Fragmentation of isoaspartyl peptides and proteins by carboxypeptidase Y: release of isoaspartyl dipeptides as a result of internal and external cleavage.
Isoaspartate-containing versions of sea urchin sperm-activating peptide, delta sleep-inducing peptide, and lactate dehydrogenase (231-242) were cleaved at internal sites by carboxypeptidase Y. Cleavage occurred between the isoaspartate and the preceding amino acid, and it was accompanied by sequential digestion of amino acids from the two resulting carboxyl termini. Because the isoaspartyl bonds were not cleaved, isoaspartyl dipeptides were among the final products. The rate of release of isoaspartyl dipeptides was different for the three peptides, a 24-h digestion yielding 0.32 mol of isoaspartylglycine/mol of isoaspartyl sperm-activating peptide, 0.50 mol of isoaspartylalanine/mol of isoaspartyl delta sleep-inducing peptide, and 1.15 mol of isoaspartylserine/mol of isoaspartyl lactate dehydrogenase (231-242). The different rates could be explained by the slow cleavage of amino acids preceded by glycine. Isoaspartyl dipeptides were not detected in digests of the corresponding aspartate- or asparagine-containing forms of the peptides. Release of isoaspartyl dipeptides by carboxypeptidase Y was used to demonstrate the presence of isoaspartylglycine sequences in deamidated adrenocorticotropin (0.54 mol/mol), in a mixture of trypic fragments of base-treated calmodulin (0.20 mol/mol), and in a mixture of tryptic fragments of base-treated triosephosphate isomerase (0.08 mol/mol). These results confirm earlier work suggesting that isoaspartylglycine formation is prevalent in proteins exposed to alkaline conditions. They also provide a methodology that should prove useful in the characterization of natural substrates for protein L-isoaspartyl methyltransferase.
Johnson
B A
BA
Department of Psychobiology, University of California, Irvine 92717.
Aswad
D W
DW
D000818:Animals; D001224:Aspartic Acid; D002268:Carboxypeptidases / Q000378:metabolism*; D003701:Delta Sleep-Inducing Peptide; D004151:Dipeptides; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D012617:Sea Urchins; D013237:Stereoisomerism; D013379:Substrate Specificity
D004151:Dipeptides; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D001224:Aspartic Acid; D003701:Delta Sleep-Inducing Peptide; C028364:speract; D007770:L-Lactate Dehydrogenase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase