Purification and properties of calmodulin-lysine N-methyltransferase from rat brain cytosol.
A S-adenosylmethionine:protein-lysine N-methyltransferase (EC 2.1.1.43) has been purified from rat brain cytosol 7,080-fold with a yield of 8%, using octopus calmodulin as a substrate. It contains a lysine residue that is not fully methylated. The enzyme was purified by ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and phosphocellulose and octopus calmodulin-Sepharose affinity chromatographies. Among protein substrates, it was highly specific toward octupus calmodulin. The Km values for octopus calmodulin and S-adenosyl-L-methionine were found to be 2.2 X 10(-8) M and 0.8 X 10(-6) M, respectively. The molecular weight was estimated to be 57,000 by gel filtration and the pH optimum was between 7.5 and 8.5. The enzyme was stimulated in the presence of 10(-7) M Mn2+ and 10(-4) M Ca2+. HPLC of the acid hydrolysate of methyl-3H-labeled calmodulin showed the formation of epsilon-N-mono, epsilon-N-di, and epsilon-N-trimethyllysine. Reverse-phase HPLC of tryptic peptides of the methyl-3H-labeled calmodulin demonstrated that the labeled N-methyllysine lies in the 107-126 peptide. These findings suggest that this enzyme methylated a specific lysine residue of octopus calmodulin.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000378:metabolism; D011232:Chemical Precipitation; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D049831:Octopodiformes; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D002147:Calmodulin; D008780:Methyltransferases; C038999:calmodulin-lysine methyltransferase; D008239:Lysine