Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia

• Published in: Biochimica et biophysica acta Vol. 1199; no. 2; pp. 183 - 194
• Main Authors: Pech, Louis L., Nelson, David L.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 02.03.1994; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Calmodulin; Calmodulin - metabolism; Calmodulin N-methyltransferase; Cattle; Chromatography, Affinity; Chromatography, Gel; Cytosol - enzymology; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Methyltransferases - isolation & purification; Methyltransferases - metabolism; Paramecium; Paramecium tetraurelia - enzymology; Protein methylase III; Protein methylation; Substrate Specificity; Transferases; Trimethyllysine; EGTA, [ethylenebis(oxythylenenitrilo)] tetraacetic acid; S2, cytosolic extract from Paramecium; MML, monomethyllysine; Trimethyllysine; CaM, calmodulin; DML, dimethyllysine; AdoMet, S-adenosyl methionine; TML, trimethyllysine; DTT, dithiothreitol; EDTAm ethylnediaminetettraacetic acid; Calmodulin N-methyltransferase; SAH, S-adenosyl L-homocysteine; Paramecium; CaBP, calcium-binding protein; PMSF, phenylmethyl-sulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, TAME, N-α-p- tosyl- L-arginine methyl ester; Protein methylase III; Protein methylation; Hepes, N-[2-hydroxy-ethyl]piperazine- N′-[2-ethanesulfonic acid]; Tris, tris(hydroxymethyl)aminomethane; Calmodulin; MES, 2-[ N-morpholino]-ethanesulfonic acid; Protozoa; Purification; Calcium; Enzyme; Paramecium tetraurelia; Transferases; Binding protein; Methyltransferases; Ciliata; Kinetic parameter; Methylation; Physicochemical properties; Calmodulin-lysine N-methyltransferase
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(94)90114-7

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 K m for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 μM 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. Ca 2+, Mg 2+, Mn 2+, and Ni 2+ stimulated calmodulin N-methyltransferase activity but Zn 2+ 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 out 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.