PubMed:8288564 JSONTXT 19 Projects

Enzymatic methylation of recombinant heterogeneous nuclear RNP protein A1. Dual substrate specificity for S-adenosylmethionine:histone-arginine N-methyltransferase. 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.