Skip to main content
SpringerLink
Log in

Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase

  • Protocol
The Protein Protocols Handbook

Part of the book series: Springer Protocols Handbooks ((SPH))

  • 333 Accesses

Abstract

The several classes of S-adenosylmethionine-dependent protein methyltrans-ferases are distinguishable by the type of amino acid they modify in a substrate protein. The protein carboxyl methyltransferases constitute the subclass of enzymes that incorporate a methyl group into a methyl ester linkage with the car-boxyl groups of proteins. Of these, protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase, EC 2.1.1.77 (PCM) specifically methyl esterifies aspartyl residues that through age-dependent alterations are in either the d-aspartyl or the l-isoaspartyl configuration (1, 2). There are two major reasons for wishing to know the identity of protein substrates for PCM. First, the proteins that are methylated by PCM in the living cell, most of which have not yet been identified, are facets in the age-dependent metabolism of cells. Second, the fact that PCM can methylate many proteins in vitro, including products of overexpres-sion systems, can be taken as evidence of spontaneous damage that has occurred in these proteins since the time of their translation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Aswad, D. W. and Deight, E. A. (1983) Endogenous substrates for protein carboxyl methyltransferase in cytosolic fractions of bovine brain. J. Neurochem. 31, 1702–1709.

    Article  Google Scholar 

  2. Lou, L. L. and Clarke, S. (1987) Enzymatic methylation of band 3 anion transporter in intact human erythrocytes. Biochemistry 26, 52–59.

    Article  CAS  PubMed  Google Scholar 

  3. Fairbanks, G. and Avruch J. (1973) Four gel systems for electrophoretic fractiona-tion of membrane proteins using ionic detergents. J. Supramol. Struct. 1, 66–75.

    Article  Google Scholar 

  4. Barber, J. R. and Clarke, S. (1984) Inhibition of protein carboxyl methylation by S-adenosyl-l-homocysteine in intact erythrocytes. J. Biol. Chem. 259 (11), 7115–7122.

    CAS  PubMed  Google Scholar 

  5. Bower, V. E. and Bates, R. G. (1955) pH Values of the Clark and Lubs buffer solutions at 25°C. J. Res. Natl. Bureau Stand. 55 (4), 197–200.

    Article  CAS  Google Scholar 

  6. Gingras, D., Menard, P., and Beliveau, R. (1991) Protein carboxyl methylation in kidney brush-border membranes. Biochim. Biophys. Acta. 1066, 261–267.

    Article  CAS  PubMed  Google Scholar 

  7. Johnson, B. A., Najbauer, J., and Aswad, D. W. (1993) Accumulation of substrates for protein l-isoaspartyl methyltransferase in adenosine dialdehyde-treated PC12 cells. J. Biol. Chem. 268 (9), 6174–6181.

    CAS  PubMed  Google Scholar 

  8. Johnson, B. A., Freitag, N. E., and Aswad, D. W. (1985) Protein carboxyl methyltransferase selectively modifies an atypical form of calmodulin. J. Biol. Chem. 260 (20), 10,913–10,916.

    CAS  Google Scholar 

  9. Lowenson, J. D. and Clarke, S. (1995) Recognition of isomerized and racemized aspartyl residues in peptides by the protein L-isoaspartate (d-aspartate) O-methyl-transferase, in Deamidation and Isoaspartate Formation in Peptides and Proteins. (Aswad, D. W., ed.), CRC, Boca Raton, pp. 47–64.

    Google Scholar 

  10. McFadden, P. N., Horwitz, J., and Clarke, S. (1983) Protein carboxyl methytrans-ferase from cow eye lens. Biochem. Biophys. Res. Comm. 113 (2), 418–424.

    Article  CAS  PubMed  Google Scholar 

  11. Neuhoff, V, Stamm, R., Pardowitz, I., Arold, N., Ehrhardt, W., and Taube, D. (1988) Essential problems in quantification of proteins following colloidal staining with Coomassie brilliant blue dyes in polyacrylamide gels, and their solutions. Electro-phoresis 9, 255–262.

    Article  CAS  Google Scholar 

  12. O’Conner, C. M. and Clarke, S. (1985) Analysis of erythrocyte protein methyl esters by two-dimensional gel electrophoresis under acidic separating conditions. Analyt. Biochem. 148, 79–86.

    Article  Google Scholar 

  13. O’Conner, C. M. and Clarke, S. (1984) Carboxyl methylation of cytosolic proteins in intact human erythrocytes. J. Biol. Chem. 259 (4), 2570–2578.

    Google Scholar 

  14. Sellinger, O. Z. and Wolfson, M. F. (1991) Carboxyl methylation affects the pro-teolysis of myelin basic protein by staphylococcus aureus V8 proteinase. Biochim. Biophys. Acta. 1080, 110–118.

    Article  CAS  PubMed  Google Scholar 

  15. MacFarlane, D. E. (1984) Inhibitors of cyclic nucleotides phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets. J. Biol. Chem. 259 (2), 1357–1362.

    CAS  PubMed  Google Scholar 

  16. Aswad, D. W. (1995) Methods for analysis of deamidation and isoaspartate formation in peptides, in Deamidation and Isoaspartate Formation in Peptides and Proteins (Aswad, D. W., ed.), CRC, Boca Raton, pp. 7–30.

    Google Scholar 

  17. Freitag, C. and Clarke, S. (1981) Reversible methylation of cytoskeletal and membrane proteins in intact human erythrocytes. J. Biol. Chem. 256 (12), 6102–6108.

    CAS  PubMed  Google Scholar 

  18. Gingras, D., Boivin, D., and Beliveau, R. (1994) Asymmetrical distribution of L-isoaspartyl protein carboxyl methyltransferases in the plasma membranes of rat kidney cortex. Biochem. J. 297, 145–150.

    CAS  PubMed Central  PubMed  Google Scholar 

  19. O’Conner, C. M., Aswad, D. W., and Clarke, S. (1984) Mammalian brain and erythrocyte carboxyl methyltranserases are similar enzymes that recognize both d-aspartyl and l-iso-aspartyl residues in structurally altered protein substrates. Proc. Natl. Acad. Sci. USA 81, 7757–7761.

    Article  Google Scholar 

  20. O’Conner, C. M. and Clarke, S. (1983) Methylation of erythrocyte membrane proteins at extracellular and intracellular d-aspartyl sites in vitro. J. Biol. Chem. 258 (13), 8485–8492.

    Google Scholar 

  21. Ohta, K., Seo, N., Yoshida, T., Hiraga, K., and Tuboi, S. (1987) Tubulin and high molecular weight microtubule-associated proteins as endogenous substrates for protein carboxyl methyltransferase in brain. Biochemie 69, 1227–1234.

    Article  CAS  Google Scholar 

  22. Barber, J. R. and Clarke, S. (1983) Membrane protein carboxyl methylation increase with human erythrocyte age. J. Biol. Chem. 258 (2), 1189–1196.

    CAS  PubMed  Google Scholar 

  23. Chamberlain, J. P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. Analyt. Biochem. 98, 132.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR

    Darin J. Weber & Philip N. McFadden

Authors
  1. Darin J. Weber
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philip N. McFadden
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. School of Life Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK

    John M. Walker

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Weber, D.J., McFadden, P.N. (2009). Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase. In: Walker, J.M. (eds) The Protein Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-198-7_164

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-198-7_164

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-474-6

  • Online ISBN: 978-1-59745-198-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation