Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia.
Calmodulin (lysine 115) N-methyltransferase was purified from the cytosolic fraction of Paramecium tetraurelia by sequential dialysis, cellulose phosphate chromatography, Reactive Red 120 agarose chromatography, and calmodulin-Sepharose affinity chromatography. The enzyme was purified 6800-fold with a 15% yield. SDS-PAGE analysis of the purified enzyme invariably revealed a major protein of 37 kDa that was reproducibly obtained and minor proteins of 35 and 28 kDa that were sometimes obtained in variable yields. The enzyme formed a mixture of mono-, di-, and trimethyllysine residues at lysine 115 of calmodulin in vitro, had a Km for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 microM and a pH optimum of about 7.5. The purified enzyme had an absolute requirement for the reductant DTT for activity, whereas the enzyme in crude fractions did not. The enzyme is a monomer with an estimated molecular mass of 33 kDa. Ca2+, Mg2+, Mn2+, and Ni2+ stimulated calmodulin N-methyltransferase activity but Zn2+ did not. Calmodulin N-methyltransferase was inhibited by its reaction product S-adenosyl homocysteine (SAH), but not by sinefungin and tubercidin. The calmodulin antagonists calmidazolium and mellitin were inhibitory but W7 was not. The enzyme was not stimulated by Triton X-100 nor by NaCl. Only calmodulins with an unmethylated lysine at residue 115, including cam2 calmodulin, were substrates. Histones and calcium-binding proteins from Paramecium other than calmodulin did not act as substrates for the purified calmodulin N-methyltransferase and no other substrates in the cytosolic fraction were observed.
Pech
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Department of Biochemistry, University of Wisconsin-Madison 53706.
Nelson
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D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D016805:Paramecium tetraurelia / Q000201:enzymology*; D013379:Substrate Specificity
D002147:Calmodulin; D008780:Methyltransferases; C026647:calmodulin methyltransferase