/ . Biochem., 80, 315-322 (1976)
Rabbit Skeletal Muscle I. Purification and Characterization1 Masaaki KURODA and Koscak MARUYAMA Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, January 28, 1976
A new regulatory protein which we have designated as f-actinin has been isolated from native thin filaments of rabbit skeletal muscle. Depolymerized native thin filaments were fractionated by salting out with ammonium sulfate, and the precipitates obtained at 40—60% ammonium sulfate saturation were further subjected to DEAE-Sephadex and Sephadex G-200 column chromatography. The purified f-actinin was shown to have a chain weight of 35,000 daltons and had a strong inhibitory action on the polymerization of G-actin. The results of amino acid analysis indicated a unique amino acid composition of r-actinin as compared with other structural proteins of muscle. Non-polar and neutral amino acid residues were abundant. One cysteine residue was contained per one molecule of r-actinin and played a critical role in the maintenance of the inhibitory activity. Pelleting of r-actinin with Factin showed that r-actinin binds to F-action.
Comparison of F-actin polymerized in vitro with native thin filaments isolated directly from myofibrils shows up some clear differences between these two types of actin filaments as regards to their physico-chemical properties (1). The observation that native thin filaments are very hard to repolymerize when once depolymerized is one of these differences {1,2). In a preceding paper, we have shown 1
This work was supported by grants from the Ministry of Education, Science and Culture, of Japan, and from the Muscular Dystrophy Associations of America, Inc. Abbreviations: SDS, sodium dodecyl sulfate ; DTT, dithiothreitol. Vol. 80, No. 2, 1976
315
that this lack of repolymerizability is only apparent (3). Further studies on the nature of repolymerizability of native thin filaments have indicated that a new protein factor exists on the thin filaments which strongly inhibits the polymerization of actin at the nucleation step (3). The present paper describes the purification procedure and some physico-chemical properties of the new protein factor. We have designated this new regulatory protein f-actinin. MATERIALS AND METHODS Preparation of Native Thin Filaments— Native thin filaments were isolated and puri-
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f-Actinin, a New Regulatory Protein from
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RESULTS
Preparation of y-Actinin—Native thin filaments were isoelectrically precipitated at pH 4.7 by the addition of 1 N HC1 and were left to stand for 1 hr at 0°. The precipitates were collected by centrifugation at 12,000 rpm for 20 min and dissolved in Buffer A (containing 0.3 raM DTT and 10 mM Tris-HCl, pH 8.0). After dialysis against Buffer A overnight at 4°, denatured actin and larger aggregates were removed by centrifugation at 30,000 rpm for 1 hr. Ammonium sulfate fractionation was carried out with the supernatant fraction at a protein concentration of 2-3 mg/ml. Solid ammonium sulfate was added up to 20% saturation and the resulting precipitates were sedimented by centrifugation at 12,000 rpm for 20 min. The fraction precipitating at 20-40% ammonium sulfate saturation was also removed by centrifugation. Soft precipitates obtained at 40-60% saturation (designated as the P 60 fraction) were collected and dialyzed against Buffer A. DEAE-Sephadex were equilibrated with Buffer A in the presence of 6 M urea
40 Tube number
50
60
Fig. 1. DEAE-Sephadex chromatography of the P 60 fraction. The P 60 fraction (150 mg) was loaded on the column in the presence of 6 M urea. For conditions, see the text. O, absorbance at 280 nm; • , absorbance at 260 nm; , KC1 concentration. / . Biochem.
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fied from rabbit skeletal muscle as described previously ( 3 ) . Preparation of Proteins—G-actin was prepared from an acetone-dried powder of rabbit skeletal muscle by the method of Mommaerts { 4). Actin free from tropomyosin was purified according to Spudich and Watt (5). Rabbit tropomyosin was purified as described by Fujii and Maruyama (6). Physico-chemical Techniques — An Edsall type apparatus (Rao Instruments Co.) was used to measure flow birefringence. Sedimentation patterns were observed with a Beckman model E ultracentrifuge. Amino acid analysis was carried out using a JEOL JLC-5A automatic amino acid analyzer. SDS polyacrylamide gel electropboresis was performed with 10% gel in the presence of 0.1% SDS according to the method of Weber and Osborn (7). The following proteins were employed as standards for the determination of molecular weight: bovine serum albumin, ovalbumin, chymotrypsinogen A, myoglobin, and cytochrome c. Protein concentration was determined by means of the biuret reaction. Sonication was performed with a generator made by Tomy Co.
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318
b
o
d
e
TROPOHYOSIN IT-ACT IN IN
O4
O6
0.8
1.0
Mobility
Fig. 3. SDS gel electrophoresis pattern at each step of preparation. For conditions, see " MATERIALS AND METHODS." a, P 60 fraction ; b, DE fraction ; c, P-2 fraction (f-actinin) ; d, P-2 fraction (f-actinin); e, 7-actinin and tropomyosin were co-raigrated on the same 10 cm gel.
with a chain weight of about 36,000 daltons, which was probably tropomyosin. The P-2 fraction, namely r-actinin showed a single band in the SDS gel electrophoresis pattern. Physico-chemical Properties—Judging from the results of SDS gel electrophoresis, the chain weight of f-actinin is estimated to be 35,000 daltons (Fig. 3, Fig. 4). When f-actinin was co-migrated with tropomyosin on the same gel, it was found that f-actinin ran a little faster than tropomyosin subunits. However, it is rather difficult to distinguish these two proteins, which have similar chain weights, on 5 cm gels. Longer gels can resolve tropomyosin subunits from f-actinin (Fig. 3e). Preliminary experiments with an analytical centrifuge indicated a sedimentation coefficient (SM.W) of about 1 S. Thus, we assume that the chain weight of the protein corresponds to the molecular weight of r-actinin in solution. The very retarded elution profile of the actinin on Sephadex G-200 column chromatography supports this assumption. The ultraviolet absorption maximum of the f-actinin was 278 nm and the A130/tt0 ratio
Fig. 4. Determination of the chain molecular weight of 7--actinin by SDS gel electrophoresis. Molecular weight calibration was carried out with bovine serum albumin, ovalbumin, chymotrypsinogen A, myoglobin, and cytochrome c as standards. The mobility of f-actinin is indicated by an arrow.
was found to be 1.5—1.6. One unit of A^ roughly corresponds to 1 mg/ml of protein as measured by the biuret method. The amino acid composition of ^-actinin was unique compared with those of other muscle proteins. As shown in Table II, yactinin is rich in neutral and non-polar amino acid residues. Proline was detected as a minor shoulder on the chart of an automatic amino acid analyzer. The number of cysteine residues is of importance since it was shown that the presence of reducing reagents such as DTT is necessary to keep isolated f-actinin active. The cysteine content of f-actinin was very small: three cysteine residues were detected per 999 amino acid residues. Therefore, it was concluded that one polypeptide chain of yactinin contains one cysteine residue. It was shown that 7--actinin is very susceptible to trypsin digestion. When f-actinin was treated with trypsin (1/100 trypsin by weight) for 5 min, the activity was reduced to one-half of the original level. On the other hand, f-actinin was fairly resistant to heat treatment and about 60% of the initial activity was retained even after treatment at 55° for 20 min. However, no activity was observed after incubation at 68° for 8 min. / . Biochtm.
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a
317
r-ACTININ. I
ratio of about 2 and the retarded minor peak (called P-2) had a ratio of 1.5-1.6. The latter peak, P-2, was collected and concentrated on. a membrane filter (Amicon UM-10). KC1 was removed by dialysis against Buffer A. This fraction is the purified 7--actinin. Although the yield at each step of the preparation varied from preparation to preparation, from 600 g of rabbit skeletal muscle,, about 9 g of native thin filaments was released by four cycle of freezing and thawing followed by sonication. After isoelectric precipitation. and clarification, about 700 mg of protein was recovered in the supernatant fraction. The amount of protein obtained as the P 60 fraction was approximately 50—60% of the total supernatant protein. When 150 mg of the P 60 fraction was subjected to DEAE-Sephadex column chromatography, about 80 mg of protein was eluted as the DE fraction and on. further chromatography on Sephadex G-200, only 2—3 mg of f-actinin was obtained as the P-2 fraction from 80 mg of the DE fraction. Thus, starting from 600 g of rabbit skeletal muscle, 5—6 mg of r-actinin was finally isolated (Table I). SDS Gel Electrophoresis Patterns—Figure 3shows the SDS gel electrophoresis patterns of each fraction obtained in the preparative procedure. While the protein composition of the P 60 fraction as determined by SDS gel electrophoresis differed slightly from preparation to preparation, the DE fraction was found to have a constant band pattern. The P-l fraction obtained by Sephadex G-200 column chromatography showed poorly resolved doublet band TABLE I. Yield of r-actinin. Preparation
60
Myofibrils Native thin filaments Clarified supernatant* P 60 fraction DE-fraction P-2 (r-Actinin)
130
Volume (ml)
Fig. 2. Sephadex G-200 chromatography of the DE fraction. Ten ml of the DE fraction (5.42 mg/ml) was applied to the column. For conditions, see the text. , absorbance at 280 nm ; , absorbance at 260 nm. Vol. 80, No. 2, 1976
1
B
500 g 5.1 g 573 mg 244 mg 147 mg 2 mg
520 g 7.5 g 639 mg 402 mg 250 mg 5 mg
600 g 9.1 g 721 mg 389 mg 234 mg 6 mg
The supernatant fraction obtained after clarification at 30,000 rpm for 1 hr.
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and charged on a 3.6x30 cm column. About 150 mg of P 60 fraction previously equilibrated with the column buffer was loaded and a linear 0-0.35 M KC1 gradient was applied in a total volume of 400 ml of the buffer solution. Columns were run at 4° at a flow rate of 10—15 ml/hr and 5 ml fractions were collected. The elution profile is shown in Fig. 1. In most cases, three or four peaks were eluted. Although the overall profile of the chromatogram varied depending upon the quality of the P 60 fraction, the material eluted at 0.2-0.23 M KC1 always appeared as the main peak. The top portion and the right-hand half of the peak eluted around 0.22 M KC1 were collected and dialyzed against Buffer A to remove urea and KC1 (designated as the DE fraction). This DE fraction was further chromatographed by gel filtration. Sephadex G-200 was equilibrated in a buffer solution consisting of 1.2 M KC1, 0.5 mM DTT, and 10 mM Tris-HCl (pH 8.0) and charged on a column (2.4x80 cm) up to 80% of the bed volume. Approximately 10—15 ml of the DE fraction (4—6 mg/ml) was applied to the column. Elution was carried out at a flow rate of 10 ml/hr. As shown in Fig. 2, two distinct peaks were observed; the major one (called P-l in this paper) had an Atso/tto
7-ACTININ. I
319
Amino acid Lys
114
His
25
Arg
18
Asp
93
Thr
48
Ser
127
Glu
160
Pro
trace
Gly
142
Ala
92
Cys
3
Val
38
Met
3
He
31
Leu
57
Tyr
31
Phe
17 999
Total
TABLE III. Relative activity of f-actinin at each step of preparation. Conditions: G-actin concentration, 0.22 mg/ml in the P60 fraction and P-2 fraction, 0.3 mg/ml in the DE fraction: for further conditions, see the text. r-Actinin added (weight percent to actin) 2%
5%
10%
20%
P 60 fraction — 34.7% 70.0% — DE fraction — 47.9% 73.0% 96.4% P-2 fraction 38.0% 56.8% 77.4% — (purified f-actinin)
100-
l
TOCO 7-Actinin added
Activity of y-Actinin—As an index of the activity of r-actinin, its inhibitory action on the polymerization of G-actin was found to be convenient. The measurement was carried out as follows : G-actin (0.2—0.3 mg/ml) was mixed with an appropriate amount of f-actinin in the presence of 0.2 mM ATP and 5 mM Trismaleate, pH 7.2, and left to stand for 5 to 10 min prior to the addition of KC1. This period, which we called as the pre-incubation period, was shown to exert a significant effect on the extent of the inhibitory activity (Kuroda, M. & Maruyama, K. (1976) / . Biochem. 80, 323). Polymerization was initiated by the addition of 0.1 M KC1. After standing for 60 min at 25°, the flow birefringence was measured (dw). The relative activity of f-actinin is expressed as a percentage as follows: X100 where An^ is the flow birefringence of actin Vol. 80, No. 2, 1976
Fig. 5. Activity curve of r-actinin. Various amount of T'-actinin (DE fraction) were added to G-actin and polymerized by the addition of 0 . 1 M KC1 or 3 mM MgCl,. For conditions, see the text. The birefringence of the control was 68° at 0.22 mg/ml of G-actin. Open symbols: polymerized by KC1, Solid symbols: polymerized by MgCl,.
polymerized in the absence of 7"-actinin. The relative activity at each step of purification is shown in Table III. It is noteworthy that the inhibitory action of f-actinin did not increase very greatly as it was purified, suggesting that loss of activity took place during purification procedures. Figure 5 shows the inhibitory activity of j-actinin at various ratios to actin. On the other hand, when G-actin was polymerized by the addition of divalent cations such as Mg*+ or Ca1+ instead of KC1, the inhibitory activity of /--actinin greatly decreased (Fig. 5). Moreover, an addition of MgCli released the inhibition attained in 0.1 M KC1. G-actin solu-
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TABLE II. Amino acid composition of f-actinin (number of amino acid residues per 999 residues). Hydrolysis, for 24 hr at 110°.
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M. KURODA and K. MARUYAMA
and G-actin polymerized completely into Factin in the same way as in the absence of yactinin. However, the effect of DTT was reversible and the addition of DTT to the inactive form of r-actinin immediately restored the activity (Table IV). Interaction with F-Actin and Tropomyosin —The interaction of f-actinin with F-actin was investigated using the DE fraction. DEAESephadex-purified f-actinin was clarified by centrifugation at 40,000 rpm for 1 hr. A reaction mixture (25 ml) containing 0.3 mg/ml of G-actin, 0.2 mM ATP, and 5 mM Trismaleate (pH 7.2) was pre-incubated in the presence of 0.06 mg/ml of the f-actinin fraction for 10 min at 25°. KC1 was added to a final concentration of 0.1 M and the mixture was allowed to stand for 1 hr. The birefringence of the mixture was confirmed to be zero. Then one-half of the reaction mixture was subjected to sonication in order to induce complete polymerization (Kuroda, M. & Maruyama, K. (1976) TABLE IV. Effect of dithiothreitol on the inhibitory activity of f-actinin. DTT was removed from the DE fraction by dialysis against 2 mM Tris buffer (pH 8.0). For measurement of the activity, 20% of DE fraction was added to 0.2 mg/ml of G-actin. For other conditions, see the text.
Fig. 6. Effect of various concentrations of MgClt on the action of 7--actinin (DE fraction). For conditions, see the text. Birefringence of the control, 51°.
?--Actinin f-Actinin dialyzed As above, +0.2 mM DTT
Birefringence
% inhibition
0°
100%
51.2°
0%
100%
0°
TABLE V. Interaction with F-actin. The repolymerizability of depolymerized pellet was checked by the addition of 0.1 M KC1 to the depolymerized solution (0.25 mg/ml). After incubation at 25° for one hour, the flow birefringence was measured at 1,030 sec"1. For other conditions, see the text. After centrifugation
Birefringence 60'
G-Actin + f-actinin G-Actin + r-actinin + KCI As above, sonicated F-Actin
82°
F-Actin + 7-actinin
80.4°
90'
0°
0°
0°
0°
0°
75°
Supernatant 0.31 0.30 0.05 0.05 0.05
mg/ml mg/ml mg/ml mg/ml mg/ml
Pellet (-) (-) (+) (+) (+)
Repolymerizability
0°
50.2° 0°
/ . Biochem.
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tion (0.2 mg/ml) was pre-incubated in the presence of 10% r-actinin, 0.2 mM ATP, and 5 mM Tris-maleate (pH 7.2) for 10 min at 25° and after the addition of 0.1 M KC1, the reaction mixtures were kept standing for 1 hr. The birefringence of the mixtures was found to be zero under these conditions. Next, various concentrations of MgCU were added to the reaction mixtures, which were further incubated for 30 min. As seen in Fig. 6, 2 mM MgClt was sufficient to release 100% inhibition almost completely. As pointed out previously, reducing reagents like DTT are required to maintain the activity of ^-actinin. When DTT was removed by dialysis, inhibitory activity was entirely lost
r-ACTININ. I
321
However, the preparation used was the DEAE-Sephadex-purified r-actinin, which con-
ACTIN X -ACTININ
Fig. 7. Pelleting of r-actinin and F-actin. G-actin (0.5 rag/ml) was polymerized in the presence of purified r-actinin. After standing overnight, the actin solution was centrifugated at 30,000 rpm for 3 hr and the pellet was check by means of SDS gel electrophoresis. Solvent conditions, as in Table IV.
Vol. 80, No. 2, 1976
tained a large amount of tropomyosin together with r-actinin. So far as examined, however, no direct interaction was detected between purified r-actinin and tropomyosin. The inhibitory effect of /-actinin was not affected by the addition of tropomyosin. Pelleting experiments with purified r-actinin and F-actin confirmed that r-actinin binds directly to F-actin (Fig. 7). DISCUSSION The previous paper suggested the presence of a protein factor which inhibits the polymerization of actin in native thin filaments (3). An attempt to isolate this protein factor revealed the existence of a new regulatory protein with a molecular weight of 35,000 daltons : we have designated this protein r-actinin. Although the precise localization of r-actinin in the myofibrils is not still clear, it is very probable that 7--actinin exists on the thin filaments. When myofibrils were treated with acetone and Gactin was extracted with water, r-actinin was apparently retained in the actin-extracted residues, since the crude preparation of native tropomyosin which was extracted from the residues with 1 M KC1 showed some inhibitory activity in the same way as r-actinin. The solubility of r-actinin in water at neutral pH was found to be poor and it was difficult to concentrate r-actinin to more than 0.5 mg/ml, at which level visible precipitates were formed on the membrane filter used for the concentration. In the presence of 1M KC1, the solubility was much improved. However, it appeared that once formed, precipitates could not be completely dissolved. The observation that r-actinin contains a considerable amount of neutral or non-polar amino acid residues may partly account for this low solubility of yactinin in water. The yield of r-actinin prepared by the present method was very low; starting from 1 kg of myofibrils, the yeild of r-actinin was less than 10 mg. This may be due partly to the low content of r-actinin in myofibrils, but it is possible that some denaturation of yactinin takes place in the course of preparation (c/. Table III). Recent observations have
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/ . Biochem. 80, 323), while the other half was left to stand without any agitation. After a further 30 min, both systems were spun at 30,000 rpm for 3 hr. Two controls were centrifugated at the same time; one was F-actin polymerized in the absence of r-actinin and the other was r-actinin added to preformed Factin (20% weight ratio). After centrifugation, the protein concentrations of the supernatants were measured. It was found that almost all the protein was sedimented in the pellet in the sonicated system, as well as in the two controls. On the other hand, in the reaction mixture which was kept standing without agitation, the birefringence of which was confirmed to be zero before centrifugation, no pellet was obtained and the whole of the protein was retained in the supernatant fraction (Table V). The pellets obtained were homogenized to depolymerize F-actin and dialyzed overnight against 0.3 mM ATP, 0.1 mM DTT, and 2 mM Tris-HCl (pH 8.0). The dialyzed solutions were clarified at 30,000 rpm for 1 hr and repolymerizability of each system was examined. As is clearly shown in Table V, the reaction mixtures sedimented in the presence of r-actinin lacked repolymerizability. These observations strongly suggest that yactinin binds to F-actin and retains its inhibitory activity.
322
The authors thank Miss F. Akiyama of Ochanomizu University for carrying out amino acid analysis. REFERENCES 1. Hama, H., Maruyama, K., & Noda, H. (1965) Biochim. Biophys. Ada 102, 249-260 2. Suzuki, S., Kawamura, M., & Maruyama, K. (1971) Comp. Biochem. Physiol. 38A, 147-155 3. Kuroda, M. & Maruyama, K. (1976) / . Biochem. 79, 249-258 4. Mommaerts, W.F.H.M. (1951) / . Biol. Chem. 188, 559-565 5. Spudich, J.A. & Watt, S. (1972) / . Biol. Chem. 246, 4866-4871 6. Fujii, T. & Maruyama, K. (1971) Sci. Pap. Coll. Gen. Educ, Univ. Tokyo 21, 45-61 7. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406-4412 8. Ebashi, S., Wakabayashi, T., & Ebashi, F. (1971) /. Biochem. 69, 441-446 9. Maruyama, K. (1971) in Contractile Proteins and Muscle (Laki, K., ed.) Vol. 2, pp. 289-313, Marcel Dekker, Inc., New York 10. Masaki, T. & Takaiti, O. (1969) / . Biochem. 66, 637-643
/ . Biochem.
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shown that 7-actinin from chicken breast muscle is rather unstable when subjected to DEAESephadex column chromatography in the presence of 6 M urea. The 35,000 daltons molecular weight of yactinin resembles those of tropomyosin subunits ( 7 ) and troponin T (8). However, the amino acid composition of f-actinin differs from those of known proteins. The most striking feature is the abundance of glycine and serine in f-actinin. Finally, we would like to mention why the present new regulatory protein was named as r-actinin. Originally, "actinin" referred to myofibrillar proteins with an amino acid composition close to that of actin(P). However, this similarity in amino acid composition was proved to be largely due to the contamination of actin and 10 S-actinin (10, Maruyama, K., unpublished observation). Hence, the definition of actinin has been changed as follows : a group of myofibrillar proteins which interacts with F-actin and affects its dynamic structure. Therefore, the present new regulatory protein is designated as f-actinin, in line with a- and y9-actinin (9).
M. KURODA and K. MARUYAMA
Rabbit Skeletal Muscle I. Purification and Characterization1 Masaaki KURODA and Koscak MARUYAMA Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, January 28, 1976
A new regulatory protein which we have designated as f-actinin has been isolated from native thin filaments of rabbit skeletal muscle. Depolymerized native thin filaments were fractionated by salting out with ammonium sulfate, and the precipitates obtained at 40—60% ammonium sulfate saturation were further subjected to DEAE-Sephadex and Sephadex G-200 column chromatography. The purified f-actinin was shown to have a chain weight of 35,000 daltons and had a strong inhibitory action on the polymerization of G-actin. The results of amino acid analysis indicated a unique amino acid composition of r-actinin as compared with other structural proteins of muscle. Non-polar and neutral amino acid residues were abundant. One cysteine residue was contained per one molecule of r-actinin and played a critical role in the maintenance of the inhibitory activity. Pelleting of r-actinin with Factin showed that r-actinin binds to F-action.
Comparison of F-actin polymerized in vitro with native thin filaments isolated directly from myofibrils shows up some clear differences between these two types of actin filaments as regards to their physico-chemical properties (1). The observation that native thin filaments are very hard to repolymerize when once depolymerized is one of these differences {1,2). In a preceding paper, we have shown 1
This work was supported by grants from the Ministry of Education, Science and Culture, of Japan, and from the Muscular Dystrophy Associations of America, Inc. Abbreviations: SDS, sodium dodecyl sulfate ; DTT, dithiothreitol. Vol. 80, No. 2, 1976
315
that this lack of repolymerizability is only apparent (3). Further studies on the nature of repolymerizability of native thin filaments have indicated that a new protein factor exists on the thin filaments which strongly inhibits the polymerization of actin at the nucleation step (3). The present paper describes the purification procedure and some physico-chemical properties of the new protein factor. We have designated this new regulatory protein f-actinin. MATERIALS AND METHODS Preparation of Native Thin Filaments— Native thin filaments were isolated and puri-
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f-Actinin, a New Regulatory Protein from
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RESULTS
Preparation of y-Actinin—Native thin filaments were isoelectrically precipitated at pH 4.7 by the addition of 1 N HC1 and were left to stand for 1 hr at 0°. The precipitates were collected by centrifugation at 12,000 rpm for 20 min and dissolved in Buffer A (containing 0.3 raM DTT and 10 mM Tris-HCl, pH 8.0). After dialysis against Buffer A overnight at 4°, denatured actin and larger aggregates were removed by centrifugation at 30,000 rpm for 1 hr. Ammonium sulfate fractionation was carried out with the supernatant fraction at a protein concentration of 2-3 mg/ml. Solid ammonium sulfate was added up to 20% saturation and the resulting precipitates were sedimented by centrifugation at 12,000 rpm for 20 min. The fraction precipitating at 20-40% ammonium sulfate saturation was also removed by centrifugation. Soft precipitates obtained at 40-60% saturation (designated as the P 60 fraction) were collected and dialyzed against Buffer A. DEAE-Sephadex were equilibrated with Buffer A in the presence of 6 M urea
40 Tube number
50
60
Fig. 1. DEAE-Sephadex chromatography of the P 60 fraction. The P 60 fraction (150 mg) was loaded on the column in the presence of 6 M urea. For conditions, see the text. O, absorbance at 280 nm; • , absorbance at 260 nm; , KC1 concentration. / . Biochem.
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fied from rabbit skeletal muscle as described previously ( 3 ) . Preparation of Proteins—G-actin was prepared from an acetone-dried powder of rabbit skeletal muscle by the method of Mommaerts { 4). Actin free from tropomyosin was purified according to Spudich and Watt (5). Rabbit tropomyosin was purified as described by Fujii and Maruyama (6). Physico-chemical Techniques — An Edsall type apparatus (Rao Instruments Co.) was used to measure flow birefringence. Sedimentation patterns were observed with a Beckman model E ultracentrifuge. Amino acid analysis was carried out using a JEOL JLC-5A automatic amino acid analyzer. SDS polyacrylamide gel electropboresis was performed with 10% gel in the presence of 0.1% SDS according to the method of Weber and Osborn (7). The following proteins were employed as standards for the determination of molecular weight: bovine serum albumin, ovalbumin, chymotrypsinogen A, myoglobin, and cytochrome c. Protein concentration was determined by means of the biuret reaction. Sonication was performed with a generator made by Tomy Co.
M. KURODA and K. MARUYAMA
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b
o
d
e
TROPOHYOSIN IT-ACT IN IN
O4
O6
0.8
1.0
Mobility
Fig. 3. SDS gel electrophoresis pattern at each step of preparation. For conditions, see " MATERIALS AND METHODS." a, P 60 fraction ; b, DE fraction ; c, P-2 fraction (f-actinin) ; d, P-2 fraction (f-actinin); e, 7-actinin and tropomyosin were co-raigrated on the same 10 cm gel.
with a chain weight of about 36,000 daltons, which was probably tropomyosin. The P-2 fraction, namely r-actinin showed a single band in the SDS gel electrophoresis pattern. Physico-chemical Properties—Judging from the results of SDS gel electrophoresis, the chain weight of f-actinin is estimated to be 35,000 daltons (Fig. 3, Fig. 4). When f-actinin was co-migrated with tropomyosin on the same gel, it was found that f-actinin ran a little faster than tropomyosin subunits. However, it is rather difficult to distinguish these two proteins, which have similar chain weights, on 5 cm gels. Longer gels can resolve tropomyosin subunits from f-actinin (Fig. 3e). Preliminary experiments with an analytical centrifuge indicated a sedimentation coefficient (SM.W) of about 1 S. Thus, we assume that the chain weight of the protein corresponds to the molecular weight of r-actinin in solution. The very retarded elution profile of the actinin on Sephadex G-200 column chromatography supports this assumption. The ultraviolet absorption maximum of the f-actinin was 278 nm and the A130/tt0 ratio
Fig. 4. Determination of the chain molecular weight of 7--actinin by SDS gel electrophoresis. Molecular weight calibration was carried out with bovine serum albumin, ovalbumin, chymotrypsinogen A, myoglobin, and cytochrome c as standards. The mobility of f-actinin is indicated by an arrow.
was found to be 1.5—1.6. One unit of A^ roughly corresponds to 1 mg/ml of protein as measured by the biuret method. The amino acid composition of ^-actinin was unique compared with those of other muscle proteins. As shown in Table II, yactinin is rich in neutral and non-polar amino acid residues. Proline was detected as a minor shoulder on the chart of an automatic amino acid analyzer. The number of cysteine residues is of importance since it was shown that the presence of reducing reagents such as DTT is necessary to keep isolated f-actinin active. The cysteine content of f-actinin was very small: three cysteine residues were detected per 999 amino acid residues. Therefore, it was concluded that one polypeptide chain of yactinin contains one cysteine residue. It was shown that 7--actinin is very susceptible to trypsin digestion. When f-actinin was treated with trypsin (1/100 trypsin by weight) for 5 min, the activity was reduced to one-half of the original level. On the other hand, f-actinin was fairly resistant to heat treatment and about 60% of the initial activity was retained even after treatment at 55° for 20 min. However, no activity was observed after incubation at 68° for 8 min. / . Biochtm.
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a
317
r-ACTININ. I
ratio of about 2 and the retarded minor peak (called P-2) had a ratio of 1.5-1.6. The latter peak, P-2, was collected and concentrated on. a membrane filter (Amicon UM-10). KC1 was removed by dialysis against Buffer A. This fraction is the purified 7--actinin. Although the yield at each step of the preparation varied from preparation to preparation, from 600 g of rabbit skeletal muscle,, about 9 g of native thin filaments was released by four cycle of freezing and thawing followed by sonication. After isoelectric precipitation. and clarification, about 700 mg of protein was recovered in the supernatant fraction. The amount of protein obtained as the P 60 fraction was approximately 50—60% of the total supernatant protein. When 150 mg of the P 60 fraction was subjected to DEAE-Sephadex column chromatography, about 80 mg of protein was eluted as the DE fraction and on. further chromatography on Sephadex G-200, only 2—3 mg of f-actinin was obtained as the P-2 fraction from 80 mg of the DE fraction. Thus, starting from 600 g of rabbit skeletal muscle, 5—6 mg of r-actinin was finally isolated (Table I). SDS Gel Electrophoresis Patterns—Figure 3shows the SDS gel electrophoresis patterns of each fraction obtained in the preparative procedure. While the protein composition of the P 60 fraction as determined by SDS gel electrophoresis differed slightly from preparation to preparation, the DE fraction was found to have a constant band pattern. The P-l fraction obtained by Sephadex G-200 column chromatography showed poorly resolved doublet band TABLE I. Yield of r-actinin. Preparation
60
Myofibrils Native thin filaments Clarified supernatant* P 60 fraction DE-fraction P-2 (r-Actinin)
130
Volume (ml)
Fig. 2. Sephadex G-200 chromatography of the DE fraction. Ten ml of the DE fraction (5.42 mg/ml) was applied to the column. For conditions, see the text. , absorbance at 280 nm ; , absorbance at 260 nm. Vol. 80, No. 2, 1976
1
B
500 g 5.1 g 573 mg 244 mg 147 mg 2 mg
520 g 7.5 g 639 mg 402 mg 250 mg 5 mg
600 g 9.1 g 721 mg 389 mg 234 mg 6 mg
The supernatant fraction obtained after clarification at 30,000 rpm for 1 hr.
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and charged on a 3.6x30 cm column. About 150 mg of P 60 fraction previously equilibrated with the column buffer was loaded and a linear 0-0.35 M KC1 gradient was applied in a total volume of 400 ml of the buffer solution. Columns were run at 4° at a flow rate of 10—15 ml/hr and 5 ml fractions were collected. The elution profile is shown in Fig. 1. In most cases, three or four peaks were eluted. Although the overall profile of the chromatogram varied depending upon the quality of the P 60 fraction, the material eluted at 0.2-0.23 M KC1 always appeared as the main peak. The top portion and the right-hand half of the peak eluted around 0.22 M KC1 were collected and dialyzed against Buffer A to remove urea and KC1 (designated as the DE fraction). This DE fraction was further chromatographed by gel filtration. Sephadex G-200 was equilibrated in a buffer solution consisting of 1.2 M KC1, 0.5 mM DTT, and 10 mM Tris-HCl (pH 8.0) and charged on a column (2.4x80 cm) up to 80% of the bed volume. Approximately 10—15 ml of the DE fraction (4—6 mg/ml) was applied to the column. Elution was carried out at a flow rate of 10 ml/hr. As shown in Fig. 2, two distinct peaks were observed; the major one (called P-l in this paper) had an Atso/tto
7-ACTININ. I
319
Amino acid Lys
114
His
25
Arg
18
Asp
93
Thr
48
Ser
127
Glu
160
Pro
trace
Gly
142
Ala
92
Cys
3
Val
38
Met
3
He
31
Leu
57
Tyr
31
Phe
17 999
Total
TABLE III. Relative activity of f-actinin at each step of preparation. Conditions: G-actin concentration, 0.22 mg/ml in the P60 fraction and P-2 fraction, 0.3 mg/ml in the DE fraction: for further conditions, see the text. r-Actinin added (weight percent to actin) 2%
5%
10%
20%
P 60 fraction — 34.7% 70.0% — DE fraction — 47.9% 73.0% 96.4% P-2 fraction 38.0% 56.8% 77.4% — (purified f-actinin)
100-
l
TOCO 7-Actinin added
Activity of y-Actinin—As an index of the activity of r-actinin, its inhibitory action on the polymerization of G-actin was found to be convenient. The measurement was carried out as follows : G-actin (0.2—0.3 mg/ml) was mixed with an appropriate amount of f-actinin in the presence of 0.2 mM ATP and 5 mM Trismaleate, pH 7.2, and left to stand for 5 to 10 min prior to the addition of KC1. This period, which we called as the pre-incubation period, was shown to exert a significant effect on the extent of the inhibitory activity (Kuroda, M. & Maruyama, K. (1976) / . Biochem. 80, 323). Polymerization was initiated by the addition of 0.1 M KC1. After standing for 60 min at 25°, the flow birefringence was measured (dw). The relative activity of f-actinin is expressed as a percentage as follows: X100 where An^ is the flow birefringence of actin Vol. 80, No. 2, 1976
Fig. 5. Activity curve of r-actinin. Various amount of T'-actinin (DE fraction) were added to G-actin and polymerized by the addition of 0 . 1 M KC1 or 3 mM MgCl,. For conditions, see the text. The birefringence of the control was 68° at 0.22 mg/ml of G-actin. Open symbols: polymerized by KC1, Solid symbols: polymerized by MgCl,.
polymerized in the absence of 7"-actinin. The relative activity at each step of purification is shown in Table III. It is noteworthy that the inhibitory action of f-actinin did not increase very greatly as it was purified, suggesting that loss of activity took place during purification procedures. Figure 5 shows the inhibitory activity of j-actinin at various ratios to actin. On the other hand, when G-actin was polymerized by the addition of divalent cations such as Mg*+ or Ca1+ instead of KC1, the inhibitory activity of /--actinin greatly decreased (Fig. 5). Moreover, an addition of MgCli released the inhibition attained in 0.1 M KC1. G-actin solu-
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TABLE II. Amino acid composition of f-actinin (number of amino acid residues per 999 residues). Hydrolysis, for 24 hr at 110°.
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M. KURODA and K. MARUYAMA
and G-actin polymerized completely into Factin in the same way as in the absence of yactinin. However, the effect of DTT was reversible and the addition of DTT to the inactive form of r-actinin immediately restored the activity (Table IV). Interaction with F-Actin and Tropomyosin —The interaction of f-actinin with F-actin was investigated using the DE fraction. DEAESephadex-purified f-actinin was clarified by centrifugation at 40,000 rpm for 1 hr. A reaction mixture (25 ml) containing 0.3 mg/ml of G-actin, 0.2 mM ATP, and 5 mM Trismaleate (pH 7.2) was pre-incubated in the presence of 0.06 mg/ml of the f-actinin fraction for 10 min at 25°. KC1 was added to a final concentration of 0.1 M and the mixture was allowed to stand for 1 hr. The birefringence of the mixture was confirmed to be zero. Then one-half of the reaction mixture was subjected to sonication in order to induce complete polymerization (Kuroda, M. & Maruyama, K. (1976) TABLE IV. Effect of dithiothreitol on the inhibitory activity of f-actinin. DTT was removed from the DE fraction by dialysis against 2 mM Tris buffer (pH 8.0). For measurement of the activity, 20% of DE fraction was added to 0.2 mg/ml of G-actin. For other conditions, see the text.
Fig. 6. Effect of various concentrations of MgClt on the action of 7--actinin (DE fraction). For conditions, see the text. Birefringence of the control, 51°.
?--Actinin f-Actinin dialyzed As above, +0.2 mM DTT
Birefringence
% inhibition
0°
100%
51.2°
0%
100%
0°
TABLE V. Interaction with F-actin. The repolymerizability of depolymerized pellet was checked by the addition of 0.1 M KC1 to the depolymerized solution (0.25 mg/ml). After incubation at 25° for one hour, the flow birefringence was measured at 1,030 sec"1. For other conditions, see the text. After centrifugation
Birefringence 60'
G-Actin + f-actinin G-Actin + r-actinin + KCI As above, sonicated F-Actin
82°
F-Actin + 7-actinin
80.4°
90'
0°
0°
0°
0°
0°
75°
Supernatant 0.31 0.30 0.05 0.05 0.05
mg/ml mg/ml mg/ml mg/ml mg/ml
Pellet (-) (-) (+) (+) (+)
Repolymerizability
0°
50.2° 0°
/ . Biochem.
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tion (0.2 mg/ml) was pre-incubated in the presence of 10% r-actinin, 0.2 mM ATP, and 5 mM Tris-maleate (pH 7.2) for 10 min at 25° and after the addition of 0.1 M KC1, the reaction mixtures were kept standing for 1 hr. The birefringence of the mixtures was found to be zero under these conditions. Next, various concentrations of MgCU were added to the reaction mixtures, which were further incubated for 30 min. As seen in Fig. 6, 2 mM MgClt was sufficient to release 100% inhibition almost completely. As pointed out previously, reducing reagents like DTT are required to maintain the activity of ^-actinin. When DTT was removed by dialysis, inhibitory activity was entirely lost
r-ACTININ. I
321
However, the preparation used was the DEAE-Sephadex-purified r-actinin, which con-
ACTIN X -ACTININ
Fig. 7. Pelleting of r-actinin and F-actin. G-actin (0.5 rag/ml) was polymerized in the presence of purified r-actinin. After standing overnight, the actin solution was centrifugated at 30,000 rpm for 3 hr and the pellet was check by means of SDS gel electrophoresis. Solvent conditions, as in Table IV.
Vol. 80, No. 2, 1976
tained a large amount of tropomyosin together with r-actinin. So far as examined, however, no direct interaction was detected between purified r-actinin and tropomyosin. The inhibitory effect of /-actinin was not affected by the addition of tropomyosin. Pelleting experiments with purified r-actinin and F-actin confirmed that r-actinin binds directly to F-actin (Fig. 7). DISCUSSION The previous paper suggested the presence of a protein factor which inhibits the polymerization of actin in native thin filaments (3). An attempt to isolate this protein factor revealed the existence of a new regulatory protein with a molecular weight of 35,000 daltons : we have designated this protein r-actinin. Although the precise localization of r-actinin in the myofibrils is not still clear, it is very probable that 7--actinin exists on the thin filaments. When myofibrils were treated with acetone and Gactin was extracted with water, r-actinin was apparently retained in the actin-extracted residues, since the crude preparation of native tropomyosin which was extracted from the residues with 1 M KC1 showed some inhibitory activity in the same way as r-actinin. The solubility of r-actinin in water at neutral pH was found to be poor and it was difficult to concentrate r-actinin to more than 0.5 mg/ml, at which level visible precipitates were formed on the membrane filter used for the concentration. In the presence of 1M KC1, the solubility was much improved. However, it appeared that once formed, precipitates could not be completely dissolved. The observation that r-actinin contains a considerable amount of neutral or non-polar amino acid residues may partly account for this low solubility of yactinin in water. The yield of r-actinin prepared by the present method was very low; starting from 1 kg of myofibrils, the yeild of r-actinin was less than 10 mg. This may be due partly to the low content of r-actinin in myofibrils, but it is possible that some denaturation of yactinin takes place in the course of preparation (c/. Table III). Recent observations have
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/ . Biochem. 80, 323), while the other half was left to stand without any agitation. After a further 30 min, both systems were spun at 30,000 rpm for 3 hr. Two controls were centrifugated at the same time; one was F-actin polymerized in the absence of r-actinin and the other was r-actinin added to preformed Factin (20% weight ratio). After centrifugation, the protein concentrations of the supernatants were measured. It was found that almost all the protein was sedimented in the pellet in the sonicated system, as well as in the two controls. On the other hand, in the reaction mixture which was kept standing without agitation, the birefringence of which was confirmed to be zero before centrifugation, no pellet was obtained and the whole of the protein was retained in the supernatant fraction (Table V). The pellets obtained were homogenized to depolymerize F-actin and dialyzed overnight against 0.3 mM ATP, 0.1 mM DTT, and 2 mM Tris-HCl (pH 8.0). The dialyzed solutions were clarified at 30,000 rpm for 1 hr and repolymerizability of each system was examined. As is clearly shown in Table V, the reaction mixtures sedimented in the presence of r-actinin lacked repolymerizability. These observations strongly suggest that yactinin binds to F-actin and retains its inhibitory activity.
322
The authors thank Miss F. Akiyama of Ochanomizu University for carrying out amino acid analysis. REFERENCES 1. Hama, H., Maruyama, K., & Noda, H. (1965) Biochim. Biophys. Ada 102, 249-260 2. Suzuki, S., Kawamura, M., & Maruyama, K. (1971) Comp. Biochem. Physiol. 38A, 147-155 3. Kuroda, M. & Maruyama, K. (1976) / . Biochem. 79, 249-258 4. Mommaerts, W.F.H.M. (1951) / . Biol. Chem. 188, 559-565 5. Spudich, J.A. & Watt, S. (1972) / . Biol. Chem. 246, 4866-4871 6. Fujii, T. & Maruyama, K. (1971) Sci. Pap. Coll. Gen. Educ, Univ. Tokyo 21, 45-61 7. Weber, K. & Osborn, M. (1969) / . Biol. Chem. 244, 4406-4412 8. Ebashi, S., Wakabayashi, T., & Ebashi, F. (1971) /. Biochem. 69, 441-446 9. Maruyama, K. (1971) in Contractile Proteins and Muscle (Laki, K., ed.) Vol. 2, pp. 289-313, Marcel Dekker, Inc., New York 10. Masaki, T. & Takaiti, O. (1969) / . Biochem. 66, 637-643
/ . Biochem.
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shown that 7-actinin from chicken breast muscle is rather unstable when subjected to DEAESephadex column chromatography in the presence of 6 M urea. The 35,000 daltons molecular weight of yactinin resembles those of tropomyosin subunits ( 7 ) and troponin T (8). However, the amino acid composition of f-actinin differs from those of known proteins. The most striking feature is the abundance of glycine and serine in f-actinin. Finally, we would like to mention why the present new regulatory protein was named as r-actinin. Originally, "actinin" referred to myofibrillar proteins with an amino acid composition close to that of actin(P). However, this similarity in amino acid composition was proved to be largely due to the contamination of actin and 10 S-actinin (10, Maruyama, K., unpublished observation). Hence, the definition of actinin has been changed as follows : a group of myofibrillar proteins which interacts with F-actin and affects its dynamic structure. Therefore, the present new regulatory protein is designated as f-actinin, in line with a- and y9-actinin (9).
M. KURODA and K. MARUYAMA
