Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase.
Measurements of ammonia release provide the first direct evidence that calmodulin becomes extensively deamidated during incubations at 37 degrees C, pH 7.4 or pH 11. A stoichiometry of 0.5 mol of NH3 released/mol of calmodulin is observed after 2 h at pH 11 or after 8-9 days at pH 7.4. These treatments also increase the ability of calmodulin to serve as a substrate for the isoaspartate-specific protein carboxyl methyltransferase from bovine brain. The stoichiometries of methylation are highly correlated with the stoichiometries of ammonia release. Deamidation and increased methyl-accepting capacity also occur in parallel for seven other proteins (aldolase, bovine serum albumin, cytochrome c, lysozyme, ovalbumin, ribonuclease A, and triosephosphate isomerase) upon incubation at pH 11. However, in comparison to calmodulin, these other proteins show very little deamidation and increased methylation capacity following incubation at pH 7.4. Deamidation of calmodulin at pH 7.4 is unaffected by the addition of 10(-7) M Ca2+; however, at 4 X 10(-6) M Ca2+, the rate of deamidation is inhibited by approximately 70%. The Ca2+-protection effect is consistent with the suggestion (B. A. Johnson, N. E. Freitag, and D. W. Aswad, (1985) J. Biol. Chem. 260, 10913-10916) that deamidation occurs preferentially at Asn-60 and/or Asn-97, each of which resides in a distinct Ca2+-binding domain.
Johnson
B A
BA
School of Biological Sciences, University of California, Irvine 92717.
Shirokawa
J M
JM
Aswad
D W
DW
D000641:Ammonia / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D011496:Protein Methyltransferases / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D000641:Ammonia; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase