title: Purification and characterization of S-adenosylmethionine-protein-arginine N-methyltransferase from rat liver.
abstract: A protein methylase I (S-adenosylmethionine-protein-arginine N-methyltransferase; EC 2.1.1.23), with a high specificity for recombinant heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1, was purified from rat liver. The purification method is simple and rapid; a single initial step of DEAE-cellulose DE-52 chromatography resulted in a 114-fold enrichment from the cytosol, and subsequent Sephadex G-200 chromatography and f.p.l.c. yielded a homogeneous preparation. Ouchterlony double-immunodiffusion analysis indicated that the rat liver enzyme is immunologically different from an analogous enzyme from the calf brain, nuclear protein/histone-specific protein methylase I [Ghosh, Paik and Kim (1988) J. Biol. Chem. 263, 19024-19033; Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1075-1082]. The purified enzyme has a molecular mass of 450 kDa on Superose chromatography and 110 kDa on SDS/PAGE, indicating that it is composed of four identical-size subunits. The Km values for protein A1 and S-adenosyl-L-methionine were 0.54 x 10(-6) and 6.3 x 10(-6) M respectively. S-Adenosyl-L-homocysteine and sinefungin were effective inhibitors of the enzyme with Ki values of 8.4 x 10(-6) M and 0.65 x 10(-6) M respectively. Bivalent metal ions such as Zn2+, Mn2+ and Ni2+ were particularly toxic to the enzyme; at 1 mM Zn2+, 99% of the activity was inhibited. In addition, 50% of the enzyme activity was lost by treatment with 0.12 mM p-chloromercuribenzoate, indicating a requirement for a thiol group for enzyme activity. Glycerol, a compound often used to prevent enzyme inactivation, inhibited over 80% of the activity when present in the reaction mixture at a concentration of 20%.
authors.affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140.
authors.forename: N
authors.initials: N
authors.lastname: Rawal
authors.forename: R
authors.initials: R
authors.lastname: Rajpurohit
authors.forename: W K
authors.initials: WK
authors.lastname: Paik
authors.forename: S
authors.initials: S
authors.lastname: Kim
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008745:Methylation; D008970:Molecular Weight; D011484:Protein-Arginine N-Methyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
chemical_list: D011484:Protein-Arginine N-Methyltransferases; D005990:Glycerol