Enzymatic methylation of calmodulin in rat brain cytosol.
The methylation of brain peptides was investigated by incubating brain high speed supernatant fractions with S-adenosyl-L-[methyl-3H]methionine and resolving the radioactive peptide products by polyacrylamide gradient gel electrophoresis. Peptides of 17,000, 21,000, 29,000, and 34,000 daltons were methylated in vitro; the 17,000-dalton peptide had the same electrophoretic mobility as authentic calmodulin and showed the shift in electrophoretic mobility in the presence of EGTA which is characteristic of calmodulin. The identification of the 17,000-dalton peptide as calmodulin was confirmed by electrophoresis under nondenaturing conditions and by two-dimensional isoelectric focusing electrophoresis. The enzyme responsible for calmodulin methylation and its substrate, the putative desmethylcalmodulin, were separated from one another by chromatography on DEAE-cellulose. When this partially purified enzyme was incubated with the substrate-containing fraction, calmodulin was the only methylated product. A test tube assay for calmodulin N-methyltransferase was developed and validated by comparison with calmodulin methylation assessed by gel electrophoresis. Both procedures showed that calmodulin methyltransferase is inhibited by EGTA and stimulated by divalent cations, with manganese giving the highest activity. Similar values for ph optimum and kinetic constants were obtained when the electrophoresis and test tube assays were compared. A rapid chromatographic procedure for the separation of methylated lysines and arginines was developed and used to demonstrate that epsilon-trimethyllysine is the radioactive amino acid formed when calmodulin is methylated in vitro. The methyltransferse, which is distinguished from previously described methylase III enzymes by its divalent cation requirement and substrate specificity, is thus designated as S-adenosyl-L-methionine calmodulin lysine methyltransfease.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D012436:S-Adenosylmethionine; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002412:Cations; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D004533:Egtazic Acid; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C026647:calmodulin methyltransferase