Abstract
We have previously reported the existence of two different molecular species of protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase, E.C. 2.1.1.23) in calf brain, one specific for myelin basic protein and the other for histone (Ghosh, S. K., Paik, W. K., and Kim, S. (1988) J. Biol. Chem. 263, 19024-19033). In the present study, however, we report that heterogeneous ribonucleoprotein particle protein A1 is most likely an in vivo substrate for the "histone-specific protein methylase I." The unmethylated recombinant protein A1 has been found to be a much superior methyl acceptor for the enzyme than histone with a Km value two orders of magnitude lower (0.19 microM) than that for histone (21 microM). Myelin basic protein, a specific inhibitor for histone protein methylase I, exhibited a lower IC50 for protein A1 methylation (IC50 = 33 microM) compared with histone methylation (IC50 = 220 microM) and competitively inhibited the former with a Ki value of 1.3 x 10(-6) M. The extent of inhibition of protein A1 and histone methylation by the polyclonal antibodies prepared against purified "histone protein methylase I" was identical. Maximally, 1.08-mol methyl groups were incorporated per mol of protein A1, which was 27-fold higher than that of histone (0.04 mol/mol of histone). HPLC analysis of the enzymatically methylated amino acid residues in protein A1 revealed the formation of NG-monomethylarginine and NG,NG-dimethylarginine. The ratio of NG,NG-dimethylarginine/NG-monomethylarginine increased as a function of incubation period; however, NG,N'G-dimethylarginine was not detectable. Proteolytic cleavage of the methyl-3H-labeled recombinant protein A1 by trypsin and Staphylococcus aureus V8 endoprotease indicated that protein A1 possesses multiple sites for methylation, one of which was identified as residue 194 arginine, which coincided with the in vivo methylation site.
Highlights
From the Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philndelphia, Pennsylvania 19140
We have previously reported the existence of two A large number of naturally occurring proteins have been different molecular species of protein methylase I (S- shown to undergo in vivo post-translational methylation on adenosy1methionine:protein-arginineN-methyltrans- some of the amino acid side chains such as lysine, arginine, ferase, E.C. 2.1.1.23) in calf brain, one specific for histidine, and aspartic/glutamic acid [1, 2]
Myelin basic protein(MBP)’ [3, 4], heterogeneous nuclear we report thatheterogeneous ribonucleoprotein parti- ribonucleoprotein particle proteins [5,6,7], sclerocle protein A 1 is most likely an in vivo substrate for derma antigen[8],chromosomal high mobility group proteins the “histone-specificprotein methylase I.”The unmeth- [9],and muscle proteins [10]
Summary
Materials-S-Adenosyl-~-[methyl-~H]methionine(specific activity, 78.5 Ci/mmol) and S-adenosyl-~-[methyl-"C]methionin(sepecific activity, 47 mCi/mmol) were obtained from DuPont NEN. Partial Digestion of [Methyl-3HlProtein A1by V8 Endoprotease and Trypsin-Mild proteolytic digestion of [rnethyl-3H]protein A1 (19.8 pg, 0.67 mol of methyl/mol of protein) was carried out in 40 pl of 50 mM Tris-HC1 buffer (pH 8.0) in the presence of 0.2 M NaCl, 1 mM phenylmethylsulfonyl fluoride, I mM EDTA, 10 mM NapS20a, and pepstatin A (1 pg/ml) as described by Kumar et al [23] by S. aureus V8 protease (1:200,1:100, and 1:50) at 22 "C for 10 min, or TPCK-treated trypsin (1:2000, 1:1000, and 1:500) at 0 "C for 5 min. A 50-pl aliquot was mixed with 25 pl of 3 X Laemmli's sample buffer and subjected to SDS-PAGE, while the other 50-p1 aliquot wasused for identification of the released amino acid as pletely inert as a substrate for the histone-specific protein methylase I and inhibited the histone methylation [25]. Inhibition of protein A1 methylation by MBP was competitive with a Ki value of 1.3 PM (Fig. 3,inset)
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