J. Biochem., 81, 977-988 (1977)
Muscle Myosin by Chymotryptic Digestion1 Koichi YAGI and Hideto KUWAYAMA Department of Chemistry, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, August 23, 1976
Subfragment-1 was prepared from pig cardiac myosin by chymotryptic digestion at low ionic strength at 0-2°C for 20 h. Two components were distinguished in polyacrylamide gel electrophoresis of subfragment-1 preparations, and were designated as S-l (CT) and S-l (CT)'. The isoelectric points were 6.70 and 6.45, respectively. The two components could not be separated by Sephadex G-200 gel filtration, but they were separated on a DEAE-cellulose (DE 32) column or a 6-aminohexylPPi-Sepharose 4B conjugate column. S-l (CT) consisted of one large polypeptide chain (the head portion of the f-chain) and one small polypeptide chain (gj. In SDS gel electrophoresis, the large polypeptide of S-l (CT) migrated at the same rate as that of S-l (CT)', but the small polypeptide of S-l (CT)' migrated at a faster rate than that of S-l (CT) The small chain of S-l (CT)' was thus designated as g/. The migration rate of the small chain of S-l (CT) was the same as that of g! of cardiac myosin, but the small chain of S-l (CT)' migrated faster than gx and slower than g, of cardiac myosin. It was therefore suggested that S-l (CT)' was produced by overdigestion of S-l (CT). The yield of S-l (CT) plus S-l (CT)' estimated on the basis of absorption at 280 nm was 36% of the weight of myosin employed. The molecular weight was 1.19 x 10" and the maximum ADP binding number was 0.48 mol using S-l (CT) 30% contaminated by S-l (CT)'. The pH-activity relationships in the presence of Ca1+ and EDTA were investigated. The ATPase activity in the presence of CaI+ at pH 7.5 was 0.3 ftmo\ mg-^min" 1 for both S-l (CT) and S-l (CT)'. The maximum ATPase activity of S-l (CT) in the presence of F-actin was 8.3 //mol mg^-min" 1 and the affinity for F-actin was about 1/20 of that of skeletal S-l (CT).
Mueller and Perry (7) were the first to recognize the presence of subfragment-1 (S-l) in heavy meromyosin (HMM) preparations from skeletal muscle myosin. Since then, methods of preparation for
S-l and its properties have been extensively investigated {2-7). Great emphasis has been placed, in recent years, on the composition of S-l preparations, because the amount of Pi liberated in the
1 This work was aided by grants from the Muscular Dystrophy Associations of America, Inc. and from the Ministry of Education, Science and Culture of Japan. Abbreviations' S-l, subfragment-1; S-l(CT) or S-l(P), S-l prepared from myosin using chymotrypsin or papain, respectively; HMM, heavy meromyosin; MOPS, morpholinopropane sulfonic acid; SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophosphate; Nbsj, 5,5'-dithio-bis(2-nitrobenzoic acid).
Vol. 81, No. 4, 1977
977
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
Preparation of Myosin Subfragment-1 from Pig Cardiac
978
raphy. The maximum number of mol of bound ADP was about 0.48 mol per 1.19xl0»g of S-l(CT). This S-l(CT) contained only one g-chain amounting to nearly 1 mol per mol. A preliminary communication has already appeared elsewhere (21). EXPERIMENTAL PROCEDURES
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
initial burst of ATPase is about a half mol per mol of S-1 (8) and the maximum amount of ADP bound to S-1 is also nearly a half mol of ADP per mol of S-1 (9). It therefore seems possible that S-1 consists of two components, one of which shows neither the initial burst nor ADP binding under the conditions used. S-1 prepared by chymotryptic digestion of skeletal myosin can be separated into two different components (pi and p2), which contain either gx or ga as the small polypeptide chain of S-1 (5). However, the maximum amount of ADP bound to pi or p2 was the same as that bound to the S-1 preparations before fractionation into pi and p2 (10). Myosin of cardiac muscle contains two different g chains in an equimolar ratio (11), while skeletal white muscle myosin contains three different g chains in various molar ratios, e.g. 1.35: 2.00 : 0.65 according to Sarkar (12). It is therefore suggested that myosin consists of two populations in various ratios. It is further suggested that pi and p2 are formed from different myosin isozymes. On the other hand, the content of g-chains in cardiac myosin suggests that one kind of myosin is present in cardiac myosin preparations, and only one kind of S-1 with respect to g chain could be obtained. Preparation of cardiac myosin has been investigated by Ellenbogen et al. (13), Mueller (14), and Barany et al. (15), and a procedure to obtain pure myosin was recently established by WikmanCoffelt et al. (16). Cardiac myosin is more resistant to trypsin than skeletal myosin (17) and Mueller et al. {18) demonstrated that an unusually high weight ratio of trypsin to myosin (1 : 25) was required for the preparation of HMM from canine cardiac myosin. According to Tada et al. (19), no significant amount of S-1 could be isolated by tryptic or chymotryptic digestion of bovine cardiac HMM, but it could be obtained from cardiac HMM and myosin by papain digestion at low temperature. Wolodko and Kay (20) recently isolated S-1 from rabbit cardiac myosin using papain. In this paper, a method for the preparation of S-1 from pig cardiac myosin is presented. It involves chymotryptic digestion at 0-2°C and at low ionic strength. An apparently homogeneous S-l(CT) preparation was obtained after purification using DEAE-cellulose (DE 32) or 6-aminohexyl PPi-Sepharose 4B conjugate column chromatog-
K. YAGI and H. KUWAYAMA
Myosin—Myosin was prepared from fresh pig heart by a method similar to that described by Wikman-Coffelt et al. (16). Myosin was stored in 50% glycerol at a temperature below — 10°C. If necessary, myosin was further purified by DEAESephadex A-50 chromatography (22) before use. Concentrations of myosin were determined from the absorbance using a value of A^Dm=4.7, which was obtained by the micro-Kjeldahl method assuming the nitrogen content to be 16%. F-Actin—Actin was extracted from acetonedried powder of rabbit skeletal muscle at 0°C and purified essentially according to the method of Mommaerts (23). Preparation of 6-AminohexylPP rSepharose 4B Conjugate Column—CNBr-activated Sepharose 4B was prepared essentially according to March et al. (24). Forty ml of 2 M Na,COs was added to wet Sepharose 4B gel (40 ml) which had previously been washed with water and then with 120 ml of 2 M Na,COs. The total volume became 80 ml. It was cooled to 5°C with gentle stirring. Four ml of CNBr dissolved in CH,CN (2 g/ml) was dropped into the gel suspension during 2 min and then the temperature was kept at 5-10°C for 3 min by adding ice. Twenty ml of chipped ice was added at once, and the cooled mixture was rapidly poured onto a cold Buchner funnel and filtered by suction. The gel on the glass filter was washed with cold water until the pH became neutral. The well packed gel was transferred to a conical beaker for reaction with PP! ligand. PPi was obtained by removing Na+ from the sodium salt on a Dowex 50 W (H+ type) column (5x40 cm). Water in the PPi was first removed using a rotary evaporator until the solution became viscous and then by evaporation under a vaccum (0.03 mmHg) at room temperature for more than 6 h with a dry ice-acetone trap. The content of water in the resulting PPi was less than 10%. 6-AminohexylPPi was synthesized by the / . Biochem.
PREPARATION OF CARDIAC MYOSIN SUBFRAGMENT-1
and 3.76 x 10"' M. Binding data were analyzed by means of the Scatchard equation
where r is the number of mol of bound ADP per mol of S-l, n is the maximum value of r, K is the dissociation constant, and (A) is the molar concentration of free ADP. Electrophoresis and Isoelectric Focusing—Verti-
cal polyacrylamide gel electrophoresis under nondissociating conditions was performed as described by Davis (28) using 5% polyacrylamide separating gel with 2.5% acrylamide stacking gel; the running buffer was 20 mM Tris-glycine buffer (pH 8.9). SDS-gel electrophoresis was performed as described by Dunker and Rueckert (29). Protein components in fractions obtained from column chromatography were analyzed by SDS-gel electrophoresis unless otherwise stated. Densitometric traces were obtained by scanning the destained gel with a Fuji Riken FD-A IV densitometer with a 600 nm filter and 0.3 x 3 mm slit. Isoelectric focusing on polyacrylamide gel was carried out by the methods described by Wrigley (50); the upper anodic vessel was filled with 5% Measurement of ATPase Activity—ATPase ethylenediamine and the lower cathodic one with activity was measured at 25°C in a reaction mixture 5% phosphoric acid. The polyacrylamide gel was containing 0.5 M KG, 5 mM CaCl,, 2 mM ATP, and polymerized with ammonium persulfate and the gel 50 mM buffer, or a mixture of 0.5 M KC1, 5 mM concentration was 4%. Carrier Ampholine, pH EDTA, 5 mM ATP, and 50 mM buffer. Tris- 3-10, was used at a concentration of 2%. Sample maleate buffer was used in the pH range between protein containing 20 % glycerol was loaded on the 5.0 and 7.0, Tris-HCl buffer between 7 0 and 9.0 and anodic gel side under Ampholine cover solution glycine buffer above pH 9.0. Aliquots of the containing 10% glycerol. Focusing was performed reaction solution were taken at appropriate in- at 4°C at voltages of 100 V for 1 h, 150 V for 2 h, tervals for Pi determination and another aliquot and then 100 V for 3 or 4 h. The gel slices were was used for pH determination after the reaction each suspended in 1 ml of water for 1 h and the pH was over. The ATPase activity in the presence of was measured at 4°C with a Radiometer PHM 26 F-actin was measured at 25°C in a reaction mixture pH meter. Molecular Weight Determinations by Analytical containing 30 mM KC1, 1 mM MgClt, 1 mM ATP, Ultracentrifugation—The determinations were done and 10 mM histidine buffer (pH 7.0). The steady-state rate was determined from the at 6°C in a Hitachi UCA-1A ultracentrifuge time course of Pi liberation and Pi was measured by equipped with a temperature control unit and Rayleigh interference optics. The meniscus deplethe method of Fiske and SubbaRow (26). Measurements of A DP Binding—The binding of tion sedimentation equilibrium method (31) was ADP to S-l was measured by the gel filtration used. The weight-average molecular weight was method (27) using Sephadex G-25 at 4°C in a me- calculated from a plot of log C, against r*/2 accorddium containing 0.5 M KC1, 10 mM MgCl,, and ing to Eq. 1 20 mM Tris-HCl buffer (pH 8.0). ADP concentration was changed in the range between 1.62x10"* Vol. 81, No. 4, 1977
M=
RT
dlnCr d(r>/2) *
(1)
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
method described by Trayer et al. (25), by mixing PPi with 6-aminohexan-l-ol (Aldrich Chemical Co.)- The white powder of 6-aminohexylPP! (0.3 g, 1 mmol) was dissolved in 10 ml of 0.2 N NaHCO, and the pH was adjusted to 9.5 with 4 N NaOH. Ten ml of cold 6-aminohexylPPi solution was added to the CNBr-activated Sepharose 4B gel in a conical beaker. The mixture was allowed to stand for 20 h at 4°C with shaking. The gel was washed with 200 ml each of 0.1 N NaHCO3, H,O, 1 mM HC1, 0.5 M NaCl, and H t O in this order and stored in 0.2 % NaN, at 4°C. The concentration of ligand in the gel was 4-5 fimo\ per ml of gel. The capacity of the 6-aminohexylPPi-Sepharose 4B conjugate gel for S-l(CT) was measured in a medium containing 30 mM KC1, 10 mM MOPS (pH 7.2), and 0.1 ITIMEDTA: it was 10 mgpermlofgel. Column chromatography was performed with medium containing 30 mM KC1, 20 mM Tris-HCl (pH 8.0), and 5 mM EDTA S-l(CT) was bound to the column loosely at pH 8.0 and eluted gradually with this medium. The column size was 1.6x11 cm. Immediately after use, the column was washed with 4 M urea in order to remove proteins remaining in the column and then with water to remove urea. The column could be used more than 6-8 times.
979
980
K. YAGI and H. KUWAYAMA
estimated from the corresponding area of the densitogram, and the weights of gi and g2 were calculated based on 2.0 x 10" g of f-chain. The mean values obtained from 10 different runs were 2.69±0.35xlO*g for gt and 2.07±0.27x 104 g for g,. It can be concluded that one myosin molecule contains 2 mol of gi and 2 mol of g,. The same conclusion was reached by Frearson and Perry (33) on the assumption that one mole of myosin contains 4 mol of g-chains. A component with a molecular weight of about 1.5 X10* is sometimes included in myosin preparations. Swynghedauw et al. (34) and Shiverick et al. (35) have described this component previously, but its properties are not known. Preparation of S-l(CT)— Myosin (15 mg/ml), which was not purified by DEAE-Sephadex A-50 chromatography, was incubated with chymotrypsin RESULTS in 30 mM KC1-20 mM Tris-HCl (pH 7.6) at 0-2°C. Molar Ratio off-Chain and g-Chains—Myosin The weight ratio of chymotrypsin to myosin was 1 to stored in 50 % glycerol and 0.3 M KC1 at — 15°C was 250. Incubation was performed for 20 h and the diluted 20-fold with ice-cold water and the precipi- reaction was stopped by adding DFP to a final tate was collected by centrifugation at 5,000 rpm for concentration of 0.1 mM. The chymotryptic digest 10 min then dissolved in 0.15 M potassium phosphate of myosin was diluted with an equal volume of buffer (pH 7.5)-10mM EDTA (pH 7.5). The 30 mM KC1 and centrifuged at 14,500 rpm for myosin was purified by DEAE-Sephadex A-50 30 min. The supernatant was carefully sucked out. column chromatography (22). The purified myo- Proteins in the supernatant and precipitate were sin was analyzed by SDS-gel electrophoresis using investigated. Nearly one-half of the protein and 5% acrylamide gel. As shown in Fig. 1, one main also of the ATPase activity was found in the resultprotein band and two minor protein bands were ing supernatant. Figure 2 shows the gel filtration pattern of the supernatant (20 ml) on a Sephadex found in the electrophoretogram. G-200 column (2.5x90 cm). Nearly 70% of the The molecular weights of the two smaller comATPase activity of the supernatant was found in the ponents (gi and g,) were estimated to be 2.7 X10* protein fractions of the second peak. Most of the 4 and 2.1 x 10 , respectively, by comparison with the protein in the second peak was probably S-l(CT), mobilities of standard proteins of known molecular judging from the elution volume. The amount of weights. The molecular weight of f-chain was taken protein in the second peak as estimated from the as 2.0 x 10s (32). The amount of each protein was absorption at 280 nm was nearly one-half of the amount of protein in the supernatant. Therefore, the amount of protein in the second peak corresponded to 1/4 of the added myosin. The protein in the second peak was concentrated by adding ammonium sulfate to 60% saturation and then s Idesalted by dialysis against 20 mM MOPS buffer 9- -S 3 (pH 7.2)-25 mM KC1. After dialysis, it was apO « 9i 92 plied to a DEAE-cellulose (DE 32) column equilibrated with 20 mM MOPS buffer (pH 7.2)-25 mM 0 1 2 3 * 5 6 7 6 KC1. Elution was performed with 20 mM MOPS Distance (cm) buffer (pH 7.2)-6O mM KC1. As shown in Fig. 3, Fig. 1. SDS-gel electrophoresis of cardiac myosin. 70% of the applied protein was obtained as a sharp The left and right sides correspond to the top and bottom peak and the rest as a retarded peak. The two of the gel, respectively.
* n
I
A_A.
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
where M is molecular weight and C, is the protein concentration at distance r. A three-channel centerpiece was used and the column height was 3 mm. Equilibrium was attained after 6 h at 25,090 rpm. Calculation were based on photographs obtained after 9 h at 23,180 rpm and after 15 h at 25,090 rpm. S-l(CT), which was excluded from Sephadex G-200 and Ultrogel ACA34 columns, was used immediately for measurement. The solvent was 0.125 M KC1, 20 IDM Tris-HCl (pH 8.0), and 0.1 mM DTT. Values of 8 of 0.735 cc/g and p of 1.012 were used for calculation. Chemicals—Chymotrypsin, DTT, SDS, ATP, and ADP were purchased from Sigma Chemical Co. Other reagents and used were of reagent grade.
981
PREPARATION OF CARDIAC MYOSIN SUBFRAGMENT-1
200
300
400
500
Elution volume (ml)
Fig. 2. Gel filtration pattern of the supernatant of the chymotryptic digest of myosin. A sample of 20 ml (/4t80=2.45) was applied to a Sephadex G-200 column (2.5x90 cm). Elution was performed with 30 mM KCl-20 mM Tris (pH 8), at a flow rate of 14 ml/h. The •same gel column was used to analyze the protein fraction excluded by DEAE-Sephadex A-50 (Fig. 6) and the results are shown by a dotted line. The inset shows the gel filtration pattern of a protein fraction obtained by ammonium sulfate fractionation of a chymotryptic •digest of myosin. The size of the Sephadex G-200 column was 2.5 x 100 cm. The flow rate was 30 ml/h and 10 ml fractions were collected. O, -4280nm. x , ATPase activity cellulose column chromatography, and then each of them was further purified by ATP-affinity column chromatography. One or two protein bands in front of the f-chain component could not be removed by these and other treatments.
Fig. 5. Separation of S-1 (CT) and S-1 (CT)' by 6-aminohexylPPi-Sepharose 4B conjugate column chromatography. The sample consisted of equal amounts of S-1 (CT) and S-1 (CT)'. The column size was 1.6x11 cm. A mixture of 30 mM KCI, 20 mM Tris-HCl (pH 8 0), and 5 mM EDTA was used to dissolve the sample proteins,, to equilibrate the column and also foi elution. The flow rate was 20 ml/h and 2.8 ml fractions were collected. 10 mM PPi containing 30 mM KCI, 20 mM TrisHCI (pH 8), and 5 mM EDTA was applied to the column at the arrow labeled PPi. Rabbit skeletal S-l(CT) was applied to the same column, as shown by a broken line for comparison. p2 and pi, which are the two components of skeletal S-l(CT), were found in the second and third peaks, respectively. Analysis was done by SDS-gel electrophoresis The first peak may consist of aggregates.
/. Biochem..
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
components were clearly distinguished by acrylamide gel electrophoresis in the absence of SDS. The component of the first peak migrated after the component of the second peak on acrylamide gel electrophoresis at pH 8.9. The polypeptide compositions of the first and second peak components were analyzed by SDS-gel electrophoresis, and as shown in Fig. 4, two protein bands, one large and one small, were observed with both. The large protein was regarded as the head portion of the fchain (f-chain component of S-1) and a molecular weight of 1.0 xlO 6 was tentatively assumed. No difference in electrophoretic mobility was detected between the large protein bands of the first and second peak fractions. A small protein band of the first peak showed an electrophoretic mobility identical with that of gx of cardiac myosin. Therefore, the first peak was regarded as being S-l(CT). The amount of g! in the purified S-l(CT) per 1.0 x 10* g of f-cham component was estimated from the areas of densitograms (23 different runs), and a value of 2.69±0.35xl0
Muscle Myosin by Chymotryptic Digestion1 Koichi YAGI and Hideto KUWAYAMA Department of Chemistry, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060 Received for publication, August 23, 1976
Subfragment-1 was prepared from pig cardiac myosin by chymotryptic digestion at low ionic strength at 0-2°C for 20 h. Two components were distinguished in polyacrylamide gel electrophoresis of subfragment-1 preparations, and were designated as S-l (CT) and S-l (CT)'. The isoelectric points were 6.70 and 6.45, respectively. The two components could not be separated by Sephadex G-200 gel filtration, but they were separated on a DEAE-cellulose (DE 32) column or a 6-aminohexylPPi-Sepharose 4B conjugate column. S-l (CT) consisted of one large polypeptide chain (the head portion of the f-chain) and one small polypeptide chain (gj. In SDS gel electrophoresis, the large polypeptide of S-l (CT) migrated at the same rate as that of S-l (CT)', but the small polypeptide of S-l (CT)' migrated at a faster rate than that of S-l (CT) The small chain of S-l (CT)' was thus designated as g/. The migration rate of the small chain of S-l (CT) was the same as that of g! of cardiac myosin, but the small chain of S-l (CT)' migrated faster than gx and slower than g, of cardiac myosin. It was therefore suggested that S-l (CT)' was produced by overdigestion of S-l (CT). The yield of S-l (CT) plus S-l (CT)' estimated on the basis of absorption at 280 nm was 36% of the weight of myosin employed. The molecular weight was 1.19 x 10" and the maximum ADP binding number was 0.48 mol using S-l (CT) 30% contaminated by S-l (CT)'. The pH-activity relationships in the presence of Ca1+ and EDTA were investigated. The ATPase activity in the presence of CaI+ at pH 7.5 was 0.3 ftmo\ mg-^min" 1 for both S-l (CT) and S-l (CT)'. The maximum ATPase activity of S-l (CT) in the presence of F-actin was 8.3 //mol mg^-min" 1 and the affinity for F-actin was about 1/20 of that of skeletal S-l (CT).
Mueller and Perry (7) were the first to recognize the presence of subfragment-1 (S-l) in heavy meromyosin (HMM) preparations from skeletal muscle myosin. Since then, methods of preparation for
S-l and its properties have been extensively investigated {2-7). Great emphasis has been placed, in recent years, on the composition of S-l preparations, because the amount of Pi liberated in the
1 This work was aided by grants from the Muscular Dystrophy Associations of America, Inc. and from the Ministry of Education, Science and Culture of Japan. Abbreviations' S-l, subfragment-1; S-l(CT) or S-l(P), S-l prepared from myosin using chymotrypsin or papain, respectively; HMM, heavy meromyosin; MOPS, morpholinopropane sulfonic acid; SDS, sodium dodecyl sulfate; DFP, diisopropyl fluorophosphate; Nbsj, 5,5'-dithio-bis(2-nitrobenzoic acid).
Vol. 81, No. 4, 1977
977
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
Preparation of Myosin Subfragment-1 from Pig Cardiac
978
raphy. The maximum number of mol of bound ADP was about 0.48 mol per 1.19xl0»g of S-l(CT). This S-l(CT) contained only one g-chain amounting to nearly 1 mol per mol. A preliminary communication has already appeared elsewhere (21). EXPERIMENTAL PROCEDURES
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
initial burst of ATPase is about a half mol per mol of S-1 (8) and the maximum amount of ADP bound to S-1 is also nearly a half mol of ADP per mol of S-1 (9). It therefore seems possible that S-1 consists of two components, one of which shows neither the initial burst nor ADP binding under the conditions used. S-1 prepared by chymotryptic digestion of skeletal myosin can be separated into two different components (pi and p2), which contain either gx or ga as the small polypeptide chain of S-1 (5). However, the maximum amount of ADP bound to pi or p2 was the same as that bound to the S-1 preparations before fractionation into pi and p2 (10). Myosin of cardiac muscle contains two different g chains in an equimolar ratio (11), while skeletal white muscle myosin contains three different g chains in various molar ratios, e.g. 1.35: 2.00 : 0.65 according to Sarkar (12). It is therefore suggested that myosin consists of two populations in various ratios. It is further suggested that pi and p2 are formed from different myosin isozymes. On the other hand, the content of g-chains in cardiac myosin suggests that one kind of myosin is present in cardiac myosin preparations, and only one kind of S-1 with respect to g chain could be obtained. Preparation of cardiac myosin has been investigated by Ellenbogen et al. (13), Mueller (14), and Barany et al. (15), and a procedure to obtain pure myosin was recently established by WikmanCoffelt et al. (16). Cardiac myosin is more resistant to trypsin than skeletal myosin (17) and Mueller et al. {18) demonstrated that an unusually high weight ratio of trypsin to myosin (1 : 25) was required for the preparation of HMM from canine cardiac myosin. According to Tada et al. (19), no significant amount of S-1 could be isolated by tryptic or chymotryptic digestion of bovine cardiac HMM, but it could be obtained from cardiac HMM and myosin by papain digestion at low temperature. Wolodko and Kay (20) recently isolated S-1 from rabbit cardiac myosin using papain. In this paper, a method for the preparation of S-1 from pig cardiac myosin is presented. It involves chymotryptic digestion at 0-2°C and at low ionic strength. An apparently homogeneous S-l(CT) preparation was obtained after purification using DEAE-cellulose (DE 32) or 6-aminohexyl PPi-Sepharose 4B conjugate column chromatog-
K. YAGI and H. KUWAYAMA
Myosin—Myosin was prepared from fresh pig heart by a method similar to that described by Wikman-Coffelt et al. (16). Myosin was stored in 50% glycerol at a temperature below — 10°C. If necessary, myosin was further purified by DEAESephadex A-50 chromatography (22) before use. Concentrations of myosin were determined from the absorbance using a value of A^Dm=4.7, which was obtained by the micro-Kjeldahl method assuming the nitrogen content to be 16%. F-Actin—Actin was extracted from acetonedried powder of rabbit skeletal muscle at 0°C and purified essentially according to the method of Mommaerts (23). Preparation of 6-AminohexylPP rSepharose 4B Conjugate Column—CNBr-activated Sepharose 4B was prepared essentially according to March et al. (24). Forty ml of 2 M Na,COs was added to wet Sepharose 4B gel (40 ml) which had previously been washed with water and then with 120 ml of 2 M Na,COs. The total volume became 80 ml. It was cooled to 5°C with gentle stirring. Four ml of CNBr dissolved in CH,CN (2 g/ml) was dropped into the gel suspension during 2 min and then the temperature was kept at 5-10°C for 3 min by adding ice. Twenty ml of chipped ice was added at once, and the cooled mixture was rapidly poured onto a cold Buchner funnel and filtered by suction. The gel on the glass filter was washed with cold water until the pH became neutral. The well packed gel was transferred to a conical beaker for reaction with PP! ligand. PPi was obtained by removing Na+ from the sodium salt on a Dowex 50 W (H+ type) column (5x40 cm). Water in the PPi was first removed using a rotary evaporator until the solution became viscous and then by evaporation under a vaccum (0.03 mmHg) at room temperature for more than 6 h with a dry ice-acetone trap. The content of water in the resulting PPi was less than 10%. 6-AminohexylPPi was synthesized by the / . Biochem.
PREPARATION OF CARDIAC MYOSIN SUBFRAGMENT-1
and 3.76 x 10"' M. Binding data were analyzed by means of the Scatchard equation
where r is the number of mol of bound ADP per mol of S-l, n is the maximum value of r, K is the dissociation constant, and (A) is the molar concentration of free ADP. Electrophoresis and Isoelectric Focusing—Verti-
cal polyacrylamide gel electrophoresis under nondissociating conditions was performed as described by Davis (28) using 5% polyacrylamide separating gel with 2.5% acrylamide stacking gel; the running buffer was 20 mM Tris-glycine buffer (pH 8.9). SDS-gel electrophoresis was performed as described by Dunker and Rueckert (29). Protein components in fractions obtained from column chromatography were analyzed by SDS-gel electrophoresis unless otherwise stated. Densitometric traces were obtained by scanning the destained gel with a Fuji Riken FD-A IV densitometer with a 600 nm filter and 0.3 x 3 mm slit. Isoelectric focusing on polyacrylamide gel was carried out by the methods described by Wrigley (50); the upper anodic vessel was filled with 5% Measurement of ATPase Activity—ATPase ethylenediamine and the lower cathodic one with activity was measured at 25°C in a reaction mixture 5% phosphoric acid. The polyacrylamide gel was containing 0.5 M KG, 5 mM CaCl,, 2 mM ATP, and polymerized with ammonium persulfate and the gel 50 mM buffer, or a mixture of 0.5 M KC1, 5 mM concentration was 4%. Carrier Ampholine, pH EDTA, 5 mM ATP, and 50 mM buffer. Tris- 3-10, was used at a concentration of 2%. Sample maleate buffer was used in the pH range between protein containing 20 % glycerol was loaded on the 5.0 and 7.0, Tris-HCl buffer between 7 0 and 9.0 and anodic gel side under Ampholine cover solution glycine buffer above pH 9.0. Aliquots of the containing 10% glycerol. Focusing was performed reaction solution were taken at appropriate in- at 4°C at voltages of 100 V for 1 h, 150 V for 2 h, tervals for Pi determination and another aliquot and then 100 V for 3 or 4 h. The gel slices were was used for pH determination after the reaction each suspended in 1 ml of water for 1 h and the pH was over. The ATPase activity in the presence of was measured at 4°C with a Radiometer PHM 26 F-actin was measured at 25°C in a reaction mixture pH meter. Molecular Weight Determinations by Analytical containing 30 mM KC1, 1 mM MgClt, 1 mM ATP, Ultracentrifugation—The determinations were done and 10 mM histidine buffer (pH 7.0). The steady-state rate was determined from the at 6°C in a Hitachi UCA-1A ultracentrifuge time course of Pi liberation and Pi was measured by equipped with a temperature control unit and Rayleigh interference optics. The meniscus deplethe method of Fiske and SubbaRow (26). Measurements of A DP Binding—The binding of tion sedimentation equilibrium method (31) was ADP to S-l was measured by the gel filtration used. The weight-average molecular weight was method (27) using Sephadex G-25 at 4°C in a me- calculated from a plot of log C, against r*/2 accorddium containing 0.5 M KC1, 10 mM MgCl,, and ing to Eq. 1 20 mM Tris-HCl buffer (pH 8.0). ADP concentration was changed in the range between 1.62x10"* Vol. 81, No. 4, 1977
M=
RT
dlnCr d(r>/2) *
(1)
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
method described by Trayer et al. (25), by mixing PPi with 6-aminohexan-l-ol (Aldrich Chemical Co.)- The white powder of 6-aminohexylPP! (0.3 g, 1 mmol) was dissolved in 10 ml of 0.2 N NaHCO, and the pH was adjusted to 9.5 with 4 N NaOH. Ten ml of cold 6-aminohexylPPi solution was added to the CNBr-activated Sepharose 4B gel in a conical beaker. The mixture was allowed to stand for 20 h at 4°C with shaking. The gel was washed with 200 ml each of 0.1 N NaHCO3, H,O, 1 mM HC1, 0.5 M NaCl, and H t O in this order and stored in 0.2 % NaN, at 4°C. The concentration of ligand in the gel was 4-5 fimo\ per ml of gel. The capacity of the 6-aminohexylPPi-Sepharose 4B conjugate gel for S-l(CT) was measured in a medium containing 30 mM KC1, 10 mM MOPS (pH 7.2), and 0.1 ITIMEDTA: it was 10 mgpermlofgel. Column chromatography was performed with medium containing 30 mM KC1, 20 mM Tris-HCl (pH 8.0), and 5 mM EDTA S-l(CT) was bound to the column loosely at pH 8.0 and eluted gradually with this medium. The column size was 1.6x11 cm. Immediately after use, the column was washed with 4 M urea in order to remove proteins remaining in the column and then with water to remove urea. The column could be used more than 6-8 times.
979
980
K. YAGI and H. KUWAYAMA
estimated from the corresponding area of the densitogram, and the weights of gi and g2 were calculated based on 2.0 x 10" g of f-chain. The mean values obtained from 10 different runs were 2.69±0.35xlO*g for gt and 2.07±0.27x 104 g for g,. It can be concluded that one myosin molecule contains 2 mol of gi and 2 mol of g,. The same conclusion was reached by Frearson and Perry (33) on the assumption that one mole of myosin contains 4 mol of g-chains. A component with a molecular weight of about 1.5 X10* is sometimes included in myosin preparations. Swynghedauw et al. (34) and Shiverick et al. (35) have described this component previously, but its properties are not known. Preparation of S-l(CT)— Myosin (15 mg/ml), which was not purified by DEAE-Sephadex A-50 chromatography, was incubated with chymotrypsin RESULTS in 30 mM KC1-20 mM Tris-HCl (pH 7.6) at 0-2°C. Molar Ratio off-Chain and g-Chains—Myosin The weight ratio of chymotrypsin to myosin was 1 to stored in 50 % glycerol and 0.3 M KC1 at — 15°C was 250. Incubation was performed for 20 h and the diluted 20-fold with ice-cold water and the precipi- reaction was stopped by adding DFP to a final tate was collected by centrifugation at 5,000 rpm for concentration of 0.1 mM. The chymotryptic digest 10 min then dissolved in 0.15 M potassium phosphate of myosin was diluted with an equal volume of buffer (pH 7.5)-10mM EDTA (pH 7.5). The 30 mM KC1 and centrifuged at 14,500 rpm for myosin was purified by DEAE-Sephadex A-50 30 min. The supernatant was carefully sucked out. column chromatography (22). The purified myo- Proteins in the supernatant and precipitate were sin was analyzed by SDS-gel electrophoresis using investigated. Nearly one-half of the protein and 5% acrylamide gel. As shown in Fig. 1, one main also of the ATPase activity was found in the resultprotein band and two minor protein bands were ing supernatant. Figure 2 shows the gel filtration pattern of the supernatant (20 ml) on a Sephadex found in the electrophoretogram. G-200 column (2.5x90 cm). Nearly 70% of the The molecular weights of the two smaller comATPase activity of the supernatant was found in the ponents (gi and g,) were estimated to be 2.7 X10* protein fractions of the second peak. Most of the 4 and 2.1 x 10 , respectively, by comparison with the protein in the second peak was probably S-l(CT), mobilities of standard proteins of known molecular judging from the elution volume. The amount of weights. The molecular weight of f-chain was taken protein in the second peak as estimated from the as 2.0 x 10s (32). The amount of each protein was absorption at 280 nm was nearly one-half of the amount of protein in the supernatant. Therefore, the amount of protein in the second peak corresponded to 1/4 of the added myosin. The protein in the second peak was concentrated by adding ammonium sulfate to 60% saturation and then s Idesalted by dialysis against 20 mM MOPS buffer 9- -S 3 (pH 7.2)-25 mM KC1. After dialysis, it was apO « 9i 92 plied to a DEAE-cellulose (DE 32) column equilibrated with 20 mM MOPS buffer (pH 7.2)-25 mM 0 1 2 3 * 5 6 7 6 KC1. Elution was performed with 20 mM MOPS Distance (cm) buffer (pH 7.2)-6O mM KC1. As shown in Fig. 3, Fig. 1. SDS-gel electrophoresis of cardiac myosin. 70% of the applied protein was obtained as a sharp The left and right sides correspond to the top and bottom peak and the rest as a retarded peak. The two of the gel, respectively.
* n
I
A_A.
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
where M is molecular weight and C, is the protein concentration at distance r. A three-channel centerpiece was used and the column height was 3 mm. Equilibrium was attained after 6 h at 25,090 rpm. Calculation were based on photographs obtained after 9 h at 23,180 rpm and after 15 h at 25,090 rpm. S-l(CT), which was excluded from Sephadex G-200 and Ultrogel ACA34 columns, was used immediately for measurement. The solvent was 0.125 M KC1, 20 IDM Tris-HCl (pH 8.0), and 0.1 mM DTT. Values of 8 of 0.735 cc/g and p of 1.012 were used for calculation. Chemicals—Chymotrypsin, DTT, SDS, ATP, and ADP were purchased from Sigma Chemical Co. Other reagents and used were of reagent grade.
981
PREPARATION OF CARDIAC MYOSIN SUBFRAGMENT-1
200
300
400
500
Elution volume (ml)
Fig. 2. Gel filtration pattern of the supernatant of the chymotryptic digest of myosin. A sample of 20 ml (/4t80=2.45) was applied to a Sephadex G-200 column (2.5x90 cm). Elution was performed with 30 mM KCl-20 mM Tris (pH 8), at a flow rate of 14 ml/h. The •same gel column was used to analyze the protein fraction excluded by DEAE-Sephadex A-50 (Fig. 6) and the results are shown by a dotted line. The inset shows the gel filtration pattern of a protein fraction obtained by ammonium sulfate fractionation of a chymotryptic •digest of myosin. The size of the Sephadex G-200 column was 2.5 x 100 cm. The flow rate was 30 ml/h and 10 ml fractions were collected. O, -4280nm. x , ATPase activity cellulose column chromatography, and then each of them was further purified by ATP-affinity column chromatography. One or two protein bands in front of the f-chain component could not be removed by these and other treatments.
Fig. 5. Separation of S-1 (CT) and S-1 (CT)' by 6-aminohexylPPi-Sepharose 4B conjugate column chromatography. The sample consisted of equal amounts of S-1 (CT) and S-1 (CT)'. The column size was 1.6x11 cm. A mixture of 30 mM KCI, 20 mM Tris-HCl (pH 8 0), and 5 mM EDTA was used to dissolve the sample proteins,, to equilibrate the column and also foi elution. The flow rate was 20 ml/h and 2.8 ml fractions were collected. 10 mM PPi containing 30 mM KCI, 20 mM TrisHCI (pH 8), and 5 mM EDTA was applied to the column at the arrow labeled PPi. Rabbit skeletal S-l(CT) was applied to the same column, as shown by a broken line for comparison. p2 and pi, which are the two components of skeletal S-l(CT), were found in the second and third peaks, respectively. Analysis was done by SDS-gel electrophoresis The first peak may consist of aggregates.
/. Biochem..
Downloaded from https://academic.oup.com/jb/article-abstract/81/4/977/893110 by Tulane University Library, Serials Acquisitions Dept. user on 17 January 2019
components were clearly distinguished by acrylamide gel electrophoresis in the absence of SDS. The component of the first peak migrated after the component of the second peak on acrylamide gel electrophoresis at pH 8.9. The polypeptide compositions of the first and second peak components were analyzed by SDS-gel electrophoresis, and as shown in Fig. 4, two protein bands, one large and one small, were observed with both. The large protein was regarded as the head portion of the fchain (f-chain component of S-1) and a molecular weight of 1.0 xlO 6 was tentatively assumed. No difference in electrophoretic mobility was detected between the large protein bands of the first and second peak fractions. A small protein band of the first peak showed an electrophoretic mobility identical with that of gx of cardiac myosin. Therefore, the first peak was regarded as being S-l(CT). The amount of g! in the purified S-l(CT) per 1.0 x 10* g of f-cham component was estimated from the areas of densitograms (23 different runs), and a value of 2.69±0.35xl0
