ANALYTICAL
BIOCHEMISTRY
95, 133-138 (1979)
The Use of Polyacrylamide Study Relations between
Gel Electrophoresis to the Chains of Myosinl
TETSU HOZUMI, KATHLEEN UE, MANUEL F. MORALES, AND JEAN BOTTS Cardiovascular
Research
Institute.
University
of Cali&nia,
San
Francisco.
San
Francism.
Calijiwnin
94143
Received October 23. 1978 Polyacrylamide gel electrophoresis (PAGE) of native proteins shows that myosin subfragment-l (S-l), prepared by ol-chymotryptic digestion of myosin, can be separated into two well-spaced bands corresponding to two S-l isozymes. One of these consists of a heavy chain fragment (HC) and light chain (LC,); the other to HC and light chain 3 (LC,). Addition of light chain 2 (LC,), lost during the digestion process, speeds up the migration of the LC,bearing S-l (HC. LC,) but leaves the L&-bearing S-l (HC.LC:,) essentially unchanged. This suggests that LC, has a stronger affinity for the former and forms with it the complex, HC’LC, ‘LC,. 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) treatment of myosin is known to remove only about half of the LCI (“DTNB light chain”). Although S-l prepared from such myosin cannot be well-resolved by DEAE-cellulose chromatography into two peaks, the beginning of the peak is largely HC LC, LC2 and the ending is largely HC LC,. as revealed by sodium dodecvl sulfate (SDS)-PAGE. Thus. the loss of LC, during DTNB treatment is- mainly from S-i bearing LC,.
It is well established (1) that each “head piece” half of the duplex myosin molecule of vertebrate striated muscle consists of a portion of “heavy chain” and two “light chains.” One of these light chains, LCp,2 is common to both head pieces, but the other can be either one of two types, LC, or LC,. When the head pieces are cleaved from myosin with papain in the presence of Mg2+ (2) and isolated, each of these so-called S-l fragments consists of a heavy chain fragment (HC) and LC, + LC, or LC, + LC,. However, when the protease is cY-chymotrypsin in the presence of EDTA, the S-1s are HC . LC, or HC . LC,- the LC2 having been degraded to fragments that may or may not remain bound to the complexes. In the experiments of others using these systems
there are two findings that have caught our attention and have seemed related: (a) The isozymic myosins as well as the isozymic S- 1s (3,4) have been resolved by chromatography on DEAE-cellulose, suggesting that they may differ with respect to charge. (b) It has been found (5) that, on treatment with excess EDTA-DTNB, LC,! is selectively but only partially removed from a conventional myosin preparation, suggesting that it may be removed from one isozyme and not the other. Because of the presumption that chymotryptically prepared HC . LC, and HC . LC3 differ in regard to charge, we studied mixtures of these and other components using polyacrylamide gel electrophoresis (PAGE) in the absence as well as in the presence of sodium dodecyl sulfate (SDS).
’ This paper is dedicated to the memory of Dr. Alvin METHODS AND MATERIALS Nason. ’ Abbreviations used: S-l, Myosin subfragment 1; EDTA, cy-chymotrypsin, papain, and HC, heavy chain fragment of myosin; LC, light chain of myosin; DTMB. 5.5’-dithiobis-(2-nitrobenzoic acid): DTNB were obtained from Sigma Chemical SDS and polyacrylamide gel, DTT, dithiothreitol: SDS, sodium dodecyl sulfate: Company: PAGE, polyacrylamide gel electrophoresis. from Bio-Rad, DEAE-cellulose (Whatman 133
0003-2697/79/070133-06$02.00/O Copyright All right\
& 1979 by Academx Prey\. Inc. of reproductmn I” any iorm rerervsd.
134
HOZUMI
ET AL.
HC.LC3
LC2 LC3
A
B
C
DEFG
1. Gel electrophoretic separation of native and SDS-treated proteins. (A-C) Native proteins (no SDS) on 3.5% polyacrylamide gel; buffer: 0.05 M Tris, 0.38 M glycine; 4”C, 150 V, 2-3 mA per tube, (D-G) SDS-treated proteins on 10% polyacrylamide SDS-gel, buffer same as above. (A,D) HC.LC, (tube No. 92, Fig. 2); (B, E) HC.LC, (tube No. 125, Fig. 2); (C, F) S-l prior to chromatographic separation; (G) myosin prior to chymotryptic digestion. FIG.
DE-52) from Whatman. All other reagents were best grade available commercially. Myosin was prepared from the back muscle of rabbit by the method of Tonomura ef al. (6). S-l for the gel electrophoresis experiments on native protein was prepared from myosin by a-chymotryptic digestion following essentially the procedure of Weeds and Taylor (4). This preparation contains
no intact LG. When S-l was prepared from DTNB-treated myosin, it was cleaved from the myosin by digestion with insoluble papain in the presence of Mg2+, according to a modified method of Lowey et al. (7). Both preparations were fractionated on DE 52 cellulose. For partial removal of LC2 prior to S-l cleavage, myosin was treated with DTNB and EDTA under conditions de-
ELECTROPHORESIS
OF MYOSIN SUBFRAGMENT-
CHAINS
.6
TUBE NUMBER FOG. 2. Chromatogram for S-l resolved into HC’LC, and HC.LCJ. Conditions: 300 mg S-l applied to DE 52 cellulose column (2.5 x 45 cm), 75 mM imidazole buffer (pH 7.0)-0.12 M KC1 (21. total), 10 ml/tube, flow rate 24 ml/h.
scribed by Gazith et al. (5). The treatment was carried out for 10 min at 0°C and terminated by addition of 10 vol of cold water to precipitate the myosin. In one kind of experiment we were interested in observing the effect of LC, on the S-l isozymes. In this case the supematant following the EDTA-DTNB treatment was collected, and the LC, that it contained was concentrated using Amicon hollow fibers DC 2. The concentrate was then dialyzed exhaustively against 20 mM Tris-HCl, pH 8.0, 2 mM DTT. To obtain the results of Fig. 3 we obtained the electrophoretograms (see below) of the chromatographitally resolved isozymic components of a chymotryptic preparation of S- 1, of the digest prior to chromatography, and of the digest after it had been incubated with LC,. This incubation was accomplished in 60 mM Tris-HCl, 0.1 mM DTT, pH 8.5, 0°C; S-l was 20 FM and LC, was 40 FM. After 66 h
electrophoretic analysis showed that almost all the HC . LC, had reacted; after 160 h no HC * LC, remained unreacted. In the other kind of experiment we were interested in the light chain composition of the residual myosin, so the precipitate following the EDTA-DTNB treatment was collected by centrifugation at 7000g for 10 min, and washed three times with 20 mM KCl, 20 mM Tris-HCl, 2 mM MgC12, pH 8.0 to remove released LC, and EDTA-DTNB. Finally the precipitate was dialyzed (and redissolved) against 0.5 M KCl, 20 mM TrisHCl, 2 mM MgC&, and 2 mM DTT for several days to remove the DTNB blocking groups. Then, as mentioned above, S-l was prepared from this myosin using Mg2+ papain so as to minimally damage its LC2 content. The concentrations of S-l and LC, were estimated using eZBO= 8.85 x lo4 and EZHO = 1 X 104, respectively. PAGE to study the “native” (no SDS)
136
HOZUMI
ET AL.
mercaptoethanol, and 0.005% bromphenol blue, pH 8.6, 0°C. The final solution contained the proteins at l-2 mg/ml. The sample loadings were IO-30 pg protein per tube. Electrophoresis was carried out at 4”C, 150 V p.d., and 2-3 mA per tube, for 3-3.5 h. Electrophoresis of HC and the LCs in 10% polyacrylamide gels, in the presence of SDS, generally was carried out according to Weber and Osborn (8) and Paterson and Strohman (9); however, the results of Fig. 1, D-G, were obtained using some variations in technique. In these variants the gels were prepared as described above for the no-SDS case except that 10% polyacrylamide was used; acrylamide:methylene bisacrylamide was 37: 1 (w/w), and 0.1% SDS replaced 10% glycerol; 0.1% SDS was also added to the electrode solution. The samples were also prepared as for the no-SDS case except that 1% SDS, 6-8 M urea replaced the 25% glycerol. Electrophoresis was carried out at room temperature, at 150 V p.d., 2-3 mA per tube.
HC-LCI
HC.LC3
RESULTS
A
B
FIG. 3. Effect of adding LC, to a mixture of HC. LC, and HC.LC,. S-l from chymotryptic (0.050 mg/dl) digestion of myosin, 10 min at 24°C. (A) Control, stored at 0°C during incubation period. (B) S-l + LC, incubated at 0°C for 160 h. [LC,]/[S-l] = 1.5. Gel conditions same as for Figs. IA-C.
isozymes of S-l was carried out in 3.5% acrylamide gels. These gels were prepared by mixing the appropriate acrylamide solution with 0.05% ammonium persulfate. Acrylamide:methylene-bisacrylamide was 20: 1 (w/w). Besides acrylamide the gels contained 0.10% (v/v) of N,N,N,N-tetramethylenediamine. The gels were 5 mm in diameter and 75-100 mm long. The electrode solution was 0.05 M Tris, 0.38 M glycine, pH 8.6. The protein samples were incubated for a few minutes with an equal volume of a solution twofold as concentrated as 0.05 M Tris, 0.38 M glycine, 25% glycerol, 1% 2-
The electrophoretogram of chymotryptitally prepared S- 1 shows two well-separated bands (Fig. 1C). If such S-l is first resolved by DE 52 cellulose chromatography (Fig. 2) following the procedure of Weeds and Taylor (4) and elution aliquots are electrophoresed, it is evident by comparison (Figs. lA-C,) that the two components resolved by PAGE are the same components resolved by chromatography, viz., HC. LC, and HC*LC3. If the isozymic mixture (Fig. 3A) is incubated with LC, and then electrophoresed, little effect is observed on the mobility of HC ’ LC3, but the mobility of the HC. LC, is increased so that the bands are now closely spaced (Fig. 3B). If instead of chymotryptically prepared S-l we use Mg-papainprepared S-l (i.e., S-l already containing LC,), the electrophoretogram is a close doublet from the beginning. When myosin was treated with EDTADTNB, PAGE in the presence of SDS
ELECTROPHORESIS
OF MYOSIN
SUBFRAGMENT-
65 70 75 79 8486 AB
C
137
CHAINS
9093
D
FIG. 4. Gel electrophoresis on SDS-treated proteins; 10% acrylamide gel containing SDS. (A) myosin; (B) DTNB-treated myosin; (C) S-l prepared from DTNB-treated myosin (digestion with insoluble papain in presence of MgZ+; (D) Representative samples (-50 pg each) from chromatographed fractions (tube No. identifications refer to Fig. 5). We believe that in papain-proteolyzed S-l, LC, is slightly more degraded (therefore runs faster) than in myosin; also in such S-l a fragment of HC coincidentally runs where LC, from myosin runs; this explains the odd labelling when myosin (A, B) is compared with S-l (C, D).
showed, as expected, that the system had lost LC,-but far from totally (Fig. 4, compare A and B). From such myosin we now prepared S- 1, using Mg-papain for the cleavage in order to minimize damage to the remaining LC, (Fig. 4C). The mixture was resolved on DE 52 cellulose (Fig. 5). Elution aliquots (indicated by tube number from Fig. 5) were electrophoresed in the presence of SDS (Fig. 4D). Although the resolution is not as good as with chymotryptically prepared S-l, it is obvious that the isozymic species are predominantly HC . LC, . LC, and HC . LC,.
CONCLUSIONS
AND DISCUSSION
The results show that PAGE reveals the isozymes of myosin S-l about as well as chromatography and also lends itself to studying associations among components. As regards our particular illustrative system, the results show that LC, binds strongly to HC.LC, but weakly to HC. LC2. Conversely, they show that LC, is hard to remove from HC- LC, . LCB but easy to remove from HC . LC, . LC,. Since Frank and Weeds (IO) showed that LC, and LC, are very similar except for an additional 41residue tail in the former, it is reasonable to think that
138
HOZUMI
ET AL.
0.3.
0.2 0
00 ON 0 0.1 -
0’
I
I 40
I Tube
I 80 No.
I
I 120
FIG. 5. DE 52-cellulose chromatography of S-l prepared from DTNB-treated myosin by digestion with insoluble papain in the presence of Mg *+. S-l, 180 mg, was applied. Other conditions were as in Fig. 2.
the strong interaction of LC, with HC.LC, involves this tail in addition to HC. Another reasonable surmise is that the presence of LC, in the S-l complex renders more difficult the resolution into isozymic components, and it is possible that residual proteolytic fragments of LC, do likewise. This is very likely the explanation of why chromatographic separation of Mg-papainprepared S-l is harder than that of chymotrypsin-prepared S-l; and, similarly, variability in the extent of LC, degradation may cause variability in the success of resolving chymotrypsin-prepared S- 1. ACKNOWLEDGMENTS This research was supported by Grants HL-06285, HL-16683, PCM-75-22698, and AHA CI-8. Tetsu Hozumi is a Fellow of the American Heart Association and Manuel F. Morales is a Career Investigator.
REFERENCES 1. Lowey, S., and Holt, J. C. (1973) Cold Spring Harbor Symp. Quant. Biol. 37, 19-28. 2. Margossian, S. S., Lowey, S., and Barshop, B. (1975) Nature (London) 258, 163-166. 3. Holt, J. C., and Lowey, S. (1977) Biochemistry 16,4398-4402.
4. Weeds, A. G., and Taylor, R. S. (1975) Nature (London) 257, 54-56. 5. Gazith, J. S., Himmelfarb, S., and Harrington, W. F. (1970) J. Biol. Chem. 245, 19-22. 6. Tonomura, Y., Appel, P., and Morales, M. F. (1966) Biochemistry 5, 515-521. 7. Lowey, S., Slayter, H. S., Weeds, A. G., and Baker, H. (1969) J. Mol. Biol. 42, l-29. 8. Weber, K., and Osbom, M. (1969) J. Biot. Chem. 244,4406-4412. 9. Paterson, B., and Strohman, R. C. (1970) Biochemistry 9,4094-4105. 10. Frank, G., and Weeda, A. G. (1974) Eur. J. Biothem. 317-334.
BIOCHEMISTRY
95, 133-138 (1979)
The Use of Polyacrylamide Study Relations between
Gel Electrophoresis to the Chains of Myosinl
TETSU HOZUMI, KATHLEEN UE, MANUEL F. MORALES, AND JEAN BOTTS Cardiovascular
Research
Institute.
University
of Cali&nia,
San
Francisco.
San
Francism.
Calijiwnin
94143
Received October 23. 1978 Polyacrylamide gel electrophoresis (PAGE) of native proteins shows that myosin subfragment-l (S-l), prepared by ol-chymotryptic digestion of myosin, can be separated into two well-spaced bands corresponding to two S-l isozymes. One of these consists of a heavy chain fragment (HC) and light chain (LC,); the other to HC and light chain 3 (LC,). Addition of light chain 2 (LC,), lost during the digestion process, speeds up the migration of the LC,bearing S-l (HC. LC,) but leaves the L&-bearing S-l (HC.LC:,) essentially unchanged. This suggests that LC, has a stronger affinity for the former and forms with it the complex, HC’LC, ‘LC,. 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) treatment of myosin is known to remove only about half of the LCI (“DTNB light chain”). Although S-l prepared from such myosin cannot be well-resolved by DEAE-cellulose chromatography into two peaks, the beginning of the peak is largely HC LC, LC2 and the ending is largely HC LC,. as revealed by sodium dodecvl sulfate (SDS)-PAGE. Thus. the loss of LC, during DTNB treatment is- mainly from S-i bearing LC,.
It is well established (1) that each “head piece” half of the duplex myosin molecule of vertebrate striated muscle consists of a portion of “heavy chain” and two “light chains.” One of these light chains, LCp,2 is common to both head pieces, but the other can be either one of two types, LC, or LC,. When the head pieces are cleaved from myosin with papain in the presence of Mg2+ (2) and isolated, each of these so-called S-l fragments consists of a heavy chain fragment (HC) and LC, + LC, or LC, + LC,. However, when the protease is cY-chymotrypsin in the presence of EDTA, the S-1s are HC . LC, or HC . LC,- the LC2 having been degraded to fragments that may or may not remain bound to the complexes. In the experiments of others using these systems
there are two findings that have caught our attention and have seemed related: (a) The isozymic myosins as well as the isozymic S- 1s (3,4) have been resolved by chromatography on DEAE-cellulose, suggesting that they may differ with respect to charge. (b) It has been found (5) that, on treatment with excess EDTA-DTNB, LC,! is selectively but only partially removed from a conventional myosin preparation, suggesting that it may be removed from one isozyme and not the other. Because of the presumption that chymotryptically prepared HC . LC, and HC . LC3 differ in regard to charge, we studied mixtures of these and other components using polyacrylamide gel electrophoresis (PAGE) in the absence as well as in the presence of sodium dodecyl sulfate (SDS).
’ This paper is dedicated to the memory of Dr. Alvin METHODS AND MATERIALS Nason. ’ Abbreviations used: S-l, Myosin subfragment 1; EDTA, cy-chymotrypsin, papain, and HC, heavy chain fragment of myosin; LC, light chain of myosin; DTMB. 5.5’-dithiobis-(2-nitrobenzoic acid): DTNB were obtained from Sigma Chemical SDS and polyacrylamide gel, DTT, dithiothreitol: SDS, sodium dodecyl sulfate: Company: PAGE, polyacrylamide gel electrophoresis. from Bio-Rad, DEAE-cellulose (Whatman 133
0003-2697/79/070133-06$02.00/O Copyright All right\
& 1979 by Academx Prey\. Inc. of reproductmn I” any iorm rerervsd.
134
HOZUMI
ET AL.
HC.LC3
LC2 LC3
A
B
C
DEFG
1. Gel electrophoretic separation of native and SDS-treated proteins. (A-C) Native proteins (no SDS) on 3.5% polyacrylamide gel; buffer: 0.05 M Tris, 0.38 M glycine; 4”C, 150 V, 2-3 mA per tube, (D-G) SDS-treated proteins on 10% polyacrylamide SDS-gel, buffer same as above. (A,D) HC.LC, (tube No. 92, Fig. 2); (B, E) HC.LC, (tube No. 125, Fig. 2); (C, F) S-l prior to chromatographic separation; (G) myosin prior to chymotryptic digestion. FIG.
DE-52) from Whatman. All other reagents were best grade available commercially. Myosin was prepared from the back muscle of rabbit by the method of Tonomura ef al. (6). S-l for the gel electrophoresis experiments on native protein was prepared from myosin by a-chymotryptic digestion following essentially the procedure of Weeds and Taylor (4). This preparation contains
no intact LG. When S-l was prepared from DTNB-treated myosin, it was cleaved from the myosin by digestion with insoluble papain in the presence of Mg2+, according to a modified method of Lowey et al. (7). Both preparations were fractionated on DE 52 cellulose. For partial removal of LC2 prior to S-l cleavage, myosin was treated with DTNB and EDTA under conditions de-
ELECTROPHORESIS
OF MYOSIN SUBFRAGMENT-
CHAINS
.6
TUBE NUMBER FOG. 2. Chromatogram for S-l resolved into HC’LC, and HC.LCJ. Conditions: 300 mg S-l applied to DE 52 cellulose column (2.5 x 45 cm), 75 mM imidazole buffer (pH 7.0)-0.12 M KC1 (21. total), 10 ml/tube, flow rate 24 ml/h.
scribed by Gazith et al. (5). The treatment was carried out for 10 min at 0°C and terminated by addition of 10 vol of cold water to precipitate the myosin. In one kind of experiment we were interested in observing the effect of LC, on the S-l isozymes. In this case the supematant following the EDTA-DTNB treatment was collected, and the LC, that it contained was concentrated using Amicon hollow fibers DC 2. The concentrate was then dialyzed exhaustively against 20 mM Tris-HCl, pH 8.0, 2 mM DTT. To obtain the results of Fig. 3 we obtained the electrophoretograms (see below) of the chromatographitally resolved isozymic components of a chymotryptic preparation of S- 1, of the digest prior to chromatography, and of the digest after it had been incubated with LC,. This incubation was accomplished in 60 mM Tris-HCl, 0.1 mM DTT, pH 8.5, 0°C; S-l was 20 FM and LC, was 40 FM. After 66 h
electrophoretic analysis showed that almost all the HC . LC, had reacted; after 160 h no HC * LC, remained unreacted. In the other kind of experiment we were interested in the light chain composition of the residual myosin, so the precipitate following the EDTA-DTNB treatment was collected by centrifugation at 7000g for 10 min, and washed three times with 20 mM KCl, 20 mM Tris-HCl, 2 mM MgC12, pH 8.0 to remove released LC, and EDTA-DTNB. Finally the precipitate was dialyzed (and redissolved) against 0.5 M KCl, 20 mM TrisHCl, 2 mM MgC&, and 2 mM DTT for several days to remove the DTNB blocking groups. Then, as mentioned above, S-l was prepared from this myosin using Mg2+ papain so as to minimally damage its LC2 content. The concentrations of S-l and LC, were estimated using eZBO= 8.85 x lo4 and EZHO = 1 X 104, respectively. PAGE to study the “native” (no SDS)
136
HOZUMI
ET AL.
mercaptoethanol, and 0.005% bromphenol blue, pH 8.6, 0°C. The final solution contained the proteins at l-2 mg/ml. The sample loadings were IO-30 pg protein per tube. Electrophoresis was carried out at 4”C, 150 V p.d., and 2-3 mA per tube, for 3-3.5 h. Electrophoresis of HC and the LCs in 10% polyacrylamide gels, in the presence of SDS, generally was carried out according to Weber and Osborn (8) and Paterson and Strohman (9); however, the results of Fig. 1, D-G, were obtained using some variations in technique. In these variants the gels were prepared as described above for the no-SDS case except that 10% polyacrylamide was used; acrylamide:methylene bisacrylamide was 37: 1 (w/w), and 0.1% SDS replaced 10% glycerol; 0.1% SDS was also added to the electrode solution. The samples were also prepared as for the no-SDS case except that 1% SDS, 6-8 M urea replaced the 25% glycerol. Electrophoresis was carried out at room temperature, at 150 V p.d., 2-3 mA per tube.
HC-LCI
HC.LC3
RESULTS
A
B
FIG. 3. Effect of adding LC, to a mixture of HC. LC, and HC.LC,. S-l from chymotryptic (0.050 mg/dl) digestion of myosin, 10 min at 24°C. (A) Control, stored at 0°C during incubation period. (B) S-l + LC, incubated at 0°C for 160 h. [LC,]/[S-l] = 1.5. Gel conditions same as for Figs. IA-C.
isozymes of S-l was carried out in 3.5% acrylamide gels. These gels were prepared by mixing the appropriate acrylamide solution with 0.05% ammonium persulfate. Acrylamide:methylene-bisacrylamide was 20: 1 (w/w). Besides acrylamide the gels contained 0.10% (v/v) of N,N,N,N-tetramethylenediamine. The gels were 5 mm in diameter and 75-100 mm long. The electrode solution was 0.05 M Tris, 0.38 M glycine, pH 8.6. The protein samples were incubated for a few minutes with an equal volume of a solution twofold as concentrated as 0.05 M Tris, 0.38 M glycine, 25% glycerol, 1% 2-
The electrophoretogram of chymotryptitally prepared S- 1 shows two well-separated bands (Fig. 1C). If such S-l is first resolved by DE 52 cellulose chromatography (Fig. 2) following the procedure of Weeds and Taylor (4) and elution aliquots are electrophoresed, it is evident by comparison (Figs. lA-C,) that the two components resolved by PAGE are the same components resolved by chromatography, viz., HC. LC, and HC*LC3. If the isozymic mixture (Fig. 3A) is incubated with LC, and then electrophoresed, little effect is observed on the mobility of HC ’ LC3, but the mobility of the HC. LC, is increased so that the bands are now closely spaced (Fig. 3B). If instead of chymotryptically prepared S-l we use Mg-papainprepared S-l (i.e., S-l already containing LC,), the electrophoretogram is a close doublet from the beginning. When myosin was treated with EDTADTNB, PAGE in the presence of SDS
ELECTROPHORESIS
OF MYOSIN
SUBFRAGMENT-
65 70 75 79 8486 AB
C
137
CHAINS
9093
D
FIG. 4. Gel electrophoresis on SDS-treated proteins; 10% acrylamide gel containing SDS. (A) myosin; (B) DTNB-treated myosin; (C) S-l prepared from DTNB-treated myosin (digestion with insoluble papain in presence of MgZ+; (D) Representative samples (-50 pg each) from chromatographed fractions (tube No. identifications refer to Fig. 5). We believe that in papain-proteolyzed S-l, LC, is slightly more degraded (therefore runs faster) than in myosin; also in such S-l a fragment of HC coincidentally runs where LC, from myosin runs; this explains the odd labelling when myosin (A, B) is compared with S-l (C, D).
showed, as expected, that the system had lost LC,-but far from totally (Fig. 4, compare A and B). From such myosin we now prepared S- 1, using Mg-papain for the cleavage in order to minimize damage to the remaining LC, (Fig. 4C). The mixture was resolved on DE 52 cellulose (Fig. 5). Elution aliquots (indicated by tube number from Fig. 5) were electrophoresed in the presence of SDS (Fig. 4D). Although the resolution is not as good as with chymotryptically prepared S-l, it is obvious that the isozymic species are predominantly HC . LC, . LC, and HC . LC,.
CONCLUSIONS
AND DISCUSSION
The results show that PAGE reveals the isozymes of myosin S-l about as well as chromatography and also lends itself to studying associations among components. As regards our particular illustrative system, the results show that LC, binds strongly to HC.LC, but weakly to HC. LC2. Conversely, they show that LC, is hard to remove from HC- LC, . LCB but easy to remove from HC . LC, . LC,. Since Frank and Weeds (IO) showed that LC, and LC, are very similar except for an additional 41residue tail in the former, it is reasonable to think that
138
HOZUMI
ET AL.
0.3.
0.2 0
00 ON 0 0.1 -
0’
I
I 40
I Tube
I 80 No.
I
I 120
FIG. 5. DE 52-cellulose chromatography of S-l prepared from DTNB-treated myosin by digestion with insoluble papain in the presence of Mg *+. S-l, 180 mg, was applied. Other conditions were as in Fig. 2.
the strong interaction of LC, with HC.LC, involves this tail in addition to HC. Another reasonable surmise is that the presence of LC, in the S-l complex renders more difficult the resolution into isozymic components, and it is possible that residual proteolytic fragments of LC, do likewise. This is very likely the explanation of why chromatographic separation of Mg-papainprepared S-l is harder than that of chymotrypsin-prepared S-l; and, similarly, variability in the extent of LC, degradation may cause variability in the success of resolving chymotrypsin-prepared S- 1. ACKNOWLEDGMENTS This research was supported by Grants HL-06285, HL-16683, PCM-75-22698, and AHA CI-8. Tetsu Hozumi is a Fellow of the American Heart Association and Manuel F. Morales is a Career Investigator.
REFERENCES 1. Lowey, S., and Holt, J. C. (1973) Cold Spring Harbor Symp. Quant. Biol. 37, 19-28. 2. Margossian, S. S., Lowey, S., and Barshop, B. (1975) Nature (London) 258, 163-166. 3. Holt, J. C., and Lowey, S. (1977) Biochemistry 16,4398-4402.
4. Weeds, A. G., and Taylor, R. S. (1975) Nature (London) 257, 54-56. 5. Gazith, J. S., Himmelfarb, S., and Harrington, W. F. (1970) J. Biol. Chem. 245, 19-22. 6. Tonomura, Y., Appel, P., and Morales, M. F. (1966) Biochemistry 5, 515-521. 7. Lowey, S., Slayter, H. S., Weeds, A. G., and Baker, H. (1969) J. Mol. Biol. 42, l-29. 8. Weber, K., and Osbom, M. (1969) J. Biot. Chem. 244,4406-4412. 9. Paterson, B., and Strohman, R. C. (1970) Biochemistry 9,4094-4105. 10. Frank, G., and Weeda, A. G. (1974) Eur. J. Biothem. 317-334.
