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A subunit of myosin
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
79,
191-19’3
(1959)
A Subunit of Myosin D. R. Kominz, W. R. Carroll, E. N. Smith and E. R. Mitchell Prong
the National
Health,
Public
Institute of Arthritis and Metabolic Diseases; National Institutes oj Health Service, United States Depaitment of Health, E&cation, c~nrl Welfare, Bethesda, Maryland
Received May 29, 1958 INTRODUCTION
On the basis of amino acid composition (I), Luki postulated that myosin is a copolymer of actin, tropomyosin, and a “third protein” which is characterized inter alia by a high phenylalanine content (2). Tsao found that prolonged treatment with urea resulted in the release from myosin of protein fragments of 14,000-16,000 molecular weight (3). Middlebrook and Szent-Gy6rgyi found that protein material is also released from myosin after brief treatment with urea (4). Tsao observed that, at pH 10.7 myosin was depolymerized to a large core of 170,000 molecular weight,, but he could not obtain the small molecules in this case (3). The present, paper deals with the release of protein material from myosin at an alkaline pH, showing that it corresponds to the material released by urea and that it could be the “third protein” postulated by Laki. METHODS Fresh rabbit skeletal muscle was extracted and the myosin precipitat,ed once a~cording to the method of Kessler and Spicer (5). The one-time precipitated myosin was dissolved in 0.5 M KC1 at a concentration of about 1% and clarified by centrifu gation at 30,000 r.p.m. in the Spinco preparative ultracentrifuge at 5%. in the r1re.q ence of millimolar adenosine triphosphate (ATP) and MgCl2 . It was reprecipitated and redissolved in 0.5 M KCl. Myosin A prepared in this way was employed in all of the subsequent procedures to be described. In addition, ultrac~entrifugal examination alone was carried out on myosin A prepared by the method of Taso (3), myosin -4 extracted from glycerinated psoas fibers, myosin B prepared by the method of Csapo from frozen muscle (6), standard 5.hr. myosin 13, and artificial artomyosin. The myosins in 0.5 M KC1 were examined in the Spinco analytical ult.racentrifuge before and after overnight dialysis at, 5°C. against 0.1 M N’a&OB The concentration of protein in t.he slow peak was estimated by superimposing the sedimentation di:igram over that, of the solvent alone, and planimetrically measuring the area between the peak and Ihe base line; area-concentration correlations were ralculated from measurements on diagrams of the purified carbonat,e subunit (see below). In addition, relat,ive peak areas were measured in t,hose cases where the total protein concentr;r.191
192
KOMINZ, CARROLL, SMITH AND MITCHELL
tion was sufficiently low so that the main peak was not hypersharp. The area of the slow peak is unaffected by the Johnston-Ogston effect (7). The area of the fast peak is diminshed by the Johnston-Ogston effect, but agreement between the results from absolute and relative peak areas indicates that this diminution is well within the error of measurement. In measurement of the concentration of protein in the slow peak, an approximately f 10% error is introduced by overlap with the main peak and with the meniscus at early sedimentation times, and by the broad base of the slow peak at later sedimentation times. Electrophoretic examination was made in the Aminco-Stern electrophoresis apparatus on a solution of rabbit myosin dialyzed against 0.1 A4 Na&03 . Paper electrophoresis was also performed at neutral pH between glass plates (8) on carbonatetreated myosin dialyzed against water. Ammonium sulfate fractionation was performed on rabbit, myosin after dialysis against 0.1 M Na&Oa or aft.er a further dialysis against water. Most of the protein precipitated between 24 and 32% saturation. The second fraction, precipitating at
5C~650/~ saturation (hereafter called “carbonate subunit”), was separated by centrifugation at 25,000r.p.m. for 20 min. and dialyzed exhaustively. Amino acid analysis was carried out by colum chromatography as previously described (9) on 18-hr., 104”C., 6 N HCl hydrolyzates. Carboxypeptidase determination of C-terminal end groups was performed as previously described (10). In the case of myosin dialyzed against 0.1 M Na&OJ and then against water, lowering the pH to 8.0 increased the viscosity to a thick gelatinous consistency; in this case the enzyme was allowed to react for 8 min. with constant stirring. Half of each sample was subjected to paper chromatography (11) and half was analyzed by column chromatography on a 50-cm.-long column of Dowex 50. For determination of the molecular weight of the carbonate subunit, a 0.66y0 solution was dialyzed against 0.1 M KCl, 0.05 I’/2 pH 8.0 phosphate buffer, and diluted to 0.44 and 0.22% with dialyzate. The three solutions were run at 42,040 r.p.m. in a synthetic boundary cell, with exposures bieng made at 16-min. intervals. Sedimentation rates were measured from the plates by a comparator. Boundary-spreading rates were measured on enlargements of the sedimentation diagrams by the sigma technique, and corrected for the wedge shape of the cell. Partial specific volume was calculated from the amino acid analysis (12). RESULTS
Unfrmtionated
Myosin
The ultracentrifugal diagram of a myosin preparation dissolved in 0.1 M Na&Os is given in Fig. 1. In all solutions of myosin A, myosin B, or synthetic actomyosin that were prepared from fresh or glycerinated muscle, a small, rapidly diffusing component with ~20of about 1.4 could be observed under these alkaline conditions (pH cu. 10). The amount of protein which is represented by this peak is in the range of 14-18 % of the total myosin protein. In the case of myosin B prepared from frozen muscle by the method of Csapo, the szo is about 1.8 and the relative amount present in the peak is 28 %. Free electrophoresis of 0.1 M Na&03-treated myosin reveals a broad slow component, heavily overlapping the myosin peak in both ascending
A SUBUNIT
OF MYOSIN
193
FIG. 1. Ultracentrifuge pattern of myosin A prepared by the method of Tsao (3) in 0.1 A4NaLQ . Concentration 0.9’%,temperature 25’C. Exposure at 72 min. after reaching speed of 59,780r.p.m., bar angle 60’. Direct’ion of sedimentation from left to right. This demonstrates the typical appearance of a slow component in myosin solutions brought to approximately pH 10by dialysis against 0.1 M Na&O, .
and descending limbs. Paper electrophoresis of the same material gives :I dark streak at the origin and a pale-staining area extending from the origin toward the anode. Paper electrophoresis of the purified carbonate subunit reveals only the pale-staining area extending from the origin t,oward the anode and forming no discrete zones. Purified Carbonate Subunit The yield of carbonate subunit obtained from the 5(r65% saturation ammonium sulfate fraction, after exhaustive dialysis to remove the ammonium sulfate, is found to be 6-7 % of the starting material. The carbonat,c subunit is monodisperse in the ultracentrifuge (Fig. 2). The amino acid composition of the carbonate subunit is given in Table I. It may be compared with the amino acid analysis carried out in this laboratory on the urea subunit of myosin prepared by Middlebrook and Szent-Gyijrgyi (4). Figures 3 and 4 plot the sedimentation and boundary-spreading data calculated from the synthetic boundary ultracentrifuge diagrams obtained at neutral pH. The extrapolated parameters are s:~,~ = 2.32 X 10-13 sec. and D&,w = 7.25 X lo-’ sq. cm./sec. From these, and v = 0.731 ct./g., a molecular weight of 29,000 is obtained. Because the slow peak
194
KOMINZ,
CARROLL,
SMITH
AND
MITCHELL
pattern of the carbonate subunit of FIQ. 2. Synthetic boundary ultracentrifuge myosin in 0.1 M KCl, p/2 0.05 pH 7.9 phosphate buffer. Concentration O.SS%, temperature 25.3%. Exposure at 51 min. after reaching speed of 42,040 r.p.m., bar angle 35”. Direction of sedimentation from left to right.
TABLE I Amino Acid Composition of Myosin Subunit (Moles/,106 g., based on 16.7oj, N)
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Amide NH*
94 34 36 126 38 55 86 33 26 30 58 14 38 8 79
Total
779
93 40 37 116 35 57 77 35 27 32 59 10 37 9 76 24 764
h
SUBUNIT
OF
195
MYOSIN
in unfractionated myosin preparations examined at alkaline pH had a considerably lower sedimentation rate than the purified carbonate subunit’ examined at neutral pH, the carbonate subunit was also examined in the synthetic-boundery cell at pH 10 in 0.1 M Na&Os . The extrapolated value of the sedimentation constant was 25% lower than at neutral pH; the diffusion rate was also about 20% lower, with a much steeper concentration dependence. The parallel drop in both parameters can be interpreted that swelling, but no drop in molecular weight, occurs at t#his alkaline pH . Degradation by Carboxypeptidasr Paper chromatography of the amino acids liberated by carboxypeptidase treatment gives qualitat’ively the same picture for 100 mg. of carbonatetreated myosin and 6 mg. of carbonate subunit: isoleucine as end-group; much less serine; and traces of alanine, valine, and methionine. The quantitative results are given in Table II; 1.4 isoleucine residues are liberated/mole myosin, and 1.0 isoleucine residue is liberated/mole carbonate subunit. s2* x IO” 2.5 r
2:i,m, 0
FIG. 3. Concentration dependence carbonate
subunit
of sedimentation from myosin.
rate of
8-
6
0
I 2
FIG. 4. Concentration carbonate
I 4
I 6
dependence of diffusion subunit from myosin.
I
h&l rate of
196
KOMINZ,
CARROLL,
SMITH
AND
MITCHELL
TABLE II C-Terminal Residues Liberated by Carboxypeptidase Carbonate Subunit
Myosin (Per 4;wy,
Isoleucine Serine Valine Methionine Alanine
9.1
1.4 0.4 -
(per 29,ooo IT.)
5 min.
15 min.
1.0 0.3 -
1.2 0.3 0.3 0.3 -
DISCUSSION
The present study and that of Middlebrook and Szent-GyQgyi (4) throw further light on the original finding of Tsao (3) that fragments small in comparison with the 420,000 molecular weight (13-15) of the parent myosin molecule can be released from it. They indicate that by different methods of denaturation, similar end products can be obtained. The sedimentation-diffusion value of 29,000 for the molecular weight of the carbonate subunit is in good agreement with the value obtained by Middlebrook and Szent-Gybrgyi for their urea subunit. This value is about twice the value found by Tsao for his urea subunit: 14,000 by osmotic pressure and 16,000 by fluorescence polarization. From the amino acid composition of the carbonate subunit, one can calculate the minimum molecular weight to be about one-half of 29,000. It is possible, therefore, that the 29,000 molecular weight represents a stable dimer. The detection by carboxypeptidase of only one C-terminal isoleucine in 29,000 molecular weight could be interpreted as a masking phenomenon (16). A simple explanation for the different estimations of molecular weight could be that the prolonged exposure to urea employed by Tsao destroyed the stability of the dimer. However, until it is demonstrated that a stable dimer of two identical components is present, it would appear reasonable to assume a 29,000 molecular weight for the carbonate subunit. The 6-7 % yield for the carbonate subunit is very similar to the 7-9 % yield obtained by Tsao for the urea subunit. However, low yields are of common occurrence, and the ultracentrifuge diagrams indicate that approximately 14% of myosin A is released into the slow peak. This corresponds to two molecules of the carbonate subunit, from which carboxypeptidase would release two C-terminal isoleucines. We find that 1.5 moles of C-terminal isoleucine is liberated from carbonate-treated myosin, and Locker obtains the same value for native myosin (17). The identity of the paper chromatogram “fingerprints” of the end groups of myosin and of its carbonate subunit suggests that all of the carboxypeptidase action in myosin is indeed upon the carbonate subunits. It is worth noting that
A
SUBUNIT
OF
TABLE Amino
Acid
Composition
197
III
of Tropomyosin-like
(Moles/W
Aspartic acid Threonine Methionine Serine Glutamic acid Glycine i41anine Valine Isoleucine J,eucine Tyrosine I’henylalanine Proline Lysine Histidine Arginine Amide NHI Charge
MYOSIX
Subunits
from
Myosin
g.)
PFA Ox. myosin fraction (18)
Denatured myosin fractmn (18)
89 45 190 26 80 25 31 93 10 9
86 36 20 45 197 22 81 25 29 91 9 6 r
87 12
8; 19
2 86 22
26 30 40 212 12 108 27 30 95 15 3 2 107 5
(1:;) 38
(1:) 32
(1::) 21
(ii, 8!)
53
LMM fraction (1Y)
82 36 18 38 204 18 84 28 32 96 9
Tropomyosin
89
two molecules of the carbonate subunit will contribute approximately the 60,000 g. protein predicted by Laki (2) to be present in the missing “third protein” of myosin. In addition to the subunit described in this paper, the use of trypsin and performic acid by Laki (18) and of trypsin by Szent-Gy6rgyi (19) has succeeded in releasing from myosin a larger tropomyosin-like subunit, from myosin, amounting to about 27% of the protein in myosin. ,4s can be seen from Table III, this differs from the classical tjropomyosin of Bailey (20, 9) primarily in the residues alanine, lysine, arginine, histidine, and amide ammonia. There is a close resemblance, however, in phenylalanine and proline. It is thus immediately apparent, that the curbonat,e subunit of myosin could not have arisen from the tropomyosin-like subunit of myosin. The large values for proline and phenylalanine in the carbonate subunit preclude such a possibility. This large value for phenylalanine is in line with Laki’s prediction (2) for the “third protein” present, in myosin. It appears likely that the tropomyosin-like subunit and the carbonate subunit make up most of the crude L-meromyosin fraction (21-23, 1) of myosin. A graphic illustration of the distinctness of these two subunits can be obtained when one allows myosin to be split by Cu(CIQ3-at an alkaline pH for several days, a procedure for split)ting disulfide bonds a+ present being studied in this laboratory (Fig. 5).
198
KOMINZ,
CARROLL,
SMITH
AND
MITCHELL
FIG. 5. Ultracentrifuge pattern of myosin treated 7 days with 0.014 Cu(CN), in 0.25 M KCl, pH 10. Concentration 1.4’j& temperature 25%. Exposure at 70 min. after reaching speed of 59,760 r.p.m., bar angle 60’. Direction of sedimentation from left to right. This illustrates the presence of both the carb0nat.e subunit (a) and the tropomyosin-like subunit (5) under these conditions.
The remarkable presence of nearly 30% slow peak in myoein B prepared from frozen muscle by the method of Csapo requires elucidation. The freezing process appears in some way to have allowed complexation of myosin with large quantities of another muscle protein which releases upon alkaline treatment material sedimenting slightly faster than the carbonate subunit. A leading possibility would be the “tropomyosin” of Hamoir (24) and A-protein of Amberson and co-workers (25), which has been shown to complex with myosin. No studies have been reported on this protein in alkaline solutions, but alterations such as have been found to occur at mildly acid conditions could reasonably be expected. Although free actin dialyzed against 0.1 M Na2C03 gives two peaks, the smaller of which corresponds in sedimentation rate to the carbonate subunit, actin when present in synthetic actomyosin behaves quite differently. Under these conditions the faster of the two actin peaks disappears and the myosin peak acquires a curvature of the leading shoulder; the slower of the actin peaks also disappears, since the area of the slow peak in synthetic actomyosin mixtures is slightly less than it is in myosin. Therefore, actin appears to be ruled out as the source of the extra slow protein in myosin obtained from frozen muscle.
.4 SUBUNIT OF MYOSIN
199
SUMMARY
1. Treatment with 0.1 M Na2C03 releases from myosin a protein of about 29,000 molecular weight, which seems to be closely related to bhe material released from myosin by concentrated urea. 2. The amino acid composition is presented. A relatively large amount of phenylalanine is present, in agreement with prediction for the subunit of myosin postulated by Laki. 3. The C-terminal residue is isoleucine. The two C-terminal isolewines of myosin are probably due to the presence of t’wo of these subunits. 4. This subunit is distinct from the tropomyosin-like subunit of myosin. REFERENCES HOUGH, A., SYMONDS, P.,a~n L~~~,K.,Arch.Biochem.Bioph!/s' 60. 148 (1954). LAKI, K., J. Cellular Comp. Physiol. 49, Suppl. 1, 249 (1957). TSAO, T.-C., Biochim. et Biophys. Acta 11, 368 (1953). MIDDLEBROOH, W. R., AND SZENT-GY~RGYI, A. G., personal communication. KESSLER, V., AND SPICER, S. S., Biochim. et Biophys. -4cta 8, 474 (1952). CSAPO, A., Nature 162,218 (1949). JOHXSTOS, J. P., AND OGSTON, A. G., Trans. Farday Sot. 42, 789 (1946). KUNKEL, H. G., AND TISELIUS, A., J. Gen. Physiol. 36, 89 (1951). KOMINZ, D. R., SAAD, F., AND LAKI, K., Proc. Conf. on the Chemistry of Muscular Contraction, Oct. 1957, Tokyo, Japan, p. 66. KOMINZ, D. R., SAAD, F., GLADNER, J. A., AND LAKI, K., .Irch. Biochem. Biophys. 70, 16 (1957). IRREVERRE, F., AND MARTIN, W., ,4naZ. Chem. 26.257 (1954). COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides.” Reinhold Publishing Company, New York, 1943. LAKI, Ii., .&ND CARROLL, W. R., Nature 176, 389 (1955). VON HIPPEI., P. H., GELLERT, M. F., AND MORALES, M. F., Proc. Conf. on the Chemistry of Muscular Contraction, Oct. 1957, Tokyo, Japan., p. 1. M~MMAERTS, W. F. H. M., AND ALDRICH, B. B., Science 126, 1294 (1957). EVANS, R. I,., AND SAHOFF, H. A., J. Biol. Chem. 228, 295 (1957). LOCKER, R. H., Biochim. et Biophys. Acta 14, 533 (1954). LAKI, Ii., Proc. Conf. on the Chemistry of Muscular Contraction, Oct. 1957, Tokyo, Japan, p. 77. SZENT-GY~RGYI, A. G., AND COHEN, C., Science 126, 697 (1957). BAILEY, K., Biochem. J. 43, 271 (1948). GERGELY, J., J. Biol. Chem. 200, 543 (1953). MIHALYI, E., AND SZENT-GY~RGYI, A. G., J. Biol. Chem. 201, 189 (1953). SZENT-GY~RGYI, A. G., Arch. Biochem. Biophys. 42, 305 (1953). HAMOIR, G., Arch. intern. physiol. et biochem. 63, Suppl. (1955). AMBERSON, W. R., WHITE, J. I., BENSUSAN, H. B., HIMMELFARB, S., AND BLANKENHORN, B. E., Am. J. Physiol. 188, 205 (1957).
1. KowNz,D.R., 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
79,
191-19’3
(1959)
A Subunit of Myosin D. R. Kominz, W. R. Carroll, E. N. Smith and E. R. Mitchell Prong
the National
Health,
Public
Institute of Arthritis and Metabolic Diseases; National Institutes oj Health Service, United States Depaitment of Health, E&cation, c~nrl Welfare, Bethesda, Maryland
Received May 29, 1958 INTRODUCTION
On the basis of amino acid composition (I), Luki postulated that myosin is a copolymer of actin, tropomyosin, and a “third protein” which is characterized inter alia by a high phenylalanine content (2). Tsao found that prolonged treatment with urea resulted in the release from myosin of protein fragments of 14,000-16,000 molecular weight (3). Middlebrook and Szent-Gy6rgyi found that protein material is also released from myosin after brief treatment with urea (4). Tsao observed that, at pH 10.7 myosin was depolymerized to a large core of 170,000 molecular weight,, but he could not obtain the small molecules in this case (3). The present, paper deals with the release of protein material from myosin at an alkaline pH, showing that it corresponds to the material released by urea and that it could be the “third protein” postulated by Laki. METHODS Fresh rabbit skeletal muscle was extracted and the myosin precipitat,ed once a~cording to the method of Kessler and Spicer (5). The one-time precipitated myosin was dissolved in 0.5 M KC1 at a concentration of about 1% and clarified by centrifu gation at 30,000 r.p.m. in the Spinco preparative ultracentrifuge at 5%. in the r1re.q ence of millimolar adenosine triphosphate (ATP) and MgCl2 . It was reprecipitated and redissolved in 0.5 M KCl. Myosin A prepared in this way was employed in all of the subsequent procedures to be described. In addition, ultrac~entrifugal examination alone was carried out on myosin A prepared by the method of Taso (3), myosin -4 extracted from glycerinated psoas fibers, myosin B prepared by the method of Csapo from frozen muscle (6), standard 5.hr. myosin 13, and artificial artomyosin. The myosins in 0.5 M KC1 were examined in the Spinco analytical ult.racentrifuge before and after overnight dialysis at, 5°C. against 0.1 M N’a&OB The concentration of protein in t.he slow peak was estimated by superimposing the sedimentation di:igram over that, of the solvent alone, and planimetrically measuring the area between the peak and Ihe base line; area-concentration correlations were ralculated from measurements on diagrams of the purified carbonat,e subunit (see below). In addition, relat,ive peak areas were measured in t,hose cases where the total protein concentr;r.191
192
KOMINZ, CARROLL, SMITH AND MITCHELL
tion was sufficiently low so that the main peak was not hypersharp. The area of the slow peak is unaffected by the Johnston-Ogston effect (7). The area of the fast peak is diminshed by the Johnston-Ogston effect, but agreement between the results from absolute and relative peak areas indicates that this diminution is well within the error of measurement. In measurement of the concentration of protein in the slow peak, an approximately f 10% error is introduced by overlap with the main peak and with the meniscus at early sedimentation times, and by the broad base of the slow peak at later sedimentation times. Electrophoretic examination was made in the Aminco-Stern electrophoresis apparatus on a solution of rabbit myosin dialyzed against 0.1 A4 Na&03 . Paper electrophoresis was also performed at neutral pH between glass plates (8) on carbonatetreated myosin dialyzed against water. Ammonium sulfate fractionation was performed on rabbit, myosin after dialysis against 0.1 M Na&Oa or aft.er a further dialysis against water. Most of the protein precipitated between 24 and 32% saturation. The second fraction, precipitating at
5C~650/~ saturation (hereafter called “carbonate subunit”), was separated by centrifugation at 25,000r.p.m. for 20 min. and dialyzed exhaustively. Amino acid analysis was carried out by colum chromatography as previously described (9) on 18-hr., 104”C., 6 N HCl hydrolyzates. Carboxypeptidase determination of C-terminal end groups was performed as previously described (10). In the case of myosin dialyzed against 0.1 M Na&OJ and then against water, lowering the pH to 8.0 increased the viscosity to a thick gelatinous consistency; in this case the enzyme was allowed to react for 8 min. with constant stirring. Half of each sample was subjected to paper chromatography (11) and half was analyzed by column chromatography on a 50-cm.-long column of Dowex 50. For determination of the molecular weight of the carbonate subunit, a 0.66y0 solution was dialyzed against 0.1 M KCl, 0.05 I’/2 pH 8.0 phosphate buffer, and diluted to 0.44 and 0.22% with dialyzate. The three solutions were run at 42,040 r.p.m. in a synthetic boundary cell, with exposures bieng made at 16-min. intervals. Sedimentation rates were measured from the plates by a comparator. Boundary-spreading rates were measured on enlargements of the sedimentation diagrams by the sigma technique, and corrected for the wedge shape of the cell. Partial specific volume was calculated from the amino acid analysis (12). RESULTS
Unfrmtionated
Myosin
The ultracentrifugal diagram of a myosin preparation dissolved in 0.1 M Na&Os is given in Fig. 1. In all solutions of myosin A, myosin B, or synthetic actomyosin that were prepared from fresh or glycerinated muscle, a small, rapidly diffusing component with ~20of about 1.4 could be observed under these alkaline conditions (pH cu. 10). The amount of protein which is represented by this peak is in the range of 14-18 % of the total myosin protein. In the case of myosin B prepared from frozen muscle by the method of Csapo, the szo is about 1.8 and the relative amount present in the peak is 28 %. Free electrophoresis of 0.1 M Na&03-treated myosin reveals a broad slow component, heavily overlapping the myosin peak in both ascending
A SUBUNIT
OF MYOSIN
193
FIG. 1. Ultracentrifuge pattern of myosin A prepared by the method of Tsao (3) in 0.1 A4NaLQ . Concentration 0.9’%,temperature 25’C. Exposure at 72 min. after reaching speed of 59,780r.p.m., bar angle 60’. Direct’ion of sedimentation from left to right. This demonstrates the typical appearance of a slow component in myosin solutions brought to approximately pH 10by dialysis against 0.1 M Na&O, .
and descending limbs. Paper electrophoresis of the same material gives :I dark streak at the origin and a pale-staining area extending from the origin toward the anode. Paper electrophoresis of the purified carbonate subunit reveals only the pale-staining area extending from the origin t,oward the anode and forming no discrete zones. Purified Carbonate Subunit The yield of carbonate subunit obtained from the 5(r65% saturation ammonium sulfate fraction, after exhaustive dialysis to remove the ammonium sulfate, is found to be 6-7 % of the starting material. The carbonat,c subunit is monodisperse in the ultracentrifuge (Fig. 2). The amino acid composition of the carbonate subunit is given in Table I. It may be compared with the amino acid analysis carried out in this laboratory on the urea subunit of myosin prepared by Middlebrook and Szent-Gyijrgyi (4). Figures 3 and 4 plot the sedimentation and boundary-spreading data calculated from the synthetic boundary ultracentrifuge diagrams obtained at neutral pH. The extrapolated parameters are s:~,~ = 2.32 X 10-13 sec. and D&,w = 7.25 X lo-’ sq. cm./sec. From these, and v = 0.731 ct./g., a molecular weight of 29,000 is obtained. Because the slow peak
194
KOMINZ,
CARROLL,
SMITH
AND
MITCHELL
pattern of the carbonate subunit of FIQ. 2. Synthetic boundary ultracentrifuge myosin in 0.1 M KCl, p/2 0.05 pH 7.9 phosphate buffer. Concentration O.SS%, temperature 25.3%. Exposure at 51 min. after reaching speed of 42,040 r.p.m., bar angle 35”. Direction of sedimentation from left to right.
TABLE I Amino Acid Composition of Myosin Subunit (Moles/,106 g., based on 16.7oj, N)
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Amide NH*
94 34 36 126 38 55 86 33 26 30 58 14 38 8 79
Total
779
93 40 37 116 35 57 77 35 27 32 59 10 37 9 76 24 764
h
SUBUNIT
OF
195
MYOSIN
in unfractionated myosin preparations examined at alkaline pH had a considerably lower sedimentation rate than the purified carbonate subunit’ examined at neutral pH, the carbonate subunit was also examined in the synthetic-boundery cell at pH 10 in 0.1 M Na&Os . The extrapolated value of the sedimentation constant was 25% lower than at neutral pH; the diffusion rate was also about 20% lower, with a much steeper concentration dependence. The parallel drop in both parameters can be interpreted that swelling, but no drop in molecular weight, occurs at t#his alkaline pH . Degradation by Carboxypeptidasr Paper chromatography of the amino acids liberated by carboxypeptidase treatment gives qualitat’ively the same picture for 100 mg. of carbonatetreated myosin and 6 mg. of carbonate subunit: isoleucine as end-group; much less serine; and traces of alanine, valine, and methionine. The quantitative results are given in Table II; 1.4 isoleucine residues are liberated/mole myosin, and 1.0 isoleucine residue is liberated/mole carbonate subunit. s2* x IO” 2.5 r
2:i,m, 0
FIG. 3. Concentration dependence carbonate
subunit
of sedimentation from myosin.
rate of
8-
6
0
I 2
FIG. 4. Concentration carbonate
I 4
I 6
dependence of diffusion subunit from myosin.
I
h&l rate of
196
KOMINZ,
CARROLL,
SMITH
AND
MITCHELL
TABLE II C-Terminal Residues Liberated by Carboxypeptidase Carbonate Subunit
Myosin (Per 4;wy,
Isoleucine Serine Valine Methionine Alanine
9.1
1.4 0.4 -
(per 29,ooo IT.)
5 min.
15 min.
1.0 0.3 -
1.2 0.3 0.3 0.3 -
DISCUSSION
The present study and that of Middlebrook and Szent-GyQgyi (4) throw further light on the original finding of Tsao (3) that fragments small in comparison with the 420,000 molecular weight (13-15) of the parent myosin molecule can be released from it. They indicate that by different methods of denaturation, similar end products can be obtained. The sedimentation-diffusion value of 29,000 for the molecular weight of the carbonate subunit is in good agreement with the value obtained by Middlebrook and Szent-Gybrgyi for their urea subunit. This value is about twice the value found by Tsao for his urea subunit: 14,000 by osmotic pressure and 16,000 by fluorescence polarization. From the amino acid composition of the carbonate subunit, one can calculate the minimum molecular weight to be about one-half of 29,000. It is possible, therefore, that the 29,000 molecular weight represents a stable dimer. The detection by carboxypeptidase of only one C-terminal isoleucine in 29,000 molecular weight could be interpreted as a masking phenomenon (16). A simple explanation for the different estimations of molecular weight could be that the prolonged exposure to urea employed by Tsao destroyed the stability of the dimer. However, until it is demonstrated that a stable dimer of two identical components is present, it would appear reasonable to assume a 29,000 molecular weight for the carbonate subunit. The 6-7 % yield for the carbonate subunit is very similar to the 7-9 % yield obtained by Tsao for the urea subunit. However, low yields are of common occurrence, and the ultracentrifuge diagrams indicate that approximately 14% of myosin A is released into the slow peak. This corresponds to two molecules of the carbonate subunit, from which carboxypeptidase would release two C-terminal isoleucines. We find that 1.5 moles of C-terminal isoleucine is liberated from carbonate-treated myosin, and Locker obtains the same value for native myosin (17). The identity of the paper chromatogram “fingerprints” of the end groups of myosin and of its carbonate subunit suggests that all of the carboxypeptidase action in myosin is indeed upon the carbonate subunits. It is worth noting that
A
SUBUNIT
OF
TABLE Amino
Acid
Composition
197
III
of Tropomyosin-like
(Moles/W
Aspartic acid Threonine Methionine Serine Glutamic acid Glycine i41anine Valine Isoleucine J,eucine Tyrosine I’henylalanine Proline Lysine Histidine Arginine Amide NHI Charge
MYOSIX
Subunits
from
Myosin
g.)
PFA Ox. myosin fraction (18)
Denatured myosin fractmn (18)
89 45 190 26 80 25 31 93 10 9
86 36 20 45 197 22 81 25 29 91 9 6 r
87 12
8; 19
2 86 22
26 30 40 212 12 108 27 30 95 15 3 2 107 5
(1:;) 38
(1:) 32
(1::) 21
(ii, 8!)
53
LMM fraction (1Y)
82 36 18 38 204 18 84 28 32 96 9
Tropomyosin
89
two molecules of the carbonate subunit will contribute approximately the 60,000 g. protein predicted by Laki (2) to be present in the missing “third protein” of myosin. In addition to the subunit described in this paper, the use of trypsin and performic acid by Laki (18) and of trypsin by Szent-Gy6rgyi (19) has succeeded in releasing from myosin a larger tropomyosin-like subunit, from myosin, amounting to about 27% of the protein in myosin. ,4s can be seen from Table III, this differs from the classical tjropomyosin of Bailey (20, 9) primarily in the residues alanine, lysine, arginine, histidine, and amide ammonia. There is a close resemblance, however, in phenylalanine and proline. It is thus immediately apparent, that the curbonat,e subunit of myosin could not have arisen from the tropomyosin-like subunit of myosin. The large values for proline and phenylalanine in the carbonate subunit preclude such a possibility. This large value for phenylalanine is in line with Laki’s prediction (2) for the “third protein” present, in myosin. It appears likely that the tropomyosin-like subunit and the carbonate subunit make up most of the crude L-meromyosin fraction (21-23, 1) of myosin. A graphic illustration of the distinctness of these two subunits can be obtained when one allows myosin to be split by Cu(CIQ3-at an alkaline pH for several days, a procedure for split)ting disulfide bonds a+ present being studied in this laboratory (Fig. 5).
198
KOMINZ,
CARROLL,
SMITH
AND
MITCHELL
FIG. 5. Ultracentrifuge pattern of myosin treated 7 days with 0.014 Cu(CN), in 0.25 M KCl, pH 10. Concentration 1.4’j& temperature 25%. Exposure at 70 min. after reaching speed of 59,760 r.p.m., bar angle 60’. Direction of sedimentation from left to right. This illustrates the presence of both the carb0nat.e subunit (a) and the tropomyosin-like subunit (5) under these conditions.
The remarkable presence of nearly 30% slow peak in myoein B prepared from frozen muscle by the method of Csapo requires elucidation. The freezing process appears in some way to have allowed complexation of myosin with large quantities of another muscle protein which releases upon alkaline treatment material sedimenting slightly faster than the carbonate subunit. A leading possibility would be the “tropomyosin” of Hamoir (24) and A-protein of Amberson and co-workers (25), which has been shown to complex with myosin. No studies have been reported on this protein in alkaline solutions, but alterations such as have been found to occur at mildly acid conditions could reasonably be expected. Although free actin dialyzed against 0.1 M Na2C03 gives two peaks, the smaller of which corresponds in sedimentation rate to the carbonate subunit, actin when present in synthetic actomyosin behaves quite differently. Under these conditions the faster of the two actin peaks disappears and the myosin peak acquires a curvature of the leading shoulder; the slower of the actin peaks also disappears, since the area of the slow peak in synthetic actomyosin mixtures is slightly less than it is in myosin. Therefore, actin appears to be ruled out as the source of the extra slow protein in myosin obtained from frozen muscle.
.4 SUBUNIT OF MYOSIN
199
SUMMARY
1. Treatment with 0.1 M Na2C03 releases from myosin a protein of about 29,000 molecular weight, which seems to be closely related to bhe material released from myosin by concentrated urea. 2. The amino acid composition is presented. A relatively large amount of phenylalanine is present, in agreement with prediction for the subunit of myosin postulated by Laki. 3. The C-terminal residue is isoleucine. The two C-terminal isolewines of myosin are probably due to the presence of t’wo of these subunits. 4. This subunit is distinct from the tropomyosin-like subunit of myosin. REFERENCES HOUGH, A., SYMONDS, P.,a~n L~~~,K.,Arch.Biochem.Bioph!/s' 60. 148 (1954). LAKI, K., J. Cellular Comp. Physiol. 49, Suppl. 1, 249 (1957). TSAO, T.-C., Biochim. et Biophys. Acta 11, 368 (1953). MIDDLEBROOH, W. R., AND SZENT-GY~RGYI, A. G., personal communication. KESSLER, V., AND SPICER, S. S., Biochim. et Biophys. -4cta 8, 474 (1952). CSAPO, A., Nature 162,218 (1949). JOHXSTOS, J. P., AND OGSTON, A. G., Trans. Farday Sot. 42, 789 (1946). KUNKEL, H. G., AND TISELIUS, A., J. Gen. Physiol. 36, 89 (1951). KOMINZ, D. R., SAAD, F., AND LAKI, K., Proc. Conf. on the Chemistry of Muscular Contraction, Oct. 1957, Tokyo, Japan, p. 66. KOMINZ, D. R., SAAD, F., GLADNER, J. A., AND LAKI, K., .Irch. Biochem. Biophys. 70, 16 (1957). IRREVERRE, F., AND MARTIN, W., ,4naZ. Chem. 26.257 (1954). COHN, E. J., AND EDSALL, J. T., “Proteins, Amino Acids and Peptides.” Reinhold Publishing Company, New York, 1943. LAKI, Ii., .&ND CARROLL, W. R., Nature 176, 389 (1955). VON HIPPEI., P. H., GELLERT, M. F., AND MORALES, M. F., Proc. Conf. on the Chemistry of Muscular Contraction, Oct. 1957, Tokyo, Japan., p. 1. M~MMAERTS, W. F. H. M., AND ALDRICH, B. B., Science 126, 1294 (1957). EVANS, R. I,., AND SAHOFF, H. A., J. Biol. Chem. 228, 295 (1957). LOCKER, R. H., Biochim. et Biophys. Acta 14, 533 (1954). LAKI, Ii., Proc. Conf. on the Chemistry of Muscular Contraction, Oct. 1957, Tokyo, Japan, p. 77. SZENT-GY~RGYI, A. G., AND COHEN, C., Science 126, 697 (1957). BAILEY, K., Biochem. J. 43, 271 (1948). GERGELY, J., J. Biol. Chem. 200, 543 (1953). MIHALYI, E., AND SZENT-GY~RGYI, A. G., J. Biol. Chem. 201, 189 (1953). SZENT-GY~RGYI, A. G., Arch. Biochem. Biophys. 42, 305 (1953). HAMOIR, G., Arch. intern. physiol. et biochem. 63, Suppl. (1955). AMBERSON, W. R., WHITE, J. I., BENSUSAN, H. B., HIMMELFARB, S., AND BLANKENHORN, B. E., Am. J. Physiol. 188, 205 (1957).
1. KowNz,D.R., 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.