitization—Kendrick-Jones et al. (9) reported that removal of more than half the total amount of EDTA-light chains from scallop myosin resulted in incomplete resensitization of desensitized myosin on re-adding EDTA-light chains. Since we obtained desensitized Akazara
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ice-cold 2 mM MgCI,, and the precipitate formed was collected by centrifugation. As shown in Fig. 12 (see also Fig. 11B, 4) EDTA-light chains rccombincd with desensitized Akazara myosin, showing EDTA-light chain and SH-light chain bands in approximately equal amounts. Moreover, resensitization was nearly complete: approximately 80% of the lost calcium sensitivity was recovered on mixing EDTA-light chains and desensitized myosin in a molar ratio of 2 : 1. It can therefore be concluded that, unlike the case with scallop adductor myosin reported by Kendrick-Jones et a/. (9), EDTA-light chains can be completely removed from and fully reintegrated in Akazara adductor myosin. We should add that these studies with Akazara adductor myosin were motivated by similar findings with myosins from other sources: calcium ions induce a UV absorption difference spectrum of squid mantle EDTA-light chains (K. Konno, K. Arai, & S. Watanabe, unpublished data), and EDTA-light chains can be completely removed from and fully reintegrated in clam foot myosin (T. Asada, G. Ashiba, & S. Watanabe, 23).
0 0
1 2 5 10 EDTA-LC(odded)/Desensitized Myosin (Molar Ratio)
Fig. 12. Resensitization of desensitized Akazara myosin by isolated EDTA-light chain preparations. Desensitized Akazara myosin (4.0 mg in 0.6 M KCI, 0.05 M Tris-maleate, pH 7.0, 2 mM MgCI,, and 0.5 mM DTT) and various amounts of Akazara EDTA-light chains (0.96 mg/ml in 40 mM NaCl, 5 mM potassium phosphate, pH 7.0, and 0.5 mM DTT) were mixed, and rabbit skeletal actin (2.0 mg in 0.6 M KCI and 0.05 M Trismaleate, pH 7.0) was added to the mixture. The ATPase activity of the combined system was measured in a medium containing 30 mM KCI, 20 mM Tris-maleate, pH 6.8, 2 mM MgCU, 0.5 mM ATP, in the presence of 0.1 mM CaCI, or 0.5 mM EGTA. Ca sensitivity is as defined in Table III: it was 50% ( • ) for the unwashed myosin we employed. MW: Desensitized myosin, 450,000; EDTA-light chain, 17,000. ( • ) +Ca; (O) + EGTA; ( A ) unwashed myosin, +Ca; ( A ) unwashed myosin, +EGTA. Urea-gel electrophoretic patterns: ratio of EDTA-light chains/desensitized myosin, a = 0 , b = 2, c-»10, and d = unwashcd myosin.
The authors wish to thank Dr. K. Arai and Mr. K. Konno, Faculty of Fisheries, Hokkaido University for valuable advice and discussions. The authors also wish to thank Mr. H. Yokota for his technical assistance in preparing Akazara myosin. REFERENCES 1. Twarog, B.M. (1954) / . Cell. Comp. Physiol. 44, 141-163 2. Johnson, W.H., Kahn, J.S., & Szent-Gydrgyi, A.G. (1959) Science 130, 160-161 3. Twarog, B.M. (1967) J. Gen. Physiol. 50, 157-168 4. Barany, M. & Barany, K. (1966) Biochem. Zeir. 345, 37-56 J. Biochem.
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' -> 3 L 5 1 2 Fig. 11. Recombination of EDTA-light chains with desensitized Akazara myosin. (A) Preparations; 1. Desensitized Akazara myosin (45 fig); 2, Akazara EDTA-light chains (50 fig); 3, Akazara SH-light chains (80 fig); 4, Akazara EDTA-light chains (60 fig); 5. Akazara SH-light chains (50 fig). 1-3, SDS-gel electrophoresis (8% gel rods); 4-5, urea-gel electrophoresis (7-5% gel rods). (B) Recombination: 1, Unwashed myosin (control) (150 fig); 2, desensitized myosin (150 fig); 3, Akazara EDTA-light chains (30 fig); 4, recombined Akazara myosin with Akazara EDTA-light chains (100 fig). Urea-gel electrophoresis (7.5% gel rods).
K. NISHITA, T. OJIMA, and S. WATANABE
MYOSIN FROM Chlamys nipponensis akazara
Vol. 86, No. 3, 1979
16. Perrie, W.T., Smillie, L.B., & Perry, S.V. (1973) Biochem. J. 135, 151-164 17. Ebashi, S. & Nonomura, Y. (1973) in The Structure and Function of Muscle (Bourne, G.H., ed.) Vol. 3, pp. 286-362, Academic Press, New York 18. Cohen, D.M. & Murphy, R.A. (1977) Fed. Proc. 36,602 19. Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77 20. Onishi, H., Yamada, Y., Ikebe, M., Suzuki, H., & Watanabe, S. (1978) / . Biochem. 83, 129-135 21. Ikebe, M., Aiba, T., Onishi, H., & Watanabe, S. (1978) / . Biochem. 83, 1643-1655 22. Gaffin, S.L. & Oplatka, A. (1974) /. Biochem. 75, 277-281 23. Asada, T., Ashiba, G., & Watanabe, S. (1979) /. Biochem. 85, 1543-1546
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5. Szent-Gydrgyi, A.G., Cohen, C , & Kendrick-Jones, J. (1971) / . Mol. Biol. 56, 239-258 6. Azuma, N., Asakura, A., & Yagi, K. (1975) / . Biochem. 77, 973-981 7. Kendrick-Jones, J., Lehman, W., & Szent-Gyorgyi, A.G. (1970) / . Mol. Biol. 54, 313-326 8. Szent-Gy6rgyi, A.G., Szentkiralyi, E.M., & Kendrick-Jones, J. (1973) / . Mol. Biol. 74, 179-203 9. Kendrick-Jones, J., Szentkiralyi, E.M., & SzentGyorgyi, A.G. (1976) / . Mol. Biol. 104, 747-775 10. Azuma, N. (1976) / . Biochem. 80, 187-189 11. Perry, S.V. & Grey, T.C. (1956) Biochem. J. 64, 184-192 12. Spudich, J.A. & Watt, S. (1971) / . Biol. Chem. 246, 4866-^*871 13. Gomori, G. (1942) /. Lab. Clin. Med. 11, 955-960 14. Gornall, A.G., Bardawill, C.S., & David, M.M. (1949) / . Biol. Chem. 177, 751-766 15. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406-4412
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