Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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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PMID:Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin. 250 76

Incubation of cytosolic extracts of bovine brain with S-adenosyl[methyl-3H]methionine results in the predominant [3H]methyl esterification of a 36-kDa polypeptide. This reaction appears to be distinct from any of the three known types of protein carboxyl methylation reactions previously established. We show here that the methylated 36-kDa polypeptide is a component of a cytosolic protein with a native molecular mass estimated at 178 kDa by gel filtration chromatography. The methyl group is not stable on the protein and is lost as [3H]methanol with a half-life of about 180 min at pH 7.0, 37 degrees C. The methyltransferase responsible for this reaction is a cytosolic protein with a native molecular mass of about 40 kDa that is readily separated from the well described protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77). The methyl ester linkage is cleaved by carboxypeptidase Y, suggesting that the 36-kDa polypeptide is methylated on its C-terminal carboxyl group. Extensive digestion of gel-purified 3H-methylated 36-kDa polypeptide with trypsin and leucine aminopeptidase results in a radioactive product that co-chromatographs with authentic L-leucine methyl ester in reverse phase high performance liquid chromatography (HPLC), thin layer chromatography, thin layer electrophoresis, and high resolution-sulfonated polystyrene cation-exchange chromatography. Additionally, the o-phthalaldehyde/beta-mercaptoethanol-derived isoindole derivative of the 3H digestion product co-migrates on HPLC with the corresponding isoindole for L-leucine methyl ester. We demonstrate that a similar methylation system is present in yeast Saccharomyces cerevisiae but not in the bacterium Escherichia coli. These results provide evidence for a new type of reversible posttranslational modification reaction that may function to modulate the activities of its methyl-accepting substrates.
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PMID:Methyl esterification of C-terminal leucine residues in cytosolic 36-kDa polypeptides of bovine brain. A novel eucaryotic protein carboxyl methylation reaction. 851 74

A previously unidentified endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain has been characterized and partially purified. This high mass methyl-accepting protein (HMAP) is concentrated in rat brain cytosol and is not detectable in rat liver, heart, lung, kidney, or skeletal muscle. HMAP is acidic and heterogeneous in size, with an average mass, as judged by size-exclusion high performance liquid chromatography, greater than 700 kDa. After partial purification from cow brain by anion-exchange chromatography, ammonium sulfate fractionation, and gel filtration, HMAP could accept 12.1 nmol of methyl groups per mg of protein, suggesting that it contains a level of isoaspartate at least 50 times greater than that of the average protein in brain cytosol. Partially purified HMAP is degraded by trypsin, verifying that it is composed, at least in part, of protein. Additional studies on this unusual macromolecule may shed important new light on mechanisms of isoaspartate formation in cells and the molecular pathology of brain aging.
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PMID:High mass methyl-accepting protein (HMAP), a highly effective endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain. 879 82