ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 281, No. 1, August 15, pp. 66-69,199O
Caldesmon from Rabbit Liver: Molecular Weight and Length by Analytical Ultracentrifugation’ Walter F. Stafford,**? Agnes Jancso,* and Philip Graceffa*$’ *Department of Muscle Research, Boston Biomedical Research Institute, 20 Staniford Street, Boston, Massachusetts 02114, and tDepartment of Neurology, Harvard Medical School, Boston, Massachusetts 02115
Received January
31,1990, and in revised form March 28,199O
Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 + 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 + 0.02 S, yields an apparent length of 53 + 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-
6098.
tion of smooth muscle contraction and nonmuscle motility. Caldesmon was first discovered in smooth muscle (1) and later found in a variety of nonmuscle tissues (2-lo), including mammalian and avian liver ( 11,12). Although muscle caldesmon migrates during SDS3-gel electrophoresis as a 140- to 150-kDa protein, its molecular mass is close to 93 kDa, as determined by sedimentation equilibrium (13), or 87 kDa (14) or 89 kDa (15) from the cDNAdeduced amino acid sequence. Nonmuscle caldesmon migrates on SDS gels as a 70- to 83-kDa protein, depending on its source. Yamashiro-Matsumura and Matsumura (9) reported that nonmuscle caldesmon is a monomer with a molecular mass of 87 kDa, calculated from its hydrodynamic properties, which suggests that the molecular weights of muscle and nonmuscle caldesmons are about the same. In contrast, Ball and Kovala (12) suggested that in nonmuscle caldesmon a central fragment (M, 44,000 from SDS-PAGE) of the muscle caldesmon is absent, which would indicate that the molecular weight of the nonmuscle protein is considerably less than that from muscle. Since Yamashiro-Matsumura and Matsumura (9) used methods which depend on the shape of the protein, we decided to try to resolve this uncertainty by determining the molecular weight of nonmuscle caldesmon by sedimentation equilibrium in the analytical ultracentrifuge, a technique which requires no assumption about the shape of the molecule. We found that rabbit liver caldesmon is a monomer with a molecular mass of 66 kDa, a value which is much smaller than that of the muscle caldesmon.
o 1990 Academic Preae, Inc.
MATERIALS
Caldesmon is an actin-binding protein which is thought to play a role in the thin filament Ca2+-regular This work was supported by National Institutes of Health Grant AR-30917 (P.G.). *To whom correspondence and reprint requests should be addressed. 66
AND
METHODS
Caldesmon, from fresh rabbit liver, was purified from the 30-50% (NH&SO, fraction of the heat stable extract (16) by chromatography on phosphocellulose P-11 (Whatman), phenyl-Sepharose CL-4B
‘Abbreviations used: SDS, sodium dodecyl sulfate; PAGE, polya&amide gel electrophoresis; Mops, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol. 0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
RABBIT TABLE
LIVER
CALDESMON
I
Amino Acid Composition of Gizzard Muscle and Liver Nonmuscle Caldesmon Gizzard”
(mol% of amino acid)
Amino acid
7.7 27.1 5.1 4.5 0.19 9.1 4.2 11.6 3.6 0.34 4.4 0.77 0.35 1.3 4.7 1.1 13.9 -
Asx Glx Ser GUY His -4% Thr Ala Pro TYr Val Met CYS Ile Leu Phe LYS Trp a Graceffa
Liver
9.0 25.4 8.3 5.7 0.7 7.4 4.9 8.7 1.6 0.55 4.3 1.6 0.61 1.4 5.8 1.7 13.1 -
et al. (13).
(Sigma), and DEAE-Sephadex A-50 (Sigma) according to Litchfield and Ball (II), with minor modifications. One modification was to influoride, 1 clude protease inhibitors (0.25 mM phenylmethylsulfonyl pg/ml leupeptin, and 0.75 mu benxamidine; all from Sigma) throughout the preparation, including chromatography. Another modification was to elute the protein from phenyl-Sepharose with a salt gradient starting with 1.5 M NaCl and ending with no salt. Caldesmon was stored on ice or frozen at -20°C in the presence of the inhibitors and 5 mM DTT. SDS-gel electrophoresis of caldesmon or molecular weight standards (Sigma) was performed in 7.5% polyacrylamide according to Laemmli (17). Amino acid composition was determined as done previously (13) with cysteine determined by the method of Hirs (18). Sedimentation equilibrium measurements in the analytical ultracentrifuge were performed as described previously (13,19). The partial specific volume of caldesmon is needed to calculate the molecular weight from equilibrium measurements. A value of 0.713 cma/g at 4°C for the partial specific volume was calculated from the amino acid composition (Table I) as we have described (13) and assuming that the Glu/Gln and Asp/Asn ratios are the same for liver as for gizzard caldesmon (14,15). Sedimentation velocity coefficient measurements were performed in a 12-mm cell on a Beckman Model E equipped with Rayleigh interference optics at a protein concentration of 0.15 mg/ml to effectively avoid nonideality from the hydrodynamic concentration dependence of the sedimentation coefficient. The sedimentation coefficient was determined by plotting the logarithm of the peak position of difference plots which were obtained by subtracting successive pairs of Rayleigh patterns to give the time derivative of the concentration distribution. Difference plots were used because baseline variations are eliminated by the subtraction process (20). Protease inhibitors, at the concentrations indicated above, were included during all ultracentrifugation runs.
RESULTS
AND
DISCUSSION
In a previous work, we determined the molecular mass of muscle caldesmon to be 93 t- 4 kDa from sedimenta-
MOLECULAR
67
WEIGHT
tion equilibrium (13). This is in reasonably good agreement with a molecular mass of about 87 kDa (14) or 89 kDa (15) calculated from the cDNA-deduced amino acid sequence. The small difference might be attributed, in part, to the tight binding of counterions to alternating regions of positive and negative charge which have been revealed in the amino acid sequence. In light of the large number of charged residues in muscle caldesmon, the number of associated counterions might contribute significantly to a reduction in the partial specific volume of caldesmon. Our calculation of the partial specific volume does not take this contribution into account. On the other hand, there is some uncertainty in the molecular weight from the cDNA sequence since two different sequences have been published. It is possible that the two sequences correspond to two isoforms of muscle caldesmon (21), but this is yet to be shown. Rabbit liver caldesmon in 0.1 M NaCl, 0.5 mM EDTA, 1 mM DTT, 5 mM Mops, pH 7.5, behaved as a single, monodisperse component with a molecular mass of 66 t 2 kDa (Fig. 1). The downward trend in molecular mass at higher concentrations is consistent with the expected nonideality for an asymmetric molecule with the dimensions of caldesmon (see below). On SDS gels this caldesmon migrated with an apparent molecular mass of 77 kDa (Fig. 2). Thus liver caldesmon, like gizzard muscle caldesmon, migrates anomalously during SDS-gel electrophoresis. However, it runs closer to its true molecular weight than does the muscle protein (Table II). Sedimentation velocity experiments, in the same buffer, showed a single component with a sedimentation coefficient of s&,~ = 2.49 + 0.02 S (Table II). A value for the Stokes radius was calculated from the molecular weight and sedimentation coefficient and found to be 67 A.
concentration,
FIG.
mglml
1. Equilibrium sedimentation of liver caldesmon in 0.1 M NaCI, 0.5 mM EDTA, 1 mM DTT, 5 mM Mops, pH 7.5, plus protease inhibitors, at 4’C; speed, 20,000 rpm for 72 h. Plot of weight average molecular weight versus local cell concentration.
68
STAFFORD,
JANCSO,
a*
b, C,
d*
4.6
20
A
B
40
60
80
Rf
FIG. 2. (A) SDS-polyacrylamide gel electrophoresis oh lane A, molecular weight standards, (a) fl-galactosidase (116 kDa), (b) phosphorylase b (97.4 kDa), (c) bovine serum albumin (66 kDa), and (d) ovalbumin (45 kDa); lane B, rabbit liver caldesmon. (B) Semilog plot of molecular weight versus mobility for standards (0). Caldesmon migrates at 77 kDa (m).
Yamashiro-Matsumura and Matsumura (9) have calculated a value of 87 kDa for the molecular mass for nonmuscle caldesmon from cultured rat fibroblasts (REF 4A cell line), from a Stokes radius of 60.5 A, obtained by gel filtration, and a sedimentation coefficient of 3.5 S, obtained by sucrose density gradient sedimentation. These values are quite different from those which we have determined for liver caldesmon. However, the methods which they used to determine the sedimentation coefficient and Stokes radius might have suffered from artifacts associated with the presence of high concentrations of sucrose (22, 23), on the one hand, and from known anomalies of migration of large asymmetric molecules on gel filtration media (24), on the other hand. The sucrose density gradient sedimentation technique is also based on the assumption that the shape and partial specific volume, of not only the protein in question but of all the standard proteins used for calibration, are the same as in more physiological buffers. Thus, these techniques may have led to erroneous values, accounting for the discrepancy with our work. However, since the caldesmons in the two studies were from different sources, they might have different physical properties. Some idea of the shape of a molecule can be obtained, as we have done previously for muscle caldesmon (13), from the frictional ratio, flfO, which can be calculated from the sedimentation coefficient and molecular weight. For liver caldesmon, f/f0 = 2.22, assuming an hydration of 0.3 g H,O/g caldesmon. From this value, we calculate that liver caldesmon has an axial ratio of 31, assuming it to be in the shape of a rod. Therefore, it is
AND
GRACEFFA
clear that liver caldesmon, like muscle caldesmon, is a highly asymmetric molecule. Assuming caldesmon to be a rod with an axial ratio of 31, one calculates a length of 53 f 2 nm and a diameter of 1.7 f 0.1 nm (Table II). We previously estimated an axial ratio of 43, a length of 74 nm, and a diameter of 1.7 nm for caldesmon from smooth muscle (13). The amino acid composition of rabbit liver caldesmon is very similar to that of gizzard muscle caldesmon (Table I). In particular, the unusually high percentage of charged residues in the gizzard muscle protein is also observed for the liver protein. The number of cysteine residues for liver caldesmon was found to be 2.8 f 0.5, indicating two or three cysteines per molecule, while gizzard muscle caldesmon has two cysteines per molecule (13-15). We have suggested (13) that the anomalous migration of muscle caldesmon on SDS gels might be due to a high content of acidic residues which could result in less than normal SDS binding. Other proteins with a high proportion of acidic residues also migrate anomalously (25-27). If the Glx and Asx content of muscle and nonmuscle caldesmon reflects the same content of acidic residues, we might expect both proteins to have a similar anomalous migration. However, muscle caldesmon migrates much more slowly than does the liver protein in relation to its size, in spite of the similarity in their overall amino acid composition. Either the Glu and Asp content of the muscle protein is much higher than that of the liver protein or other factors, e.g., the distribution of acidic residues, are also important in determining mobility. According to our results, the molecular mass of nonmuscle caldesmon (from rabbit liver) is about 27 kDa smaller than that of the chicken gizzard smooth muscle caldesmon. By comparing the cyanogen bromide cleavage pattern of chicken liver and gizzard caldesmons, Ball and Kovala (12) concluded that the two molecules have similar N- and C-terminal regions, but that the nonmuscle caldesmon lacks a 44-kDa (by gel electrophoresis) peptide located in the middle of the muscle isoform. However, from the amino acid sequence and assuming 100% cleavage, one does not expect any product of this
TABLE Caldesmon
Sedimentation coeff. (Svedbergs) Molecular mass (kDa) Apparent mol. mass (kDa, SDS gel) Length (nm) Diameter (nm) Stokes radius (A) ’ Graceffa
et al. (13).
II
Characterization Liver
Muscle a
2.49 f 0.02 66+2 77 53 + 2 1.7 + 0.1 67
2.65 f 0.05 93+4 140 74 f 2 1.7 * 0.1 87
RABBIT
LIVER
CALDESMON
size. The best candidate for this peptide would be the one from Leu 231 to Met 386 (from the sequence of Bryan et al. (14)) or to Met 401 (from the sequence of Hayashi et al. (15)), with a calculated molecular mass of 18.4 or 20.2 kDa, respectively, consistent with our measurements. However, since the molecular mass of this peptide is somewhat smaller than the molecular mass difference between the two proteins, then this peptide may be only part of the complete “difference peptide.” Since the amino acid composition (38% acidic and 31% basic residues) and sequence of this peptide are very unusual (14, 15), it is possible that its mobility on SDS gels is abnormal. An absence of this peptide would also result in a lower acidic residue content for the liver caldesmon and thus might account for its less anomalous electrophoretie migration compared to the muscle protein (see above). The function of this central region of the muscle caldesmon is presently unknown. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. ACKNOWLEDGMENTS We thank Dr. Renne C. Lu and Anna Wong for determining amino acid composition and Dr. John Gergely for a critical reading of the manuscript.
REFERENCES 1. Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, Natl. Acad. Sci. USA ‘78,5652-5655.
S. (1981) Proc.
A., and Lynch, W. (1985) J. Cell Biol. 100,1656-1663.
4. Sobue, K., Tanaka, T., Kanda, K., Ashino, N., and Kakiuchi, (1985) Proc. Natl. Acad. Sci. USA 82,5025-5029.
S.
69
WEIGHT
R. D., Cheek, T. R., and Norman, K-M. (1986) Nature (London) 319,68-70. Dingus, J., Hwo, S., and Bryan, J. (1986) J. Cell Biol. 102,17481757. Pho, D. B., Desbruyeres, E., Der Terrossian, E., and Olomucki, A. (1986) FEBS L&t. 202,117-121. Ueki, N., Sobue, K., Kanda, K., Hada, T., and Higashino, K. (1987) Proc. Natl. Acad. Sci. USA 84,9049-9053. Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol.
5. Burgoyne, 6. 7. 8. 9.
106,1973-1983. 10. Der Terrossian, E., Deprette, C., and Cassoly, R. (1989) Biochem. Biophys. Res. Commun. 159,395-401. 11. Litchfield, D. W., and Ball, E. H. (1987) J. Biol. Chem. 262,8056-
8060. 12. Ball, E. H., and Kovala, T. (1988) Biochemistry 27,6093-6098. 13. Graceffa, P., Wang, C-L. A., and Stafford, W. F. (1988) J. Biol. Chem. 263,14,196-14,202. 14. Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264,13,873-13,879. 15. Hayashi, K., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164,503-511. 16. Bretscher, A. (1984) J. Bill. Chem. 269,12,873-12,880. 17. Laemmli, U. K. (1970) Nature (London) 227,680-685. 18. Him, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., Ed.), Vol. 11, pp. 59-62, Academic Press, San Diego. 19. Yphantis, D. A. (1964) Biochemistry 3,297-317. 20. Cohen, R. (1976) Biophys. Chem. 5.77-96. 21. Lynch, W. P., Riseman, V. M., and Bretscher, A. (1987) J. Biol. Chem. 262,7429-7437. 22. Na, G. C., and Timasheff, S. N. (1980) Biochemistry 19, 13551365. 23. Arakawa, T., and Timasheff, S. N. (1983) Arch. Biochem. Biophys.
244,169-177. 24. Meredith, S. C., and Nathans, 234-243. 9.5
2. Owada, M. K., Hakura, A., Iida, K., Yahara, I., Sobue, K., and Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81,3133-3137. 3. Bretscher,
MOLECULAR
I”’
Cozens, B., and Reithmeier, 62486252
G. R. (1982) Anal. Biochem.
121,
R. A. F. (1984) J. Biol. Chem. 259,
26. Takano, E., Maki, M., Mori, H., Hatanaka, M., Marti, T., Titani, K., Kannagi, R., Ooi, T., and Murachi, T. (1988) Biochemistry 27, 1964-1972. 27. Bryan, J. (1989) J. Muscle Res. Cell Motil. 10.95-96.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 281, No. 1, August 15, pp. 66-69,199O
Caldesmon from Rabbit Liver: Molecular Weight and Length by Analytical Ultracentrifugation’ Walter F. Stafford,**? Agnes Jancso,* and Philip Graceffa*$’ *Department of Muscle Research, Boston Biomedical Research Institute, 20 Staniford Street, Boston, Massachusetts 02114, and tDepartment of Neurology, Harvard Medical School, Boston, Massachusetts 02115
Received January
31,1990, and in revised form March 28,199O
Although smooth muscle caldesmon migrates as a 140- to 150-kDa protein during sodium dodecyl sulfate-gel electrophoresis, its molecular mass is around 93 kDa as determined by sedimentation equilibrium (P. Graceffa, C-L. A. Wang, and W. F. Stafford, 1988, J. Biol. Chem. 263, 14,196-14,202). Nonmuscle caldesmon migrates during electrophoresis with a molecular mass close to 77 kDa, about half that of the muscle isoform. However, it is controversial whether the molecular weight of nonmuscle caldesmon is the same or much less than that of the muscle protein. Therefore we have now determined the molecular mass of rabbit liver caldesmon by sedimentation equilibrium and found a value of 66 + 2 kDa, a value much smaller than that of muscle caldesmon. This new value of the molecular weight, together with a sedimentation coefficient of 2.49 + 0.02 S, yields an apparent length of 53 + 2 nm and a diameter of 1.7 nm for the liver protein. We previously estimated a length of 74 nm and a diameter of 1.7 nm for the muscle caldesmon. We have also determined the amino acid composition of liver caldesmon and found it to be similar to that of the muscle protein. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. The difference in molecular weight between the two caldesmons is consistent with the nonmuscle protein lacking a central peptide of the muscle isoform, as suggested by E. H. Ball, and T. Kovala, (1988, Biochemistry 27, 6093-
6098.
tion of smooth muscle contraction and nonmuscle motility. Caldesmon was first discovered in smooth muscle (1) and later found in a variety of nonmuscle tissues (2-lo), including mammalian and avian liver ( 11,12). Although muscle caldesmon migrates during SDS3-gel electrophoresis as a 140- to 150-kDa protein, its molecular mass is close to 93 kDa, as determined by sedimentation equilibrium (13), or 87 kDa (14) or 89 kDa (15) from the cDNAdeduced amino acid sequence. Nonmuscle caldesmon migrates on SDS gels as a 70- to 83-kDa protein, depending on its source. Yamashiro-Matsumura and Matsumura (9) reported that nonmuscle caldesmon is a monomer with a molecular mass of 87 kDa, calculated from its hydrodynamic properties, which suggests that the molecular weights of muscle and nonmuscle caldesmons are about the same. In contrast, Ball and Kovala (12) suggested that in nonmuscle caldesmon a central fragment (M, 44,000 from SDS-PAGE) of the muscle caldesmon is absent, which would indicate that the molecular weight of the nonmuscle protein is considerably less than that from muscle. Since Yamashiro-Matsumura and Matsumura (9) used methods which depend on the shape of the protein, we decided to try to resolve this uncertainty by determining the molecular weight of nonmuscle caldesmon by sedimentation equilibrium in the analytical ultracentrifuge, a technique which requires no assumption about the shape of the molecule. We found that rabbit liver caldesmon is a monomer with a molecular mass of 66 kDa, a value which is much smaller than that of the muscle caldesmon.
o 1990 Academic Preae, Inc.
MATERIALS
Caldesmon is an actin-binding protein which is thought to play a role in the thin filament Ca2+-regular This work was supported by National Institutes of Health Grant AR-30917 (P.G.). *To whom correspondence and reprint requests should be addressed. 66
AND
METHODS
Caldesmon, from fresh rabbit liver, was purified from the 30-50% (NH&SO, fraction of the heat stable extract (16) by chromatography on phosphocellulose P-11 (Whatman), phenyl-Sepharose CL-4B
‘Abbreviations used: SDS, sodium dodecyl sulfate; PAGE, polya&amide gel electrophoresis; Mops, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol. 0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
RABBIT TABLE
LIVER
CALDESMON
I
Amino Acid Composition of Gizzard Muscle and Liver Nonmuscle Caldesmon Gizzard”
(mol% of amino acid)
Amino acid
7.7 27.1 5.1 4.5 0.19 9.1 4.2 11.6 3.6 0.34 4.4 0.77 0.35 1.3 4.7 1.1 13.9 -
Asx Glx Ser GUY His -4% Thr Ala Pro TYr Val Met CYS Ile Leu Phe LYS Trp a Graceffa
Liver
9.0 25.4 8.3 5.7 0.7 7.4 4.9 8.7 1.6 0.55 4.3 1.6 0.61 1.4 5.8 1.7 13.1 -
et al. (13).
(Sigma), and DEAE-Sephadex A-50 (Sigma) according to Litchfield and Ball (II), with minor modifications. One modification was to influoride, 1 clude protease inhibitors (0.25 mM phenylmethylsulfonyl pg/ml leupeptin, and 0.75 mu benxamidine; all from Sigma) throughout the preparation, including chromatography. Another modification was to elute the protein from phenyl-Sepharose with a salt gradient starting with 1.5 M NaCl and ending with no salt. Caldesmon was stored on ice or frozen at -20°C in the presence of the inhibitors and 5 mM DTT. SDS-gel electrophoresis of caldesmon or molecular weight standards (Sigma) was performed in 7.5% polyacrylamide according to Laemmli (17). Amino acid composition was determined as done previously (13) with cysteine determined by the method of Hirs (18). Sedimentation equilibrium measurements in the analytical ultracentrifuge were performed as described previously (13,19). The partial specific volume of caldesmon is needed to calculate the molecular weight from equilibrium measurements. A value of 0.713 cma/g at 4°C for the partial specific volume was calculated from the amino acid composition (Table I) as we have described (13) and assuming that the Glu/Gln and Asp/Asn ratios are the same for liver as for gizzard caldesmon (14,15). Sedimentation velocity coefficient measurements were performed in a 12-mm cell on a Beckman Model E equipped with Rayleigh interference optics at a protein concentration of 0.15 mg/ml to effectively avoid nonideality from the hydrodynamic concentration dependence of the sedimentation coefficient. The sedimentation coefficient was determined by plotting the logarithm of the peak position of difference plots which were obtained by subtracting successive pairs of Rayleigh patterns to give the time derivative of the concentration distribution. Difference plots were used because baseline variations are eliminated by the subtraction process (20). Protease inhibitors, at the concentrations indicated above, were included during all ultracentrifugation runs.
RESULTS
AND
DISCUSSION
In a previous work, we determined the molecular mass of muscle caldesmon to be 93 t- 4 kDa from sedimenta-
MOLECULAR
67
WEIGHT
tion equilibrium (13). This is in reasonably good agreement with a molecular mass of about 87 kDa (14) or 89 kDa (15) calculated from the cDNA-deduced amino acid sequence. The small difference might be attributed, in part, to the tight binding of counterions to alternating regions of positive and negative charge which have been revealed in the amino acid sequence. In light of the large number of charged residues in muscle caldesmon, the number of associated counterions might contribute significantly to a reduction in the partial specific volume of caldesmon. Our calculation of the partial specific volume does not take this contribution into account. On the other hand, there is some uncertainty in the molecular weight from the cDNA sequence since two different sequences have been published. It is possible that the two sequences correspond to two isoforms of muscle caldesmon (21), but this is yet to be shown. Rabbit liver caldesmon in 0.1 M NaCl, 0.5 mM EDTA, 1 mM DTT, 5 mM Mops, pH 7.5, behaved as a single, monodisperse component with a molecular mass of 66 t 2 kDa (Fig. 1). The downward trend in molecular mass at higher concentrations is consistent with the expected nonideality for an asymmetric molecule with the dimensions of caldesmon (see below). On SDS gels this caldesmon migrated with an apparent molecular mass of 77 kDa (Fig. 2). Thus liver caldesmon, like gizzard muscle caldesmon, migrates anomalously during SDS-gel electrophoresis. However, it runs closer to its true molecular weight than does the muscle protein (Table II). Sedimentation velocity experiments, in the same buffer, showed a single component with a sedimentation coefficient of s&,~ = 2.49 + 0.02 S (Table II). A value for the Stokes radius was calculated from the molecular weight and sedimentation coefficient and found to be 67 A.
concentration,
FIG.
mglml
1. Equilibrium sedimentation of liver caldesmon in 0.1 M NaCI, 0.5 mM EDTA, 1 mM DTT, 5 mM Mops, pH 7.5, plus protease inhibitors, at 4’C; speed, 20,000 rpm for 72 h. Plot of weight average molecular weight versus local cell concentration.
68
STAFFORD,
JANCSO,
a*
b, C,
d*
4.6
20
A
B
40
60
80
Rf
FIG. 2. (A) SDS-polyacrylamide gel electrophoresis oh lane A, molecular weight standards, (a) fl-galactosidase (116 kDa), (b) phosphorylase b (97.4 kDa), (c) bovine serum albumin (66 kDa), and (d) ovalbumin (45 kDa); lane B, rabbit liver caldesmon. (B) Semilog plot of molecular weight versus mobility for standards (0). Caldesmon migrates at 77 kDa (m).
Yamashiro-Matsumura and Matsumura (9) have calculated a value of 87 kDa for the molecular mass for nonmuscle caldesmon from cultured rat fibroblasts (REF 4A cell line), from a Stokes radius of 60.5 A, obtained by gel filtration, and a sedimentation coefficient of 3.5 S, obtained by sucrose density gradient sedimentation. These values are quite different from those which we have determined for liver caldesmon. However, the methods which they used to determine the sedimentation coefficient and Stokes radius might have suffered from artifacts associated with the presence of high concentrations of sucrose (22, 23), on the one hand, and from known anomalies of migration of large asymmetric molecules on gel filtration media (24), on the other hand. The sucrose density gradient sedimentation technique is also based on the assumption that the shape and partial specific volume, of not only the protein in question but of all the standard proteins used for calibration, are the same as in more physiological buffers. Thus, these techniques may have led to erroneous values, accounting for the discrepancy with our work. However, since the caldesmons in the two studies were from different sources, they might have different physical properties. Some idea of the shape of a molecule can be obtained, as we have done previously for muscle caldesmon (13), from the frictional ratio, flfO, which can be calculated from the sedimentation coefficient and molecular weight. For liver caldesmon, f/f0 = 2.22, assuming an hydration of 0.3 g H,O/g caldesmon. From this value, we calculate that liver caldesmon has an axial ratio of 31, assuming it to be in the shape of a rod. Therefore, it is
AND
GRACEFFA
clear that liver caldesmon, like muscle caldesmon, is a highly asymmetric molecule. Assuming caldesmon to be a rod with an axial ratio of 31, one calculates a length of 53 f 2 nm and a diameter of 1.7 f 0.1 nm (Table II). We previously estimated an axial ratio of 43, a length of 74 nm, and a diameter of 1.7 nm for caldesmon from smooth muscle (13). The amino acid composition of rabbit liver caldesmon is very similar to that of gizzard muscle caldesmon (Table I). In particular, the unusually high percentage of charged residues in the gizzard muscle protein is also observed for the liver protein. The number of cysteine residues for liver caldesmon was found to be 2.8 f 0.5, indicating two or three cysteines per molecule, while gizzard muscle caldesmon has two cysteines per molecule (13-15). We have suggested (13) that the anomalous migration of muscle caldesmon on SDS gels might be due to a high content of acidic residues which could result in less than normal SDS binding. Other proteins with a high proportion of acidic residues also migrate anomalously (25-27). If the Glx and Asx content of muscle and nonmuscle caldesmon reflects the same content of acidic residues, we might expect both proteins to have a similar anomalous migration. However, muscle caldesmon migrates much more slowly than does the liver protein in relation to its size, in spite of the similarity in their overall amino acid composition. Either the Glu and Asp content of the muscle protein is much higher than that of the liver protein or other factors, e.g., the distribution of acidic residues, are also important in determining mobility. According to our results, the molecular mass of nonmuscle caldesmon (from rabbit liver) is about 27 kDa smaller than that of the chicken gizzard smooth muscle caldesmon. By comparing the cyanogen bromide cleavage pattern of chicken liver and gizzard caldesmons, Ball and Kovala (12) concluded that the two molecules have similar N- and C-terminal regions, but that the nonmuscle caldesmon lacks a 44-kDa (by gel electrophoresis) peptide located in the middle of the muscle isoform. However, from the amino acid sequence and assuming 100% cleavage, one does not expect any product of this
TABLE Caldesmon
Sedimentation coeff. (Svedbergs) Molecular mass (kDa) Apparent mol. mass (kDa, SDS gel) Length (nm) Diameter (nm) Stokes radius (A) ’ Graceffa
et al. (13).
II
Characterization Liver
Muscle a
2.49 f 0.02 66+2 77 53 + 2 1.7 + 0.1 67
2.65 f 0.05 93+4 140 74 f 2 1.7 * 0.1 87
RABBIT
LIVER
CALDESMON
size. The best candidate for this peptide would be the one from Leu 231 to Met 386 (from the sequence of Bryan et al. (14)) or to Met 401 (from the sequence of Hayashi et al. (15)), with a calculated molecular mass of 18.4 or 20.2 kDa, respectively, consistent with our measurements. However, since the molecular mass of this peptide is somewhat smaller than the molecular mass difference between the two proteins, then this peptide may be only part of the complete “difference peptide.” Since the amino acid composition (38% acidic and 31% basic residues) and sequence of this peptide are very unusual (14, 15), it is possible that its mobility on SDS gels is abnormal. An absence of this peptide would also result in a lower acidic residue content for the liver caldesmon and thus might account for its less anomalous electrophoretie migration compared to the muscle protein (see above). The function of this central region of the muscle caldesmon is presently unknown. In conclusion, muscle and nonmuscle caldesmons appear to have similar overall amino acid composition and tertiary structure with the smaller nonmuscle protein having a correspondingly smaller length. ACKNOWLEDGMENTS We thank Dr. Renne C. Lu and Anna Wong for determining amino acid composition and Dr. John Gergely for a critical reading of the manuscript.
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