Advertisement

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

  • Darin J. Weber
  • Philip N. McFadden
Protocol
  • 78 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The several classes of S-adenosylmethionine-dependent protein methyltransferases 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 carboxyl 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 overexpression systems, can be taken as evidence of spontaneous damage that has occurred in these proteins since the time of their translation.

Keywords

Scintillation Vial Scintillation Fluid Solubilization Buffer Electrode Buffer Cationic Detergent 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 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.CrossRefGoogle Scholar
  2. 2.
    Lou, L. L. and Clarke, S. (1987) Enzymatic methylation of band 3 anion transporter in intact human erythrocytes. Biochemistry 26, 52–59.PubMedCrossRefGoogle Scholar
  3. 3.
    Fairbanks, G. and Avruch, J. (1973) Four gel systems for electrophoretic fractionation of membrane proteins using ionic detergents. J. Supramol. Struct. 1, 66–75.CrossRefGoogle Scholar
  4. 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.PubMedGoogle Scholar
  5. 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.Google Scholar
  6. 6.
    Gingras, D., Menard, P., and Beliveau, R. (1991) Protein carboxyl methylation in kidney brush-border membranes. Biochim. Biophys. Acta. 1066, 261–267.PubMedCrossRefGoogle Scholar
  7. 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.PubMedGoogle Scholar
  8. 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.PubMedGoogle Scholar
  9. 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-methyltransferase, in Deamidation and Isoaspartate Formation in Peptides and Proteins. (Aswad, D. W., ed.), CRC, Boca Raton, pp. 47–64.Google Scholar
  10. 10.
    McFadden, P. N., Horwitz, J., and Clarke, S. (1983) Protein carboxyl methytransferase from cow eye lens. Biochem. Biophys. Res. Comm. 113(2), 418–424.PubMedCrossRefGoogle Scholar
  11. 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. Electrophoresis 9, 255–262.PubMedCrossRefGoogle Scholar
  12. 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.CrossRefGoogle Scholar
  13. 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. 14.
    Sellinger, O. Z. and Wolfson, M. F. (1991) Carboxyl methylation affects the proteolysis of myelin basic protein by staphylococcus aureus V8 proteinase. Biochim. Biophys. Acta. 1080, 110–118.PubMedCrossRefGoogle Scholar
  15. 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.PubMedGoogle Scholar
  16. 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. 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.PubMedGoogle Scholar
  18. 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.PubMedGoogle Scholar
  19. 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-isoaspartyl residues in structurally altered protein substrates. Proc. Natl. Acad. Sci. USA 81, 7757–7761.CrossRefGoogle Scholar
  20. 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. 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.CrossRefGoogle Scholar
  22. 22.
    Barber, J. R. and Clarke, S. (1983) Membrane protein carboxyl methylation increase with human erythrocyte age. J. Biol. Chem. 258(2), 1189–1196.PubMedGoogle Scholar
  23. 23.
    Chamberlain, J. P. (1979) Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor, sodium salicylate. Analyt. Biochem. 98, 132.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Darin J. Weber
    • 1
  • Philip N. McFadden
    • 1
  1. 1.Department of Biochemistry and BiophysicsOregon State UniversityCorvallis

Personalised recommendations