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Keywords:

  • Calmodulin-lysine N-methyltransferase;
  • Octopus calmodulin;
  • Rat brain cytosol;
  • Purification)

Abstract: A S-adenosylmethionineiprotein-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 × 10−8M and 0.8 × 10−6M, 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 10−7M Mn2+ and 10−4M Ca2+. HPLC of the acid hydrolysate of methyl-3H-labeled calmodulin showed the formation of ε-N-mono-, ε-N-di-, and ε-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.