Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin.
We have previously shown that the D-aspartyl/L-isoaspartyl protein carboxyl methyltransferase recognizes two major sites in affinity-purified preparations of bovine brain calmodulin that arise from spontaneous degradation reactions. These sites are derived from aspartyl residues at positions 2 and 78, which are located in apparently flexible regions of calmodulin. We postulated that this flexibility was an important factor in the nonenzymatic formation and enzymatic recognition of D-aspartyl and/or L-isoaspartyl residues. Because removal of Ca2+ ions from this protein may also lead to increased flexibility in the four Ca2+ binding regions, we have now characterized the sites of methylation that occur when calmodulin is incubated in buffers with or without the calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,-N',N'-tetraacetic acid (EGTA). Calmodulin was treated at pH 7.4 for 13 days at 37 degrees C under these conditions and was then methylated with erythrocyte D-aspartyl/L-isoaspartyl methyltransferase isozyme I and S-adenosyl-L-[methyl-3H]methionine. The 3H-methylated calmodulin product was purified by reverse-phase HPLC and digested with various proteases including trypsin, chymotrypsin, endoproteinase Lys-C, clostripain, and Staphylococcus aureus V8 protease, and the resulting peptides were separated by reverse-phase HPLC. Peptides containing Asp-2 and Asp-78, as well as calcium binding sites II, III, and IV, were found to be associated with radiolabel under these conditions. When calmodulin was incubated under the same conditions in the presence of calcium, methylation at residues in the Ca2+ binding regions was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Clarke
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D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004533:Egtazic Acid / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D011487:Protein Conformation; D011496:Protein Methyltransferases / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000378:metabolism*
D002147:Calmodulin; D010446:Peptide Fragments; D001224:Aspartic Acid; D004533:Egtazic Acid; D001216:Asparagine; D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase; D010447:Peptide Hydrolases; D002118:Calcium