Optimal conditions for the use of protein L-isoaspartyl methyltransferase in assessing the isoaspartate content of peptides and proteins.
Protein L-isoaspartyl methyltransferase provides a basis for enzymatic measurement of atypical, isoaspartyl linkages which make a major contribution to protein microheterogeneity. The low Vmax of the methyltransferase reaction and the instability of the methyl ester can hinder accurate determinations, and different laboratories using different conditions have achieved discrepant values for the isoaspartate content of the same proteins. To investigate the effects of these conditions, and to optimize the assay, isoaspartyl delta sleep-inducing peptide was methylated under a variety of conditions. We found that 1 microM methyltransferase was required to obtain stoichiometric modification of 2 microM peptide in 40-min reactions at pH 6.2 and 30 degrees C. A computer model utilizing kinetic constants obtained from studies on initial rates of methylation predicted the same requirement for enzyme concentration. Carrier protein was necessary for optimal methyltransferase activity at enzyme concentrations below 0.4 microM. Stoichiometric methylation required concentrations of S-adenosylmethionine to be in substantial excess over those of peptide; 50 microM S-adenosylmethionine is the minimum needed for complete modification of 10 microM peptide. Spontaneous demethylation was significant under all conditions tested, so that the methyl ester itself never reached a ratio of 1 mol/mol of total peptide. These results demonstrate that the most accurate measurements of isoaspartate are obtained when reactions are carried out at low peptide concentrations, high S-adenosylmethionine concentrations, and high enzyme concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Johnson
B A
BA
School of Biological Sciences, University of California, Irvine 92717.
Aswad
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000032:analysis* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D003701:Delta Sleep-Inducing Peptide / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D010455:Peptides; D011506:Proteins; D001224:Aspartic Acid; D003701:Delta Sleep-Inducing Peptide; C068904:delta sleep-inducing peptide, isoAsp(5)-; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase