Biochimica et Biophysica Acta, 1035 (1990) 109-112
109
Elsevier
BBA Report
BBAGEN20266
Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle William A. L a F r a m b o i s e 1,2 M o n i c a J. D a o o d 2, R o b e r t D. G u t h r i e 2 Paolo M o r e t t i 3 Stefano Schiaffino 3 a n d M a r c i a Ontell 1 I Department of Neurobiology, Anatomy and Cell Science and the 2 Department of Pediatrics, University of Pittsburgh School of Medicine Pittsburgh, PA (U.S.A.) and 3 Institut of General Pathology and CNR Unit for Muscle Biology and Physiopathology, University of Padova, Padooa (Italy)
(Received9 March1990)
Key words: Myosinheavychain; Skeletalmuscle; Electrophoresis;(Rat)
One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis [1,2]. Eiectrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to he: 2A < 2X < 2B < t / s l o w . Identification of sarcomeric myosin heavy chain isoforms has become increasingly important with the recognition that the maximal velocity of shortening of muscle fibers [3,4] is correlated with their myosin heavy chain composition. Electrophoretic separation of fast and slow MHC isoforms was first obtained in avian muscle by Rushbrook and Stracher [5] and in mammalian muscle by Carraro and Catani [6] using 5 or 7.5% SDS-PAGE. Danieli-Betto et al. [7] increased the gel glycerol content to achieve further separation of fast heavy chains in rat skeletal muscle into 2A and 2B isoforms with 6% SDS-PAGE. Immunocytochemical evidence for the existence of an additional fast myosin heavy chain isoform, 2X, was obtained by Schiaffino et al. [1,2]; however, this isoform could not be electrophoretically separated from the 2A band with 6% SDSPAGE [8]. Using a 5-8% linear gradient of acrylamide, Bar and Pette [9] reported electrophoretic separation of three fast bands from rat skeletal muscles. The slowest migrating band was designated as type IId. This interpretation was subsequently revised after analysis of histochemically characterized individual muscle fibers, and it was suggested that 2A was the slowest migrating
Abbreviation: MHC,myosinheavychain. Correspondence: M. Ontell, Department of Neurobiologyand Cell Science, Universityof Pittsburgh, Schoolof Medicine,Pittsburgh, PA 15261, U.S.A.
band while IId migrated between the 2A and 2B bands [10]. However, unambiguous determination of the relative mobility of these bands using monoclonal antibodies specific to these myosin heavy chains with immunoblotting remained to be established. A simple method for electrophoretic separation of the 2X myosin heavy chain isoform, using a single percentage separating gel is reported here. Adjustment of the Bis-acrylamide ratio and modification of the voltage-time regimen for electrophoresis has allowed consistent separation of four myosin heavy chains which were identified, in order of decreasing mobility, as fl/slow, 2B, 2X and 2A by immunoblotting analysis with specific myosin heavy chain antibodies. E x t r a c t i o n procedure. Costal diaphragm, tibialis anterior and soleus muscles were removed from male rats at 60 and 115-148 days of age. Muscles were extracted on ice for 40 min in 4 vol. of buffer (pH 6.5) as previously described [11]. The extracted myosin was diluted in 9 vol. of 1 mM EDTA and 0.1%2-mercaptoethanol (v/v), and stored overnight at 4 ° C to allow precipitation of myosin filaments. The filament solution was subsequently centrifuged (13000 × g for 30 min in Model 235C, Fisher Scientific microfuge) to form a pellet which was then dissolved in myosin sample buffer (0.5 M NaCI, 10 mM NaH2PO4) followed by dilution 1 : 100 in SDS sample buffer (62.5 mM Tris-HC1, 2% (w/v) SDS, 10% glycerol, 5% (v/v) 2-mercaptoethanol and 0.001% (w/v) Bromophenol blue) at pH 6.8 [121. The samples were boiled for 2 min and stored at - 8 0 o C.
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
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Gel preparation. Gels were prepared either from a stock solution of 30% acrylamide: 29.2 % ( w / v ) acrylamide and 0.8% ( w / v ) Bis (N,N'-bismethylene acrylamide) according to Laemmli [12] or from a 30% stock solution containing 28.5% ( w / v ) acrylamide and 1.5% (w/v) Bis to achieve minimum pore diameter [13]. Electrophoresis was carried out on slabs (18 cm × 16 cm × 0.75 nun thick) consisting of an 11.5 cm separating gel and a 4.5 cm stacking gel. Separating gels of T = 5 and 6% (T--- total concentration of acrylamide + Bis) were tested with stacking gels of 3 and 4%, respectively, at C = 2.7 and 5% ( C = percentage of total monomer due to Bis). Final concentrations of Tris-HC1 in both stacking and separating gels were as previously described [12] and glycerol (25-33%, v / v ) was routinely added to the separating gel [6,7,14]. Polymerization was activated by addition of 0.03% ( w / v ) ammonium persulfate and 0.10% ( v / v ) (when T = 5%) or 0.17% ( v / v ) (when T = 6%) tetramethylethylenediamine (TEMED). Studies were performed with separating gels that had been allowed to polymerize at least 1 h at room temperature and either used that day or stored 1-5 days at 4°C. Electrophoretic protocol. Electrophoresis was performed with Tris-glycine running buffer (pH 8.3) [12] with the upper chamber buffer filtered through a nitrocellulose membrane (0.45 /~m). Volumes of myosin extract (1-3/~1) containing 500-1500 ng of protein were loaded on the gels. After the plates were placed in the electrophoresis apparatus (Hoefer Scientific Instruments, SE 600 vertical slab gel unit) the power supply (E-C Apparatus, EC400) and the cooling unit (Neslab, Endocal RBC-3) were turned on. The temperature of the buffer was maintained at 15 ° C. Three voltage-time regimens were tested: (1) 100 V during stacking of the proteins and then 200 V until the Bromophenol blue dye front reached the bottom of the gel, i.e., approx. 7 h
[15]; (2) 50 V for 22-24 [7]; or (3) 120 V for 22-24 [9]. Separating gels were silver stained [16]. Immunoblot technique. Immunoblot analysis was performed after electrophoretic transfer of protein from unstained gels to nitrocellulose sheets [17] with two different monoclonal antibodies to myosin heavy chains [8]. The blots were incubated first with monoclonal antibody BF-32 which reacts with the fl/slow and 2A isoforms [8]. Binding of the primary antibody was visualized through the use of a peroxidase-conjugated secondary antibody (goat anti-mouse Ig, Dakopatts) followed by development with diaminobenzidine in the presence of imidazole [18]. The blot was then reacted with antibody RT-D9, specific for myosin heavy chains 2B and 2X [8], which was subsequently visualized using the same procedure. lsoforrn separation. Electrophoresis of myosin preparations in 5 or 6% separating gels with lower Bis content ( C = 2.7%) typically yielded results similar to those previously reported [7,8,14]. Three myosin heavy chain bands were resolved: (1) a high mobility band, corresponding to fl/slow, which is the major component in the soleus muscles [7,8]; (2) an intermediate-mobility band, corresponding to 2B, which is the major component in the tibialis anterior muscle [8]; and (3) a lowmobility band corresponding to 2A and 2X that comigrate under these conditions [8] (Fig. 1A). The band with lowest mobility was especially prominent in all diaphragm and tibialis anterior muscles. The band containing the 2B myosin heavy chain was barely visible or completely absent in diaphragm muscles from male rats 115-148 days old. However, the 2B band was found to be present in significant amounts in diaphragm muscles from male rats at 60 days of age. Decreasing the polymerization time for 6% gels run overnight at 120 V occasionally resolved the slow migrating band into a doublet. However, this result was
Fig. 1. Electrophoreticseparation of myosin heavy chain isoforms by SDS-PAGE. Myosin preparations from diaphragm (DIA) of 60-day-old rat (a) and DIA (b), tibialis anterior (TA) and soleus (SOL) muscles from 148-day-oldrat were loaded onto the gel. Gels were silver stained [16]. (A) shows the separation of bands achieved with the majority of attempted protocols. (B) displays the separation routinely achieved with a 5% acrylamide separation gel and a 3% stacking gel (C = 5% for both gels). The separating gels were allowed to polymerizefor 2 h prior to pouring the stacking gel and eleetrophoresiswas carried out for 22 h at 120 V. With the modified protocol three fast bands (2A, 2B and 2X) can be separated.
111 reproducible only about 20% of the time. Variations in polymerization time, buffer temperature, duration of electrophoresis and acrylamide composition of the gels were systematically tested in an attempt to achieve consistent resolution of the four myosin heavy chain isoforms. The concentration of Bis appeared to be a critical factor. Predictable separation of four bands was obtained on separating gels polymerized for 2 h at room temperature with C = 5% and T = 5% prior to pouring the stacking gel (Fig. 1B). A 3% stacking gel (C = 5%) was used, and electrophoresis was carried out for 22 h at 120 V with the buffer cooled to 15-20°C. This combination of parameters resulted in the fl/slow isoform migrating approx. 6.5-7.0 cm. into the separating gel with consistent separation of the three fast isoforms. Confirmation of isoform type. Immunoblotting analysis demonstrated that monoclonal antibody BF-32, which reacts with fl/slow and 2A myosin heavy chains [8], stained both the slowest and the fastest migrating bands (Fig. 2B). It had been established previously that the rat soleus muscle contained two myosin heavy chain isoforms, fl/slow myosin in abundance and 2A as a minor component [7,8], and that the fl/slow myosin has the greatest mobility in SDS-PAGE of the myosin isoforms found in rat muscle [7]. On this basis, it is possible to distinguish the band containing 2A myosin heavy chain from that containing fl/slow myosin heavy chain. Monoclonal antibody RT-D9, which reacts with 2B and 2X myosin heavy chains [8], was subsequently applied to the same blot. It stained two bands which
were not stained with BF-32, a band which migrated immediately ahead of the 2A band, as well as a band which migrated midway between the fl/slow and 2A bands (Fig. 2C). This latter band was present in the tibialis anterior muscle, which had been shown by immunocytochemical analysis to contain abundant 2B myosin heavy chain [8], but was absent in the diaphragm muscles of l15-148-day-old rats (in agreement with reports indicating that 2B is absent or only minimally present at this stage) [8]. On this basis, it was possible to identify this band as containing myosin heavy chain 2B. While the 2B band is absent in diaphragm muscle of rats aged older than 115 days, it is transiently expressed in the diaphragm muscles of young adult rats (Fig. 1). The remaining band which stained with RT-D9 was identified as myosin heavy chain 2X. These results demonstrate that 2A is the slowest migrating band, while 2X migrates slightly faster in this electrophoretic system. Significance. The present study details a reliable protocol that allows resolution of the four myosin heavy chains found in adult rat skeletal muscle. This increased resolution has been achieved by modifying the acrylamide gel composition, the polymerization time and the electrophoresis conditions previously described [6,7,8]. Precise identification of the myosin heavy chain isoforms was obtained by electrophoresis of muscles whose myosin isoform content had been previously determined with immunocytochemistry and by a double immunoblotting procedure, in which the same blot was reacted
Fig. 2. Electrophoretic separation and immunoblot of myosin heavy chain isoforms of the diaphragm (DIA) and tibialis anterior (TA) muscles of a 148-day-old rat. Eiectrophoresis was performed as in Fig. lB. (A) silver stained [16]. (B) Nitrocellulose electrotransfer of DIA and TA obtained from the same gel as in A, but from adjacent lanes. The transfer was stained with monoclonal antibody BF-32, which reacts with the fl/slow and 2A myosin heavy chains [8]. (C) The same nitrocellulose sheet as in B after exposure to a second monoclonal antibody, RT-D9, which reacts with the 2X and 2B isoforms [8]. Immunoperoxidase staining. Muscle of the 148-day-old rat, which has been shown by immunocytochemistry to lack the 2B isoform [8], fails to exhibit a band midway between the 2A and fl/slow band, while this band (2B) is clearly present in the TA which has been shown by immunocytochemistry to have a substantial population of myofibers with the 2B isoform [2,8,9]. The 2X isoform migrates immediately ahead of the 2A isoform.
112
first with an antibody specific for the B/slow and 2A isoforms followed by another antibody specific for the 2X and 2B isoforms. The results clearly demonstrate that 2X myosin heavy chain migrates faster than the isoform 2A, but slower than myosin heavy chain 2B. A crucial factor for the reproducible resolution of the four myosin heavy chain isoforms appears to be the relative concentration of the Bis component with respect to total acrylamide in the gels. Adjustment of the Bis component from 2.7 to 5.0% of total acrylamide provided consistent separation of the band containing myosin heavy chain 2X from that band containing myosin heavy chain 2A at T = 5%. A C = 5% yields a minimum pore size in the range of T = 5-10% used to separate MHCs, and both increasing and decreasing C from this limiting value increases pore size [13]. The results emphasize the important influence of Bis crosslinkage and polymerization kinetics in the electrophoretic migration of proteins. Moreover they provide positive identification of members of the myosin heavy chain family found within typical adult rat skeletal muscles and establish their electrophoretic mobility as 2A < 2X < 2B < B/slow. This paper was supported by a grant from the National Institutes of Health (NIHAR36294). References 1 Schiaffino, S., Saggin, L., Viel, A. and Gorza, L. (1985) J. Muscle Res. Cell Motil. 6, 60-61. 2 Schiaffino, S., Saggin, L., Viel, A., Ausoni, S., Sartore, S. and
3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18
Gorza, L. (1986) in Biochemical Aspects of Physical Exercise (Benzi, G., Packer, L. and Siliprandi, N., eds.), pp. 27-34, Elsevier Science Publishers, Amsterdam. Reiser, P.J., Moss, R.L., Giulian, G.G. and Greaser, M.L. (1985) J. Biol. Chem. 260, 9077-9080. Schiaffino, S., Ausoni, S., Gorza, L., Saggin, L., Gundersen, K. and Lomo, T. (1988) Acta Physiol. Scand. 135, 575-576. Rushbrook, J.I. and Stracher, A. (1979) Proc. Natl. Acad. Sci. USA 76, 4331-4334. Carraro, U. and Catani, C. (1983) Biochem. Biophys. Res. Commun. 116, 793-802. Danieli-Betto, D., Zerbato, E. and Betto, R. (1986) Biochem. Biophys. Res. Commun. 138, 981-987. Schiaffino, S., Gorza, L., Sartore, S., Saggin, L., Ausoni, S., Vianello, M., Gundersen, K. and Lomo, T. (1989) J. Muscle Res. Cell Motil. 10, 197-205. Bar, A. and Pette, D. (1988) FEBS Lett. 235, 153-155. Terrain, A., Staron, R.S. and Pette, D. (1989) Histochemistry 92, 453-457. Butler-Browne, G.S. and Whalen, R.G. (1984) Dev. Biol. 102, 324-334. Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. Cooper, T.G. (1977) The Tools of Biochemistry, pp. 195-200, John Wiley&Sons, New York. Biral, D., Betto, R., Danieli-Betto, D. and Salviati, G. (1988) Biochem. J. 250, 307-308. Parry, D.J. and Knight, S. (1988) in Sarcomeric and Nonsarcomeric Muscles: Basic and Applied Research Prospects for the 90's (Carraro, U., ed.), pp. 193-198, Unipress, Padova. Oakley, B.R., Kirsch, D.R. and Morris, N.R. (1980) Anal. Biochem. 105, 265-275. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. So. USA 76, 4350-4354. Trojanowski, J.Q., Obrocka, M.A. and Lee, V.M.Y. (1983) J. Histochem. Cytochem. 31, 1217-1223.
109
Elsevier
BBA Report
BBAGEN20266
Electrophoretic separation and immunological identification of type 2X myosin heavy chain in rat skeletal muscle William A. L a F r a m b o i s e 1,2 M o n i c a J. D a o o d 2, R o b e r t D. G u t h r i e 2 Paolo M o r e t t i 3 Stefano Schiaffino 3 a n d M a r c i a Ontell 1 I Department of Neurobiology, Anatomy and Cell Science and the 2 Department of Pediatrics, University of Pittsburgh School of Medicine Pittsburgh, PA (U.S.A.) and 3 Institut of General Pathology and CNR Unit for Muscle Biology and Physiopathology, University of Padova, Padooa (Italy)
(Received9 March1990)
Key words: Myosinheavychain; Skeletalmuscle; Electrophoresis;(Rat)
One slow and three fast myosin heavy chains have been described in typical skeletal muscles of the adult rat using immunocytochemical analysis [1,2]. Eiectrophoretic isolation and immunochemical identification of these four isoforms has not been achieved. An electrophoretic procedure is described which, by altering the cross-linkage and polymerization kinetics of 5% polyacrylamide gels, allows resolution of these four distinct myosin heavy chains. Using specific monoclonal antibodies and double immunoblotting analysis, the identity and electrophoretic migration order of the myosin heavy chains was established to he: 2A < 2X < 2B < t / s l o w . Identification of sarcomeric myosin heavy chain isoforms has become increasingly important with the recognition that the maximal velocity of shortening of muscle fibers [3,4] is correlated with their myosin heavy chain composition. Electrophoretic separation of fast and slow MHC isoforms was first obtained in avian muscle by Rushbrook and Stracher [5] and in mammalian muscle by Carraro and Catani [6] using 5 or 7.5% SDS-PAGE. Danieli-Betto et al. [7] increased the gel glycerol content to achieve further separation of fast heavy chains in rat skeletal muscle into 2A and 2B isoforms with 6% SDS-PAGE. Immunocytochemical evidence for the existence of an additional fast myosin heavy chain isoform, 2X, was obtained by Schiaffino et al. [1,2]; however, this isoform could not be electrophoretically separated from the 2A band with 6% SDSPAGE [8]. Using a 5-8% linear gradient of acrylamide, Bar and Pette [9] reported electrophoretic separation of three fast bands from rat skeletal muscles. The slowest migrating band was designated as type IId. This interpretation was subsequently revised after analysis of histochemically characterized individual muscle fibers, and it was suggested that 2A was the slowest migrating
Abbreviation: MHC,myosinheavychain. Correspondence: M. Ontell, Department of Neurobiologyand Cell Science, Universityof Pittsburgh, Schoolof Medicine,Pittsburgh, PA 15261, U.S.A.
band while IId migrated between the 2A and 2B bands [10]. However, unambiguous determination of the relative mobility of these bands using monoclonal antibodies specific to these myosin heavy chains with immunoblotting remained to be established. A simple method for electrophoretic separation of the 2X myosin heavy chain isoform, using a single percentage separating gel is reported here. Adjustment of the Bis-acrylamide ratio and modification of the voltage-time regimen for electrophoresis has allowed consistent separation of four myosin heavy chains which were identified, in order of decreasing mobility, as fl/slow, 2B, 2X and 2A by immunoblotting analysis with specific myosin heavy chain antibodies. E x t r a c t i o n procedure. Costal diaphragm, tibialis anterior and soleus muscles were removed from male rats at 60 and 115-148 days of age. Muscles were extracted on ice for 40 min in 4 vol. of buffer (pH 6.5) as previously described [11]. The extracted myosin was diluted in 9 vol. of 1 mM EDTA and 0.1%2-mercaptoethanol (v/v), and stored overnight at 4 ° C to allow precipitation of myosin filaments. The filament solution was subsequently centrifuged (13000 × g for 30 min in Model 235C, Fisher Scientific microfuge) to form a pellet which was then dissolved in myosin sample buffer (0.5 M NaCI, 10 mM NaH2PO4) followed by dilution 1 : 100 in SDS sample buffer (62.5 mM Tris-HC1, 2% (w/v) SDS, 10% glycerol, 5% (v/v) 2-mercaptoethanol and 0.001% (w/v) Bromophenol blue) at pH 6.8 [121. The samples were boiled for 2 min and stored at - 8 0 o C.
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
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Gel preparation. Gels were prepared either from a stock solution of 30% acrylamide: 29.2 % ( w / v ) acrylamide and 0.8% ( w / v ) Bis (N,N'-bismethylene acrylamide) according to Laemmli [12] or from a 30% stock solution containing 28.5% ( w / v ) acrylamide and 1.5% (w/v) Bis to achieve minimum pore diameter [13]. Electrophoresis was carried out on slabs (18 cm × 16 cm × 0.75 nun thick) consisting of an 11.5 cm separating gel and a 4.5 cm stacking gel. Separating gels of T = 5 and 6% (T--- total concentration of acrylamide + Bis) were tested with stacking gels of 3 and 4%, respectively, at C = 2.7 and 5% ( C = percentage of total monomer due to Bis). Final concentrations of Tris-HC1 in both stacking and separating gels were as previously described [12] and glycerol (25-33%, v / v ) was routinely added to the separating gel [6,7,14]. Polymerization was activated by addition of 0.03% ( w / v ) ammonium persulfate and 0.10% ( v / v ) (when T = 5%) or 0.17% ( v / v ) (when T = 6%) tetramethylethylenediamine (TEMED). Studies were performed with separating gels that had been allowed to polymerize at least 1 h at room temperature and either used that day or stored 1-5 days at 4°C. Electrophoretic protocol. Electrophoresis was performed with Tris-glycine running buffer (pH 8.3) [12] with the upper chamber buffer filtered through a nitrocellulose membrane (0.45 /~m). Volumes of myosin extract (1-3/~1) containing 500-1500 ng of protein were loaded on the gels. After the plates were placed in the electrophoresis apparatus (Hoefer Scientific Instruments, SE 600 vertical slab gel unit) the power supply (E-C Apparatus, EC400) and the cooling unit (Neslab, Endocal RBC-3) were turned on. The temperature of the buffer was maintained at 15 ° C. Three voltage-time regimens were tested: (1) 100 V during stacking of the proteins and then 200 V until the Bromophenol blue dye front reached the bottom of the gel, i.e., approx. 7 h
[15]; (2) 50 V for 22-24 [7]; or (3) 120 V for 22-24 [9]. Separating gels were silver stained [16]. Immunoblot technique. Immunoblot analysis was performed after electrophoretic transfer of protein from unstained gels to nitrocellulose sheets [17] with two different monoclonal antibodies to myosin heavy chains [8]. The blots were incubated first with monoclonal antibody BF-32 which reacts with the fl/slow and 2A isoforms [8]. Binding of the primary antibody was visualized through the use of a peroxidase-conjugated secondary antibody (goat anti-mouse Ig, Dakopatts) followed by development with diaminobenzidine in the presence of imidazole [18]. The blot was then reacted with antibody RT-D9, specific for myosin heavy chains 2B and 2X [8], which was subsequently visualized using the same procedure. lsoforrn separation. Electrophoresis of myosin preparations in 5 or 6% separating gels with lower Bis content ( C = 2.7%) typically yielded results similar to those previously reported [7,8,14]. Three myosin heavy chain bands were resolved: (1) a high mobility band, corresponding to fl/slow, which is the major component in the soleus muscles [7,8]; (2) an intermediate-mobility band, corresponding to 2B, which is the major component in the tibialis anterior muscle [8]; and (3) a lowmobility band corresponding to 2A and 2X that comigrate under these conditions [8] (Fig. 1A). The band with lowest mobility was especially prominent in all diaphragm and tibialis anterior muscles. The band containing the 2B myosin heavy chain was barely visible or completely absent in diaphragm muscles from male rats 115-148 days old. However, the 2B band was found to be present in significant amounts in diaphragm muscles from male rats at 60 days of age. Decreasing the polymerization time for 6% gels run overnight at 120 V occasionally resolved the slow migrating band into a doublet. However, this result was
Fig. 1. Electrophoreticseparation of myosin heavy chain isoforms by SDS-PAGE. Myosin preparations from diaphragm (DIA) of 60-day-old rat (a) and DIA (b), tibialis anterior (TA) and soleus (SOL) muscles from 148-day-oldrat were loaded onto the gel. Gels were silver stained [16]. (A) shows the separation of bands achieved with the majority of attempted protocols. (B) displays the separation routinely achieved with a 5% acrylamide separation gel and a 3% stacking gel (C = 5% for both gels). The separating gels were allowed to polymerizefor 2 h prior to pouring the stacking gel and eleetrophoresiswas carried out for 22 h at 120 V. With the modified protocol three fast bands (2A, 2B and 2X) can be separated.
111 reproducible only about 20% of the time. Variations in polymerization time, buffer temperature, duration of electrophoresis and acrylamide composition of the gels were systematically tested in an attempt to achieve consistent resolution of the four myosin heavy chain isoforms. The concentration of Bis appeared to be a critical factor. Predictable separation of four bands was obtained on separating gels polymerized for 2 h at room temperature with C = 5% and T = 5% prior to pouring the stacking gel (Fig. 1B). A 3% stacking gel (C = 5%) was used, and electrophoresis was carried out for 22 h at 120 V with the buffer cooled to 15-20°C. This combination of parameters resulted in the fl/slow isoform migrating approx. 6.5-7.0 cm. into the separating gel with consistent separation of the three fast isoforms. Confirmation of isoform type. Immunoblotting analysis demonstrated that monoclonal antibody BF-32, which reacts with fl/slow and 2A myosin heavy chains [8], stained both the slowest and the fastest migrating bands (Fig. 2B). It had been established previously that the rat soleus muscle contained two myosin heavy chain isoforms, fl/slow myosin in abundance and 2A as a minor component [7,8], and that the fl/slow myosin has the greatest mobility in SDS-PAGE of the myosin isoforms found in rat muscle [7]. On this basis, it is possible to distinguish the band containing 2A myosin heavy chain from that containing fl/slow myosin heavy chain. Monoclonal antibody RT-D9, which reacts with 2B and 2X myosin heavy chains [8], was subsequently applied to the same blot. It stained two bands which
were not stained with BF-32, a band which migrated immediately ahead of the 2A band, as well as a band which migrated midway between the fl/slow and 2A bands (Fig. 2C). This latter band was present in the tibialis anterior muscle, which had been shown by immunocytochemical analysis to contain abundant 2B myosin heavy chain [8], but was absent in the diaphragm muscles of l15-148-day-old rats (in agreement with reports indicating that 2B is absent or only minimally present at this stage) [8]. On this basis, it was possible to identify this band as containing myosin heavy chain 2B. While the 2B band is absent in diaphragm muscle of rats aged older than 115 days, it is transiently expressed in the diaphragm muscles of young adult rats (Fig. 1). The remaining band which stained with RT-D9 was identified as myosin heavy chain 2X. These results demonstrate that 2A is the slowest migrating band, while 2X migrates slightly faster in this electrophoretic system. Significance. The present study details a reliable protocol that allows resolution of the four myosin heavy chains found in adult rat skeletal muscle. This increased resolution has been achieved by modifying the acrylamide gel composition, the polymerization time and the electrophoresis conditions previously described [6,7,8]. Precise identification of the myosin heavy chain isoforms was obtained by electrophoresis of muscles whose myosin isoform content had been previously determined with immunocytochemistry and by a double immunoblotting procedure, in which the same blot was reacted
Fig. 2. Electrophoretic separation and immunoblot of myosin heavy chain isoforms of the diaphragm (DIA) and tibialis anterior (TA) muscles of a 148-day-old rat. Eiectrophoresis was performed as in Fig. lB. (A) silver stained [16]. (B) Nitrocellulose electrotransfer of DIA and TA obtained from the same gel as in A, but from adjacent lanes. The transfer was stained with monoclonal antibody BF-32, which reacts with the fl/slow and 2A myosin heavy chains [8]. (C) The same nitrocellulose sheet as in B after exposure to a second monoclonal antibody, RT-D9, which reacts with the 2X and 2B isoforms [8]. Immunoperoxidase staining. Muscle of the 148-day-old rat, which has been shown by immunocytochemistry to lack the 2B isoform [8], fails to exhibit a band midway between the 2A and fl/slow band, while this band (2B) is clearly present in the TA which has been shown by immunocytochemistry to have a substantial population of myofibers with the 2B isoform [2,8,9]. The 2X isoform migrates immediately ahead of the 2A isoform.
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first with an antibody specific for the B/slow and 2A isoforms followed by another antibody specific for the 2X and 2B isoforms. The results clearly demonstrate that 2X myosin heavy chain migrates faster than the isoform 2A, but slower than myosin heavy chain 2B. A crucial factor for the reproducible resolution of the four myosin heavy chain isoforms appears to be the relative concentration of the Bis component with respect to total acrylamide in the gels. Adjustment of the Bis component from 2.7 to 5.0% of total acrylamide provided consistent separation of the band containing myosin heavy chain 2X from that band containing myosin heavy chain 2A at T = 5%. A C = 5% yields a minimum pore size in the range of T = 5-10% used to separate MHCs, and both increasing and decreasing C from this limiting value increases pore size [13]. The results emphasize the important influence of Bis crosslinkage and polymerization kinetics in the electrophoretic migration of proteins. Moreover they provide positive identification of members of the myosin heavy chain family found within typical adult rat skeletal muscles and establish their electrophoretic mobility as 2A < 2X < 2B < B/slow. This paper was supported by a grant from the National Institutes of Health (NIHAR36294). References 1 Schiaffino, S., Saggin, L., Viel, A. and Gorza, L. (1985) J. Muscle Res. Cell Motil. 6, 60-61. 2 Schiaffino, S., Saggin, L., Viel, A., Ausoni, S., Sartore, S. and
3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18
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