The amino terminus of PKA catalytic subunit - A site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes

Conserved deamidation of PKA catalytic subunit isozymes C alpha and C beta -more than 25% at Asn2 in vivo in both cases-has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, Konig N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457-469). Isoasp artate formation in proteins in vivo is indicative of succinimide intermedi ates involved in both the initial deamidation reaction as well as the "repa ir" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O- methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succ inimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomer s with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-te rminal tryptic peptides (T1) of the enzyme were analyzed following procedur es refined specifically with a set of corresponding synthetic peptides. The methods combined hi,oh resolution high-performance liquid chromatography a nd a new mass spectrometric procedure for the discrimination between Asp- a nd isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstr ate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in additi on to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L -Asp and D-Asp isomers argues for an effective action of PIMT present in ca rdiac tissue.