Abstract
Their large size and their relative resistance to proteolytic cleavage (1) make myosins particularly difficult substrates for the acquisition of their peptide sequences by standard protocols. For this reason, instead of identifying myosins first according to their biochemical activity and then obtaining their sequences, PCR and other DNA-based techniques exploiting the highly conserved sequences in the amino end, the “head” domain, have been used to find new myosins (2–5). However, such identification of myosins by sequence leaves open the question of their function. If peptide sequence could be obtained from myosin proteins whose biochemical behavior was known, then the gap between function and sequence could be bridged. We describe here a method that enabled us to acquire peptide sequences of semi-purified myosins (6).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bálint M., Szilágyi L., Fekete G., Blazsó M., and Biró N. A. (1968) Studies on proteins and protein complexes of muscle by means of proteolysis: V. Fragmentation of light meromyosin by trypsin. J. Mol. Biol. 37, 317–330.
Garces J. and Gavin R. H. (1998) A PCR screen identifies a novel, unconventional myosin heavy chain gene (MYO1) in Tetrahymena thermophyla. J. Euk. Micro. 45, 252–259.
Goodson H. and Spudich J. A. (1995). Identification and molecular characterization of a yeast myosin I. Cell Motil Cytoskeleton 30, 73–84.
Bement W. M., Hasson T., Wirth J. A., Cheney R. E., and Mooseker M. S. (1994) Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types. Proc. Natl. Acad. Sci. USA 91, 6549–6553.
Titus M. A., Kuspa A., and Loomis W. F. (1994) Discovery of myosin genes by physical mapping in Dictyostelium. Proc. Natl. Acad. Sci. USA 91, 9446–9450.
Medeiros N., Reese T. S., Jaffe H., DeGiorgis J. A., and Bearer E. L. (1998) Primary peptide sequences from squid muscle and optic lobe myosin IIs: A strategy to identify an organelle motor. Cell. Biol. Int. 22, 161–173.
Bearer E. L., DeGiorgis J. A., Bodner R. A., Kao A., and Reese T. S. (1993) Evidence for myosin motors on organelles in squid axoplasm. Proc. Natl. Acad. Sci. USA 90, 11,252–11,256.
Bearer E. L., DeGiorgis J. A., Jaffe H., Medeiros N. A., and Reese T. S. (1996) An axoplasmic myosin with a calmodulin-like light chain. Proc. Natl. Acad. Sci. USA 93, 6064–6068.
Bearer E. L., DeGiorgis J. A., Medeiros N. A., and Reese T. S. (1996) Actin-based motility of isolated axoplasmic organelles. Cell. Motil. Cytoskel. 33, 106–114.
Bearer E. L., Jaffe H., Medeiros N., and Reese T. S. (1996) Purification and amino acid sequencing of two high molecular weight myosins from squid muscle and brain. Molec. Biol. Cell 7, 37a.
Nyitray L., Goodwin E. B., and Szent-Györgyi A. G. (1991) Complete primary structure of a scallop striated muscle myosin heavy chain. Sequence comparison with other heavy chains reveals regions that might be critical for regulation. J. Biol. Chem. 266, 18,469–18,476.
Matulef K., Sirokman K., Perreault-Micale C. L., and Szent-Györgyi A. G. (1998) The amino acid sequnce of squid syphon muscle myosin heavy chain. J. Musc. Res. Cell. Motil. 19, 705–712.
Medeiros N. A., DeGiorgis J. A., Reese T. S., and Bearer E. L. (1997) Obtaining primary amino acid sequences from squid brain myosins. Biol. Bull. 193, 198.
DeGiorgis J. A., Reese T. S., and Bearer E. L. (2000) Myosin II and V: Implications for exonal transport, submitted.
Tabb J. S., Molyneaux B. J., Cohen D. L., Kuznetsov S. A., and Langford G. M. (1998) Transport of ER vesicles on actin filaments in neurons by myosin V. J. Cell Sci. 111, 3221–3234.
Stone K. L., Lopresti M. B., and Williams K. R. (1990) Enzymatic digestion of proteins and HPLC isolation in the subnanomole range. In Laboratory Methodology in Biochemistry (edited by Fini C. F.), pp. 181–205. Boca Raton, FL: CRC Press.
Riviere L. R., Fleming M., Elicone C., and Tempst P. (1991) Study and applications of the effects of detergents and chaotropes on enzymatic proteolysis. In Techniques in Protein Chemistry II (edited by Villafranca J. J.), pp. 171–179. San Diego, CA: Academic Press.
Fernandez J., Demott M., Atherton D., and Mische S. M. (1992) Internal protein sequence analysis: Enzymatic digestion for less than 10 μg of protein bound to polyvinylidene difluoride or nitrocellulose membranes. Anal. Biochem. 201, 255–264.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Bearer, E.L. (2001). Microsequencing of Myosins for PCR Primer Design. In: Gavin, R.H. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology™, vol 161. Humana Press. https://doi.org/10.1385/1-59259-051-9:009
Download citation
DOI: https://doi.org/10.1385/1-59259-051-9:009
Publisher Name: Humana Press
Print ISBN: 978-0-89603-771-7
Online ISBN: 978-1-59259-051-3
eBook Packages: Springer Protocols