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Purification of mouse myosin by gel filtration
SHORT COMMUNICATIONS
18,7
sc 2393
Purification of mouse myosin by gel filtration Myosin has most commonly been prepared b y the technique of SZENT-GYORGYI1 based on the solubility of the protein in o.3 M KCI and insolubility in o.o 3 M KC1. Modifications of this method have included (NHd)~S04 fractionation 2 and more recently cellulose ion-exchange chromatography 8-~. Since the newly developed, loosely crosslinked dextran gel, Sephadex G-2oo (ref. 6), seemed well-suited to the separation of myosin from smaller molecules, gel filtration was tried as a purification step in place of DEAE-celhilose chromatography. Because our ultimate purpose is the study of myosin of dystrophic mice of strain 129 Jackson Memorial Laboratory (Bar I-tarbor, Maine) this strain was used in the present investigation. The ATP was purchased from the Pabst Company (Milwaukee, Wisc.) and the Sephadex G-2oo, lot number TO 3o17, from Pharmacia (Uppsala). For eacl~ preparation, tell mice were killed b y cervical dislocation and then skinned, eviscerated, and chilled in cracked ice. The carcasses were ground once in a meat grinder (2-ram holes) and the mince of about 80 g was extracted for IO rain with 3 vol. of 0.3 M KC1 containing 0.05 M histidine-HC1-KOH buffer (pH 6.8) and 2 mM ATP as suggested b y AsAI 5. Deionized water was used throughout and the preparation was kept at o-4 ° at all times. The extract was centrifuged at 6ooo × g for IO min and the supernatant fluid filtered through cheese cloth to remove fat. The filtrate was diluted with IO vol. of water and the myosin precipitate collected b y centrifugation and redissolved in o.5 M KC1 containing I mM Tris-HC1 buffer (pH 7.0). This solution will be called Stage I (Table I). Actomyosin (myosin B) was separated from myosin b y diluting the solution to o.3 M KC1 and centrifuging for I h at 35ooo × g. The m y o s i n was reprecipitated b y diluting the supernatant to o.o4 M KC1, collected b y centrifugation, and dissolved in o. 5 M KC1 containing I m M Tris HC1 (pH 7.o) (Stage 2). Since further repetition of t h i s solubility cycle in a number of preparations failed to increase the specific ATPase activity or the A ~9/A 26a absorbancy ratio of the protein, no further cycles were used in later experiments. Following centrifugation at ioo ooo × g for 2 h, the myosin solution (Stage 3) was placed on a Sephadex G-2oo column that had been equilibrated with o. 5 M KC1, I mM Tris-HC1 buffer (pH 7.o). Gel filtration was carried out in the cold with the same buffer at an elution rate of lO-2O ml/h. The ehiate was collected in 5 - I o - m l fractions and the most active were pooled (Stage 4). Protein concentrations greater than 1% markedly slowed the flow and greater than 1.5 % stopped it. Adequate flow rates were obtairled with o.7-1% solution. All preparations were completed and analyzed within 36 h. The protein concentration was determined spectrophotometrically assuming that the A~% of pure myosin at 279 m/~ is 5.60 as suggested b y SMALL et al. 7. The absorbancy at 32o m/~ was subtracted from that at 279 m/~ to eliminate non-specific light-scattering effects. This correction was minimal in Stages 3 and 4The ATPase was measured b y incubating a o.I % sohition of the myosin for 5 min at 22 ° in 0.5 M KC1, 0.06 M Tris-HC1 (pH 9), 5 mM CaCI~ and 5 mM ATP. After the reaction was stopped with trichloroacetic acid, inorganic phosphate was determined b y a modification of tile Fiske-SubbaRow method using FeSO 4 as a reducing agent. D a t a on the yield and purity of myosin at the four stages of isolation are shown :
: , ~ ,
,~
.-
Biochim.
Biophys.
Acts,
86 (1964) 187-i89
I88
SHORT COMMUNICATIONS TABLE I SUMMARY
0I ~ A
.~IYOSIN
ISOLATION
S~age*
Total protein (rag)
I
I73 o
0.85
z6
2
4
314 I7O IO 5
0.82 0.97 I.e6
47 74 96
Peak io-ml fraction
47
1.29
3
Ultraviolet absorbancy ratio A 2w/A ~so* *
A TPase specific activity (pg P/mg protein~5 i:ui*~/
I°5
* T h e s t a g e s 05 p u r i f i c a t i o n are d i s c u s s e d in t h e t e x t . ** T h e At20 v a l u e w a s first s u b t r a c t e d . I t was a b o u t ha!5 as h i g h as t h e A279 v a l u e in S t a ge i, a n d no m o r e t h a n 3 % of t h e A av9 v a l u e b y Stage 3.
for a typical preparation in Table I. The ATPase specific activity increased 8I 3/o between Stages I and 2. High-speed centrifugation of the myosin solution resulted in a further 58 % increase (Stage ~ to 3). The elution pattern from the Sephadex 0-2o0 coiumn (Fig. I) shows an initial major peak identified as myosin by its high specific ATPase activity and high Amg/A26 o ratio, followed b y material with very low enzyme activity and a low A ~79/A 2eo ratio. As expected, the iarge myosin molecule is apparently excluded from the interstitial volume of the G-~oo beads and is rapidly eluted. The low A ~79/A~0 ratio of the retarded material suggests that it is rich in nucleotide derivatives. This observation supports previous studies implicating them as major contaminants of myosin preparations4, 8. The first 3o ml of the major peak (Stage 4, Table I) contained 62 % of the protein applied to the column and 8I % of the enzyme units which represents a 3 ° % increase in specific activity over the pre-column preparation. A summary of column purifi-
Ioo
2
o cu
t
,¢
_z
(
°[]
z_
A~
ao g
2
I o
J
~'o ELUTION
D
,oo
"o..q,~
V O L U M E , {ML)
Fig. x. GeI f i l t r a t i o n e l u t i o n d i a g r a m of m o u s e m y o s i n p r e p a r a t i o n . 2z m l of a 0.77 % s o l u t i o n of t h e p a r t i a l l y purified p r e p a r a t i o n were a p p l i e d to a a6 × 4.5 c m c o l u m n of S e p h a d e x G-2oo p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 5 M KCI c o n t a i n i n g I mM T r i s - H C 1 buffer (pH 7.o). The chrom a t o g r a p h y w a s c a r r i e d o u t in t h e cold room. F r a c t i o n s o5 5 - I o ml of e l u a t e were a n a l y z e d . O - - O , Ae79; ± - - ~ , Azvg/A26e; [~--,3, A T P a s e specific a c t i v i t y .
Bioch4m. Biophys. Acla, 86 (I964) I 8 7 - 1 8 9
SHORT COMMUNICATIONS TABLE RESULTS
OF
SEPHADEX
G-2oo GEL
II
FILTRATION
Preparation
189
IN
FIVE
MYOSIN
PREPARATIONS
Maximal increase in sl)ecific activity*
(%)
i
26
2
20
3
33
4
54
5**
4°
* ATPase specific a c t i v i t y of the peak io-ml fraction as c o m p a r e d w i t h t h a t of t h e s a m p l e applied to the column. ** The p r e p a r a t i o n s h o w n in Table I.
cation of five different preparations (Table II) shows that the ATPase specific activity of the most active Io-ml fraction averaged 35 % higher than the material applied to the column, On the average, pooled fractions from the major elution peak of each preparation contained 7 ° % of the applied protein in one-half its original concentration with a specific ATPase activity 25 % higher than the we-column solution. The total protein eluted from the column was generally 9O-lOO % of that applied. Comparison of the Sephadex column purification technique with the DEAEcellulose ion-exchange column method as recently reported by BRAI-IMSa, PERRY¢, and AsAI5 shows a similar increase in specific ATPase activity. A major advantage of the Sephadex technique is greater Speed, 5-7 h for a complete elution resulting in consistent purification and yield comparable with the most successful of the DEAEcellulose methods. Additional advantages are the avoidance of excessive dilution of the myosin preparation and the need of only one elution buffer. Furthermore, this buffer can contain 0.5 M KC1, therefore minimizing the tendency of myosin to aggregate and precipitate on the column. Such a high ionic strength is Unsuitable for DEAE-cellulose chromatography. One of us (M.S.) holds a postdoctorM research fellowship from the Muscular Dystrophy Association of America.
Department of Biochemistry, University of California Medical Center, San Francisco, Calif. (U.S.A.)
MARVIN SMOLLER RICHARD A. F I N E B E R G
1 A. SZENT-GY6RGYI, Chemistry of Muscular Contraction, Academic Press, New York, 2nd Ed., 1951 , p. 146. 2 W. W. KI•LLEY AND L. B. BRADLEY, J . Biol, Chem., 218 (1956) 653. J. BRAHMS, J. Am. Chem. Soc., 81 (1959) 4997. 4 S. V. PERRY, Biochem. J., 74 (196o) 94. H, ASAI, Biochemistry, 2 (1963) 458. 0 p. FLODIN AND J. I~ILLANDJ~R, Biochim. Biophys, Acta, 63 (1962) 403. 7 p. A. SMALL, W. F. HARRINGTON AND W. W. KIELLEY, Bioehim. Biophys. Acta, 49 (1961) 462. s E. MIHALYI, Ig. LAKI AND M. I. I4NOLLER, Arch. Biochem. Biophys., 68 (1957) 13o.
Received October 26th, 1963 Biochim. Biophys. Acta, 86 (1964) 187-189
18,7
sc 2393
Purification of mouse myosin by gel filtration Myosin has most commonly been prepared b y the technique of SZENT-GYORGYI1 based on the solubility of the protein in o.3 M KCI and insolubility in o.o 3 M KC1. Modifications of this method have included (NHd)~S04 fractionation 2 and more recently cellulose ion-exchange chromatography 8-~. Since the newly developed, loosely crosslinked dextran gel, Sephadex G-2oo (ref. 6), seemed well-suited to the separation of myosin from smaller molecules, gel filtration was tried as a purification step in place of DEAE-celhilose chromatography. Because our ultimate purpose is the study of myosin of dystrophic mice of strain 129 Jackson Memorial Laboratory (Bar I-tarbor, Maine) this strain was used in the present investigation. The ATP was purchased from the Pabst Company (Milwaukee, Wisc.) and the Sephadex G-2oo, lot number TO 3o17, from Pharmacia (Uppsala). For eacl~ preparation, tell mice were killed b y cervical dislocation and then skinned, eviscerated, and chilled in cracked ice. The carcasses were ground once in a meat grinder (2-ram holes) and the mince of about 80 g was extracted for IO rain with 3 vol. of 0.3 M KC1 containing 0.05 M histidine-HC1-KOH buffer (pH 6.8) and 2 mM ATP as suggested b y AsAI 5. Deionized water was used throughout and the preparation was kept at o-4 ° at all times. The extract was centrifuged at 6ooo × g for IO min and the supernatant fluid filtered through cheese cloth to remove fat. The filtrate was diluted with IO vol. of water and the myosin precipitate collected b y centrifugation and redissolved in o.5 M KC1 containing I mM Tris-HC1 buffer (pH 7.0). This solution will be called Stage I (Table I). Actomyosin (myosin B) was separated from myosin b y diluting the solution to o.3 M KC1 and centrifuging for I h at 35ooo × g. The m y o s i n was reprecipitated b y diluting the supernatant to o.o4 M KC1, collected b y centrifugation, and dissolved in o. 5 M KC1 containing I m M Tris HC1 (pH 7.o) (Stage 2). Since further repetition of t h i s solubility cycle in a number of preparations failed to increase the specific ATPase activity or the A ~9/A 26a absorbancy ratio of the protein, no further cycles were used in later experiments. Following centrifugation at ioo ooo × g for 2 h, the myosin solution (Stage 3) was placed on a Sephadex G-2oo column that had been equilibrated with o. 5 M KC1, I mM Tris-HC1 buffer (pH 7.o). Gel filtration was carried out in the cold with the same buffer at an elution rate of lO-2O ml/h. The ehiate was collected in 5 - I o - m l fractions and the most active were pooled (Stage 4). Protein concentrations greater than 1% markedly slowed the flow and greater than 1.5 % stopped it. Adequate flow rates were obtairled with o.7-1% solution. All preparations were completed and analyzed within 36 h. The protein concentration was determined spectrophotometrically assuming that the A~% of pure myosin at 279 m/~ is 5.60 as suggested b y SMALL et al. 7. The absorbancy at 32o m/~ was subtracted from that at 279 m/~ to eliminate non-specific light-scattering effects. This correction was minimal in Stages 3 and 4The ATPase was measured b y incubating a o.I % sohition of the myosin for 5 min at 22 ° in 0.5 M KC1, 0.06 M Tris-HC1 (pH 9), 5 mM CaCI~ and 5 mM ATP. After the reaction was stopped with trichloroacetic acid, inorganic phosphate was determined b y a modification of tile Fiske-SubbaRow method using FeSO 4 as a reducing agent. D a t a on the yield and purity of myosin at the four stages of isolation are shown :
: , ~ ,
,~
.-
Biochim.
Biophys.
Acts,
86 (1964) 187-i89
I88
SHORT COMMUNICATIONS TABLE I SUMMARY
0I ~ A
.~IYOSIN
ISOLATION
S~age*
Total protein (rag)
I
I73 o
0.85
z6
2
4
314 I7O IO 5
0.82 0.97 I.e6
47 74 96
Peak io-ml fraction
47
1.29
3
Ultraviolet absorbancy ratio A 2w/A ~so* *
A TPase specific activity (pg P/mg protein~5 i:ui*~/
I°5
* T h e s t a g e s 05 p u r i f i c a t i o n are d i s c u s s e d in t h e t e x t . ** T h e At20 v a l u e w a s first s u b t r a c t e d . I t was a b o u t ha!5 as h i g h as t h e A279 v a l u e in S t a ge i, a n d no m o r e t h a n 3 % of t h e A av9 v a l u e b y Stage 3.
for a typical preparation in Table I. The ATPase specific activity increased 8I 3/o between Stages I and 2. High-speed centrifugation of the myosin solution resulted in a further 58 % increase (Stage ~ to 3). The elution pattern from the Sephadex 0-2o0 coiumn (Fig. I) shows an initial major peak identified as myosin by its high specific ATPase activity and high Amg/A26 o ratio, followed b y material with very low enzyme activity and a low A ~79/A 2eo ratio. As expected, the iarge myosin molecule is apparently excluded from the interstitial volume of the G-~oo beads and is rapidly eluted. The low A ~79/A~0 ratio of the retarded material suggests that it is rich in nucleotide derivatives. This observation supports previous studies implicating them as major contaminants of myosin preparations4, 8. The first 3o ml of the major peak (Stage 4, Table I) contained 62 % of the protein applied to the column and 8I % of the enzyme units which represents a 3 ° % increase in specific activity over the pre-column preparation. A summary of column purifi-
Ioo
2
o cu
t
,¢
_z
(
°[]
z_
A~
ao g
2
I o
J
~'o ELUTION
D
,oo
"o..q,~
V O L U M E , {ML)
Fig. x. GeI f i l t r a t i o n e l u t i o n d i a g r a m of m o u s e m y o s i n p r e p a r a t i o n . 2z m l of a 0.77 % s o l u t i o n of t h e p a r t i a l l y purified p r e p a r a t i o n were a p p l i e d to a a6 × 4.5 c m c o l u m n of S e p h a d e x G-2oo p r e v i o u s l y e q u i l i b r a t e d w i t h 0. 5 M KCI c o n t a i n i n g I mM T r i s - H C 1 buffer (pH 7.o). The chrom a t o g r a p h y w a s c a r r i e d o u t in t h e cold room. F r a c t i o n s o5 5 - I o ml of e l u a t e were a n a l y z e d . O - - O , Ae79; ± - - ~ , Azvg/A26e; [~--,3, A T P a s e specific a c t i v i t y .
Bioch4m. Biophys. Acla, 86 (I964) I 8 7 - 1 8 9
SHORT COMMUNICATIONS TABLE RESULTS
OF
SEPHADEX
G-2oo GEL
II
FILTRATION
Preparation
189
IN
FIVE
MYOSIN
PREPARATIONS
Maximal increase in sl)ecific activity*
(%)
i
26
2
20
3
33
4
54
5**
4°
* ATPase specific a c t i v i t y of the peak io-ml fraction as c o m p a r e d w i t h t h a t of t h e s a m p l e applied to the column. ** The p r e p a r a t i o n s h o w n in Table I.
cation of five different preparations (Table II) shows that the ATPase specific activity of the most active Io-ml fraction averaged 35 % higher than the material applied to the column, On the average, pooled fractions from the major elution peak of each preparation contained 7 ° % of the applied protein in one-half its original concentration with a specific ATPase activity 25 % higher than the we-column solution. The total protein eluted from the column was generally 9O-lOO % of that applied. Comparison of the Sephadex column purification technique with the DEAEcellulose ion-exchange column method as recently reported by BRAI-IMSa, PERRY¢, and AsAI5 shows a similar increase in specific ATPase activity. A major advantage of the Sephadex technique is greater Speed, 5-7 h for a complete elution resulting in consistent purification and yield comparable with the most successful of the DEAEcellulose methods. Additional advantages are the avoidance of excessive dilution of the myosin preparation and the need of only one elution buffer. Furthermore, this buffer can contain 0.5 M KC1, therefore minimizing the tendency of myosin to aggregate and precipitate on the column. Such a high ionic strength is Unsuitable for DEAE-cellulose chromatography. One of us (M.S.) holds a postdoctorM research fellowship from the Muscular Dystrophy Association of America.
Department of Biochemistry, University of California Medical Center, San Francisco, Calif. (U.S.A.)
MARVIN SMOLLER RICHARD A. F I N E B E R G
1 A. SZENT-GY6RGYI, Chemistry of Muscular Contraction, Academic Press, New York, 2nd Ed., 1951 , p. 146. 2 W. W. KI•LLEY AND L. B. BRADLEY, J . Biol, Chem., 218 (1956) 653. J. BRAHMS, J. Am. Chem. Soc., 81 (1959) 4997. 4 S. V. PERRY, Biochem. J., 74 (196o) 94. H, ASAI, Biochemistry, 2 (1963) 458. 0 p. FLODIN AND J. I~ILLANDJ~R, Biochim. Biophys, Acta, 63 (1962) 403. 7 p. A. SMALL, W. F. HARRINGTON AND W. W. KIELLEY, Bioehim. Biophys. Acta, 49 (1961) 462. s E. MIHALYI, Ig. LAKI AND M. I. I4NOLLER, Arch. Biochem. Biophys., 68 (1957) 13o.
Received October 26th, 1963 Biochim. Biophys. Acta, 86 (1964) 187-189