Localization of Protein Regions Involved in the Interaction between Calponin and Myosin*

(Received for publication, October 1, 1996, and in revised form, January 22, 1997)

Pawel T. Szymanski Dagger § and Terence Tao Dagger par

From the Dagger  Muscle Research Group, Boston Biomedical Research Institute, Boston, Massachusetts 02114, the  Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, and the par  Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Calponin is a 33-kDa smooth muscle-specific protein that has been suggested to play a role in muscle contractility. It has previously been shown to interact with actin, tropomyosin, and calmodulin. More recently we showed that calponin also interacts with myosin (Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256-259). In the present study we used a combination of co-sedimentation and fluorescence assays to localize the regions in myosin and calponin that are involved in the interaction between these two proteins. We found that recombinant chicken gizzard alpha -calponin co-sediments with myosin rod and, to a lesser extent, with light meromyosin. Fluorescently labeled recombinant calponin shows interaction with heavy meromyosin and myosin subfragment 2 but not subfragment 1. A fragment comprising residues 7-182 and a synthetic peptide spanning residues 146-176 of calponin co-sediment with myosin, but fragments comprising residues 7-144 and 183-292 do not. Our results indicate that there are calponin binding sites in the subfragment 2 and light meromyosin regions of myosin, and that the region comprising residues 145-182 of calponin mediates its interaction with myosin.


INTRODUCTION

Calponin (CaP)1 is a 33-kDa smooth muscle-specific, thin filament-associated protein that has been suggested to play a role in muscle contractility (2, 3). It is capable of binding to actin (2, 4-6), tropomyosin (7-10), and calmodulin (4, 6, 11). The binding sites for these proteins are all located in the NH2-terminal portion of CaP (residues 7-182) (12).

CaP inhibits the actin-activated myosin ATPase in solution (3, 13), unloaded shortening velocity, and, to a lesser extent, isometric force in permeabilized smooth muscle fibers (14-17). It is commonly assumed that these effects are exerted via CaP binding to actin and competition of CaP with myosin heads for binding to actin. On the other hand, observations that CaP inhibits actin translocation over myosin heads (18, 19) and enhances the affinity between actin and myosin in in vitro motility assays (19) suggest that CaP interacts simultaneously with both actin and myosin. Previous studies showed that CaP indeed binds to isolated smooth muscle myosin (1, 20). This interaction was found to be reversible upon the addition of Ca2+-calmodulin (1) and partially abolished upon phosphorylation of CaP by protein kinase C.2

In this study we further investigated the interaction between CaP and myosin. Using a combination of co-sedimentation and fluorescence assays, we localized the regions in these two proteins that are involved in their interaction. Fragments of chicken gizzard myosin and recombinant chicken gizzard alpha -CaP (Ralpha CaP) were generated by proteolytic digestion. This allowed us to isolate the major functional portions of these two proteins, viz. S1, S2, rod, HMM, and LMM of myosin and the NH2-terminal fragment (residues 7-182), the NH2-terminal fragment without the central portion (residues 7-144), and the COOH-terminal fragment (residues 183-292) of CaP.

Our data show that there are CaP binding sites in the S2 and LMM regions of myosin and that the region in CaP that contains a so-called actin-binding domain (residues 145-176) (4) is primarily involved in the interaction of CaP with myosin.


EXPERIMENTAL PROCEDURES

Materials

alpha -Chymotrypsin, papain, and other commonly used reagents were from Sigma. Precasted polyacrylamide gradient (4-20%) Tris-glycine gels were from Novex. The rapid-Ag-stain kit was from ICN, and other materials for gel electrophoresis were from Bio-Rad.

Proteins

Chicken gizzard myosin was prepared according to Ikebe et al. (21). Fragmentation of myosin into rod and S1 by digestion with papain and into LMM and HMM by digestion with alpha -chymotrypsin was performed as described (22). S2 was generated from HMM by alpha -chymotrypsin cleavage (23).

Expression and purification of Ralpha CaP was as described previously (24). Digestion of Ralpha CaP with alpha -chymotrypsin using a protease to substrate weight ratios of 1:1000 and 1:100 were performed according to Mezgueldi et al. (4). Labeling of Ralpha CaP with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (Aldrich) was carried out by incubating Ralpha CaP (40 µM) with a 3-fold molar excess of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine in 20 mM Hepes, 0.1 M NaCl, pH 7.5, for 3 h at room temperature. Dithiothreitol (1 mM) was added to quench the reaction, followed by dialysis to remove excess reagents.

The polypeptide EKQQRRFQPEKLREGRNIIGLQMGTNKFAC corresponding to residues 146-176 of CaP and a COOH-terminal Cys was synthesized on an ABI protein synthesizer and purified by high pressure liquid chromatography using conventional methods.

Protein concentrations were determined spectrophotometrically at 280 nm, using A (1%, 1 cm) values of 7.4 for Ralpha CaP, 4.5 for smooth muscle myosin, 2.2 for rod, 3.0 for LMM, 6.5 for HMM, 7.0 for S2, and 7.7 for S1; an extinction coefficient of 5600 M-1 cm-1 was used for the synthetic polypeptide.

Binding Assays

For sedimentation binding assays Ralpha CaP or its fragments were incubated with myosin or its insoluble fragments (rod or LMM) in 20 mM Hepes, 50 mM NaCl, 2 mM NaN3, 1 mM dithiothreitol, pH 7.5, for 20 min at 4 °C and then centrifuged at 80,000 rpm for 20 min at 4 °C in a Beckman Instruments TL100 ultracentrifuge using the TL100.2 rotor. Reaction mixtures before centrifugation and pellets solubilized with Laemmli buffer (25) were subjected to gradient (4-20%) SDS-PAGE. The amounts of materials were quantified by densitometry of Coomassie Blue- or silver-stained gels using a laboratory-built image-analysis system as described earlier (1). Standard curve for Ralpha CaP was constructed to establish the linear concentration range.

For binding assays in solution, DAN-Ralpha CaP (1 µM) was incubated with increasing concentrations (0-5 µM) of the soluble myosin fragments (HMM, S1, and S2) in 20 mM Hepes, 50 mM NaCl, 2 mM NaN3, 1 mM dithiothreitol, pH 7.5, for 20 min at 4 °C. Fluorescence-intensity measurements were carried out on an I.S.S. K2 fluorometer (Champaign, IL) using wavelengths of 377 and 480 nm for excitation and emission, respectively.

Statistical Analysis

Student's t test was used for statistical analysis, and a confidence level of p < 0.05 was chosen as an indication of a statistically significant difference.


RESULTS AND DISCUSSION

Fluorescence Titration

Addition of increasing concentrations of HMM to a solution containing DAN-Ralpha CaP produced a concentration-dependent and saturable increase of the label's fluorescence intensity (Fig. 1). In contrast, S1 did not cause any change in the fluorescence of DAN-Ralpha CaP. These data indicate that CaP interacts with HMM but not with S1 and indirectly implicate the S2 region of myosin as a CaP binding site. The fluorescence titration curve of S2 is very similar to that of HMM (Fig. 1). When the data were fitted by a nonlinear regression method (26), we obtained apparent binding constants of 4.0 ± 0.3 × 106 M-1 (n = 3) and 4.7 ± 0.4 × 106 M-1 (n = 3) (all uncertainty values are S.E.; n refers to the number of determinations) for HMM and S2, respectively. Thus, isolated S2 binds DAN-Ralpha CaP with virtually the same affinity as HMM, providing more direct evidence that the S2 region of myosin contains a CaP binding site.


Fig. 1. Fluorometric titration of DAN-Ralpha CaP with the soluble fragments of myosin. Increasing concentrations (0-5 µM) of HMM (bullet ), S2 (black-triangle), and S1 (black-square) were added to DAN-Ralpha CaP (1 µM). Each data point represents the average of three or more independent experiments. The line represents the best fit obtained by nonlinear regression. Experimental details are given under "Experimental Procedures."
[View Larger Version of this Image (14K GIF file)]


Since it is possible for S1 to bind DAN-Ralpha CaP without affecting the label's fluorescence, steady-state polarization and anisotropy decay measurements were also carried out; neither showed any changes with S1 (data not shown). Also, the binding of DAN-Ralpha CaP to myosin was found to be the same as that of unlabeled Ralpha CaP using the co-sedimentation assay (see below; data not shown), indicating that the label does not affect the capacity of Ralpha CaP to interact with myosin.

Co-sedimentation

Addition of increasing concentrations of myosin, myosin rod, and LMM to a solution of Ralpha CaP followed by high speed centrifugation produced a concentration-dependent increase in sedimentation of Ralpha CaP that approaches saturation at high concentrations of added proteins (Fig. 2). As was found previously for myosin (1), some amounts of Ralpha CaP remain in the supernatant even at the highest concentrations of the proteins used, and about 10-20% of Ralpha CaP sedimented in the absence of added proteins. Nonlinear regression analysis of the data in Fig. 2 yielded apparent binding constants of 2.6 ± 0.3 × 106 M-1 (n = 6), 1.0 ± 0.3 × 106 M-1 (n = 7), and 1.5 ± 0.5 × 106 M-1 (n = 5) for LMM, intact myosin, and rod, respectively. These binding constants are not statistically different (p < 0.05) from each other. We noted, however, that the fraction of Ralpha CaP bound at high concentrations of added proteins is significantly lower for LMM compared with intact myosin and rod. The analysis procedure yielded maximal fraction bound values of 0.3, 0.7, and 0.5 for LMM, myosin, and rod, respectively. We stress that our experimental conditions were such that the binding curves have not reached saturation, so that the fraction of Ralpha CaP bound at saturation could only be determined approximately.


Fig. 2. Binding of Ralpha CaP to the insoluble fragments of myosin. Increasing concentrations (0-5 µM) of myosin (bullet ), myosin rod (black-diamond ), and LMM (black-triangle) were added to Ralpha CaP (0.75 µM). The abscissa gives the molar ratio of the amount of Ralpha CaP that sedimented to the total amount of Ralpha CaP. Other information is as in Fig. 1.
[View Larger Version of this Image (15K GIF file)]


The co-sedimentation results described above show that the CaP binding function of myosin resides entirely in the rod segment, a conclusion that is consistent with our finding that S1 does not bind CaP. One of the CaP binding sites in the rod must be in the S2 region, as we concluded from the fluorescence titration results. Furthermore, we found that LMM, which lacks the S2 segment, also binds CaP. Thus, there must be another CaP binding site in the LMM portion of the myosin rod. Our conclusion that there are two CaP binding sites in myosin or myosin rod and only one in LMM is consistent with our finding that the maximal amount of bound Ralpha CaP for LMM is roughly half the amount for myosin or rod.

Binding of Ralpha CaP Fragments to Myosin

As reported previously for gizzard CaP (4), limited digestion of Ralpha CaP with alpha -chymotrypsin at a low protease to substrate weight ratio of 1:1000 for 17 min at 25 °C produced two major fragments (Fig. 3A, lane 4). Their molecular masses are 21.5 and 13.7 kDa based on electrophoretic mobilities. These two fragments remain stable up to about 20 min of digestion, and then further cleavage takes place (Fig. 3A, lane 5). The 21.5-kDa fragment represents the NH2-terminal portion of Ralpha CaP spanning the region between Asn7 and Tyr182, and the 13.7-kDa fragment spans the region between Gly183 and the COOH terminus of intact Ralpha CaP (4).


Fig. 3. A, limited alpha -chymotrypsin digestion of Ralpha CaP as a function of time. Coomassie Blue-stained gradient SDS-PAGE. Lane 1, molecular mass standards: 204, 121, 82, 50.2, 34.2, 28.1, 19.4, and 7.3 kDa, corresponding to myosin, beta -galactosidase, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme, and aprotinin, respectively; lanes 2-5, Ralpha CaP cleavage pattern after 0, 1, 17, and 60 min of digestion, respectively. Arrowheads indicate the positions of two complementary fragments of Ralpha CaP with apparent molecular masses of 21.5 and 13.7 kDa. B, sedimentation of the limited chymotrypsin digest of Ralpha CaP in the absence and presence of myosin. T, incubates before centrifugation; P and S, pellets and supernatants, respectively, after centrifugation. Lane 6, molecular mass standards, as in A; lanes 7-9, Ralpha CaP digest alone; lanes 10-12, Ralpha CaP digest plus myosin. Protein concentrations are 3 µM. Arrowheads indicate the positions of the 21.5- and 13.7-kDa fragments (residues 7-182 and 183-292, respectively) of Ralpha CaP. HC, LC20, and LC17 indicate myosin heavy chain and the 20- and 17-kDa myosin light chains, respectively.
[View Larger Version of this Image (37K GIF file)]


When the limited alpha -chymotryptic Ralpha CaP digest was centrifuged together with an equimolar concentration of myosin, 76.6 ± 3.2% (n = 6) and 12.2 ± 1.0% (n = 6) of the 21.5- and 13.7-kDa fragments, respectively, were sedimented (Fig. 3B, lanes 10-12). When the same Ralpha CaP digest was centrifuged without myosin, 17.2 ± 0.9% (n = 6) and 11.3 ± 1.8% (n = 6) of 21.5- and 13.7-kDa fragments, respectively, were found in the pellets (Fig. 3B, lanes 7-9). Thus, although a significant amount (~55%) of the 21.5-kDa fragment sedimented via binding to myosin, virtually none of the 13.7-kDa fragment did. These data indicate that the myosin binding function of CaP resides within residues 7-182 and not residues 183-292. It is interesting to note that actin (4, 5), tropomyosin (4, 5, 8, 9), calmodulin (5, 6, 11), and caltropin (27) binding sites are located in the same NH2-terminal portion of CaP.

As for native CaP (4), digestion of Ralpha CaP with alpha -chymotrypsin at a high protease to substrate weight ratio (1:100) produced further cleavage (Fig. 4A). After digestion for 30 min at 25 °C, the 13.7-kDa COOH-terminal fragment was completely digested. The 21.5-kDa NH2-terminal fragment (residues 7-182) was cleaved between Tyr144 and Ala145 into a 15.5-kDa fragment that spans residues 7-144 and smaller segments.


Fig. 4. A, extensive chymotrypsin digestion of Ralpha CaP as a function of time. Coomassie Blue-stained gradient SDS-PAGE. Lane 1, molecular mass standards as in Fig. 3A; lanes 2-7, Ralpha CaP cleavage pattern after 0, 1, 5, 10, 15, and 30 min of digestion, respectively. Arrowheads indicate the positions of the 21.5-, 15.5-, and 13.7-kDa fragments. B, sedimentation of the extensive chymotrypsin digest of Ralpha CaP in the absence and presence of myosin. T, P, and S are as in Fig. 3B. Lane 8, molecular mass standards as in Fig. 3A; lanes 9-11, Ralpha CaP digest alone; lanes 12-14, Ralpha CaP digest (3 µM, based on the concentration of Ralpha CaP before digestion) plus myosin (3 µM). Arrowhead indicates the position of the 15.5-kDa fragment (residues 7-144). Symbols are as in Fig. 3B.
[View Larger Version of this Image (32K GIF file)]


When this extensive chymotryptic digest of Ralpha CaP was centrifuged with an equimolar concentration of myosin, 15.4 ± 1.4% (n = 6) of the 15.5-kDa fragment was sedimented compared with the total unsedimented material (Fig. 4B, lanes 12-14). When the same digest was centrifuged without myosin, 13.5 ± 1.0% (n = 6) of the 15.5-kDa Ralpha CaP fragment was sedimented (Fig. 4B, lanes 9-11). These data clearly indicate that the Ralpha CaP fragment that spans residues 7-144 does not bind myosin. Since the 21.5-kDa NH2-terminal fragment (residues 7-182) does bind myosin, our data suggest that the myosin binding site in CaP is located within residues 145-182.

When a synthetic peptide that contains residues 146-176 of CaP was centrifuged with and without myosin, 51.0 ± 2.2% (n = 6) and 14.7 ± 0.9% (n = 5), respectively, of the peptide was sedimented (Fig. 5A). These data show more directly that the central region of CaP spanning residues 145-182 is responsible for binding to myosin.


Fig. 5. A, sedimentation of the synthetic peptide in the absence and presence of myosin. Silver-stained gradient SDS-PAGE. T, P, and S are as in Fig. 3B. Lane 1, molecular mass standards as in Fig. 3A; lanes 2-4, synthetic peptide (residues 146-176 of CaP plus a Cys at the COOH terminus) alone; lanes 5-7, synthetic peptide (3 µM) plus myosin (3 µM). B, sedimentation of synthetic peptide with myosin in the presence of intact Ralpha CaP. Lanes 8 and 9, incubate before sedimentation and supernatant after sedimentation, respectively, of the synthetic peptide (3 µM) plus myosin (3 µM); lanes 10-12, pellets after sedimentation of peptide plus myosin in the presence of 0, 1.5, and 3 µM Ralpha CaP, respectively. SP indicates the position of the synthetic peptide. All other symbols are as in Fig. 3B.
[View Larger Version of this Image (55K GIF file)]


To ascertain whether the binding of our synthetic CaP peptide to myosin is specific, we centrifuged the preformed complex between myosin and the peptide (each at 3 µM) in the presence of increasing concentrations of Ralpha CaP. As shown in Fig. 5B (lanes 8-12), addition of Ralpha CaP resulted in a concentration-dependent displacement of the synthetic peptide from myosin. Specifically, the amount of peptide that sedimented with myosin decreased from 48.3 ± 2.1% (n = 3) in the absence of Ralpha CaP to 29.7 ± 2.0% (n = 3) and 21.8 ± 1.5% (n = 3) in the presence of 1.5 and 3.0 µM Ralpha CaP, respectively. Our data show that the peptide competes with Ralpha CaP for binding to myosin, indicating that the interaction between the peptide and myosin is specific.


CONCLUSIONS

Our results show that there are CaP binding regions in the S2 and LMM portions of myosin and that the CaP segment comprising residues 146-182 constitutes a myosin binding region. We noted that caldesmon (28-31) and telokin (32), two proteins that have been shown to regulate smooth muscle contractility (33) and myosin assembly (34), also bind to myosin in the S2 region. More interestingly, the same stretch of residues (145-182) in CaP has previously been shown to be a region of interaction with actin (4, 5). Thus, myosin and actin share (or partially share) a common binding region in CaP, so it is not likely that CaP functions as a linker between myosin and actin.

The physiological significance of the interaction between CaP and myosin is not clear at this point. It was recently reported that CaP is primarily localized in the cytoskeletal regions of chicken gizzard cells (35). However, it was recognized that some of the CaP are found near myosin filaments, suggesting that CaP may serve as a link between cytoskeletal extensions and contractile regions (35). We may speculate that a physiological function of CaP might be to link a component of the cytoskeleton (e.g. desmin intermediate filaments) with myosin in the contractile region. Under such a circumstance the local concentrations of both CaP and myosin are likely to be quite high, so that the interaction might be able to occur in situ even though it is relatively weak at physiological NaCl concentrations in vitro (1). Clearly, much additional work will be required before the true physiological role of CaP can be understood.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant PO1-AR41637 (to T. T.) and American Heart Association Grant 13-523-934 (to P. T. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Harvard University, West Roxbury VA Medical Center, 1400 VFW Pkwy., Research 151, Bldg. 3, Rm. 2B102, West Roxbury, MA 02132. Tel: 617-323-7700 ext. 6195; Fax: 617-363-5592; E-mail: tao{at}bbri.harvard.edu.
1   The abbreviations used are: CaP, calponin; Ralpha CaP, recombinant alpha -isoform of chicken gizzard calponin; HMM, heavy meromyosin; LMM, light meromyosin; S1, myosin subfragment 1; S2, myosin subfragment 2; DAN-Ralpha CaP, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled Ralpha CaP; PAGE, polyacrylamide gel electrophoresis; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
2   P. T. Szymanski and T. Tao, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. John Gergely for critically reviewing the manuscript and Bing Li for the preparation of Ralpha CaP and synthetic peptide.


REFERENCES

  1. Szymanski, P. T., and Tao, T. (1993) FEBS Lett. 331, 256-259 [CrossRef]
  2. Takahashi, K., Hiwada, K., and Kokobu, T. (1986) Biochem. Biophys. Res. Commun. 141, 20-26 [Medline] [Order article via Infotrieve]
  3. Winder, S. J., and Walsh, M. P. (1990) J. Biol. Chem. 265, 10148-10155 [Abstract/Free Full Text]
  4. Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951 [Abstract/Free Full Text]
  5. Mezgueldi, M., Mendre, C., Calas, B., Kassab, R., and Fattoum, A. (1995) J. Biol. Chem. 270, 8867-8876 [Abstract/Free Full Text]
  6. Winder, S. J., and Walsh, M. P. (1990) Biochem. Int. 22, 335-341 [CrossRef][Medline] [Order article via Infotrieve]
  7. Takahashi, K., Abe, M., Hiwada, K., and Kokobu, T. (1988) J. Hypertens. 6, S40-S43
  8. Vancompernolle, K., Gimona, M., Herzog, M., Van Damme, J., Vandekerckhove, J., and Small, V. (1990) FEBS Lett. 274, 146-150 [CrossRef][Medline] [Order article via Infotrieve]
  9. Childs, T. J., Watson, M. W., Novy, R. E., Lin, J. J., and Mak, A. S. (1992) Biochim. Biophys. Acta 1121, 41-46 [Medline] [Order article via Infotrieve]
  10. Nakamura, F., Mino, T., Yamamoto, J., Naka, M., and Tanaka, T. (1993) J. Biol. Chem. 268, 6194-6201 [Abstract/Free Full Text]
  11. Takahashi, K., Hiwada, K., and Kokobu, T. (1988) J. Hypertens. 11, 620-626
  12. Gimona, M., and Small, V. J. (1996) in Biochemistry of Smooth Muscle Contraction (Barany, M., ed), pp. 91-101, Academic Press, San Diego, CA
  13. Abe, M., Takahashi, K., and Hiwada, K. (1990) J. Biochem. 108, 835-838 [Abstract]
  14. Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M., and Tanaka, T. (1995) J. Biol. Chem. 270, 20400-20403 [Abstract/Free Full Text]
  15. Jaworowski, A., Anderson, K. J., Arner, A., Engstrom, M., Gimona, M., Strasser, P., and Small, V. J. (1995) FEBS Lett. 365, 161-171
  16. Obara, K., Szymanski, P. T., Tao, T., and Paul, R. J. (1996) Am. J. Physiol. 270, C481-C487 [Abstract/Free Full Text]
  17. Horowitz, A., Clement-Chomienne, O., Walsh, M. P., Tao, T., Katsuyama, H., and Morgan, K. G. (1996) Am. J. Physiol. 270, H1858-H1863 [Abstract/Free Full Text]
  18. Shirinsky, V. P., Biryukov, K. G., Hettasch, J. H., and Sellers, J. R. (1992) J. Biol. Chem. 267, 15886-15892 [Abstract/Free Full Text]
  19. Haeberle, J. R. (1994) J. Biol. Chem. 269, 12424-12431 [Abstract/Free Full Text]
  20. Lin, Y., Ye, L. H., Ishikawa, R., Fujita, K., and Kohama, K. (1993) J. Biochem. 113, 643-645 [Abstract]
  21. Ikebe, M., Aiba, T., Onishi, H., and Watanabe, S. (1978) J. Biochem. 83, 1643-1655 [Abstract]
  22. Seidel, J. C. (1980) J. Biol. Chem. 255, 4355-4361 [Free Full Text]
  23. Margossian, S. S., and Lowey, S. (1982) Methods Enzymol. 85, 55-71 [Medline] [Order article via Infotrieve]
  24. Gong, B. J., Mabuchi, K., Takahashi, K., Nadal-Ginard, B., and Tao, T. (1993) J. Biochem. 114, 453-456 [Abstract]
  25. Laemmli, U. K. (1990) Nature 227, 680-685
  26. Morris, E. P., and Lehrer, S. S. (1984) Biochemistry 23, 2214-2220 [Medline] [Order article via Infotrieve]
  27. Wills, F. L., McCubbin, W. D., Gimona, M., Strasser, P., and Kay, C. M. (1994) J. Protein Sci. 3, 2311-2321
  28. Ikebe, M., and Reardon, S. (1988) J. Biol. Chem. 263, 3055-3058 [Abstract/Free Full Text]
  29. Marston, S. B., Pinter, K., and Bennett, P. M. (1992) J. Muscle Res. Cell Motil. 31, 206-218
  30. Hemric, M. E., and Chalovich, J. M. (1990) J. Biol. Chem. 265, 19672-19678 [Abstract/Free Full Text]
  31. Hemric, M. E., Lu, F. W. M., Shrager, R., Carey, J., and Chalovich, J. M. (1993) J. Biol. Chem. 268, 15305-15311 [Abstract/Free Full Text]
  32. Shirinsky, V. P., Vorotnikov, A. V., Birukov, K. G., Nanaev, A. K., Collinge, M., Lukas, T. J., Sellers, J. R., and Watterson, D. M. (1993) J. Biol. Chem. 268, 16578-16583 [Abstract/Free Full Text]
  33. Katsuyama, H., Wang, C.-L. A., and Morgan, K. G. (1992) J. Biol. Chem. 267, 14555-14558 [Abstract/Free Full Text]
  34. Katayama, E., Scott-Woo, G., and Ikebe, M. (1995) J. Biol. Chem. 270, 3919-3925 [Abstract/Free Full Text]
  35. Mabuchi, K., Li, Y. L., Tao, T., and Wang, A. C.-L. (1996) J. Muscle Res. Cell Motil. 17, 243-260 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.