Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Basic fibroblast growth factor (bFGF) is a heparin-binding angiogenic polypeptide mitogen. Protein sequence analysis of bFGF isolated from tissue sources initially established that it is composed of 146 amino acids (apparent Mr 18,000). More recently larger apparent molecular weight forms have been identified and partially characterized. In addition, these high molecular weight forms (apparent Mr 22,000 and 25,000) have been shown to localize preferentially to nuclear fractions of transfected cells. In this report we demonstrate that the higher molecular weight, amino terminally extended forms of bFGF contain methylated arginine residues. The demonstration is based on 1) amino acid sequence analysis of a protein known to contain methylated arginine (myelin basic protein) and a comparison with amino acid sequence analysis of trypsin-derived fragments of the high molecular weight bFGF purified from guinea pig brain and 2) the ability to label in vivo the high molecular weight forms of bFGF with S-adenosyl-L-(methyl-3H)-methionine, the substrate of arginine-protein methylase I. These results are suggestive of a role of arginine methylation in directing nuclear localization of certain forms of bFGF.
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PMID:Direct evidence for methylation of arginine residues in high molecular weight forms of basic fibroblast growth factor. 171 85

CNBr treatment of calf thymus [methyl-14C]histone H4, methylated in vitro with S-adenosyl-L-[methyl-14C]methionine by a highly histone-specific wheat germ protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), produced two peptide fragments corresponding to residues 1-83 and 84-102, with the former being radioactive. Two-dimensional peptide mapping of the chymotryptic and tryptic digest of [methyl-14C]histone H4 and analysis of the chymotryptic digest on HPLC have shown that only a single peptide is radiolabeled. In order to define the exact site of methylation (arginine residue), the radioactive peptide from the chymotryptic digest of [methyl-14C]histone H4 was further purified on HPLC by linear and then isocratic elution. The purified chymotryptic peptide was then digested with trypsin and purified on HPLC, and its amino acid composition was determined on HPLC. These results indicate that the peptide corresponding to residues 24-35 of histone H4 is radiolabeled. Since this peptide contains a single arginine residue at position 35, we have concluded that the enzyme is specific not only to the protein substrate but also to the methylation site.
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PMID:Site specificity of histone H4 methylation by wheat germ protein-arginine N-methyltransferase. 371 61

The enzymatic methylation of polypeptides on the guanidino group of internal arginine residues by S-adenosylmethionine:protein arginine N-methyltransferase (protein methylase I) yields NG-monomethylarginine, NG,NG-dimethylarginine and NG,NG-dimethylarginine. It has commonly been observed that these arginine residues are present in glycine-and-arginine rich motifs. To understand structural features which are essential for serving as the methyl acceptor for protein methylase I, we have investigated substrate capacities of several synthetic oligopeptides whose sequences are homologous and/or analogous to the methyl acceptor region of the naturally occurring arginine-methylated proteins. These studies have led to the following conclusions. (i) The preferred amino-acid sequence of methyl-accepting peptides was shown to be an arginine-containing peptide with glycine in both the N- and C-flanking positions. While a tetrapeptide with such a sequence (residues 106-109 of bovine myelin basic protein) exhibited almost negligible substrate activity, an overlapping hexapeptide was a moderate substrate. (ii) Substitution of the C-flanking glycine in GKGRGL (residues 104-109 of myelin basic protein) with histidine, phenylalanine, lysine or aspartic acid completely abolished the ability of these hexapeptides to serve as substrates. (iii) A heptapeptide with a repeated glycine-arginine motif (GRGRGRG) was an excellent substrate for the enzyme. (iv) A cyclic octapeptide (CGKGRGLC), which was formed by cyclization of GKGRGL by introduction of disulfide bridge to cross-link N- and C-terminus of the hexapeptide, was an even better substrate than the hexapeptide. (v) Upon HPLC amino-acid analysis, all enzymatically methyl-14C-labeled oligopeptides were found to yield predominantly NG-monomethylarginine with a minor fraction of NG,NG-dimethylarginine in certain peptide samples. However, no NG,NG-dimethylarginine formation was detectable. (vi) The recombinant hnRNP protein A1 (residues 1-320) is known to be methylated at arginine-194 by nuclear-protein/histone protein methylase I (Rajpurohit et al. (1994) J. Biol. Chem. 269, 1079-1082). However, the hexapeptide (SSSQRG) which corresponds to residues 189-194 of protein A1 containing the methylatable arginine residue was relatively inert as a substrate. Furthermore, the N-terminal fragment of protein A1 (residues 1-196) generated by controlled trypsin digestion was also completely inactive as a substrate for the enzyme. These results indicate that the remainder of the A1 protein molecule plays an important though not yet understood role in enzymatic methylation of the arginine-194.
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PMID:Structural specificity of substrate for S-adenosylmethionine:protein arginine N-methyltransferases. 753 38

Recombinant unmethylated heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1 was enzymatically methylated by nuclear protein/histone protein methylase I [Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1057-1082] and the effect of methylation on several physiocochemical properties was studied. The relative binding-affinity of methylated and unmethylated protein A1 to nucleic acid was quite different. This was observed by the elution behaviour of the protein A1 on a single-stranded DNA/cellulose column; the concentration of NaCl required to release the bound protein A1 was 0.59 M for the methylated and 0.63 M for the unmethylated, respectively. Employing isoelectrofocusing, pI values of the methylated and unmethylated proteins were found to be 9.41 and 9.48, respectively. Maximum fluorescence quenching of protein A1 in the presence of coliphage MS2-RNA was found to be 40% with methylated and 45% with unmethylated. When both species of protein A1 were subjected to controlled trypsin digestion, t1/2 of the methylated protein was 1.31 min and the unmethylated, 1.63 min. The difference in their t1/2 values was much greater in the presence of MS2-RNA; 2.4 min for the former and 4.3 min for the latter, indicating that the methylated species was less stabilized by the RNA than the unmethylated. All of the above results consistently suggested that the binding-property of hnRNP protein A1 to single-stranded nucleic acid was significantly reduced subsequent to its arginine-methylation. The biological significance of this observation is discussed.
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PMID:Effect of enzymic methylation of heterogeneous ribonucleoprotein particle A1 on its nucleic-acid binding and controlled proteolysis. 781 96

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-L-[methyl-3H]methionine (8-N3-Ado[methyl-3H]Met), the binding sites for S-adenosyl-L-methionine(AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC 2.1.1.23); AdoMet:histone-arginine N-methyltransferase (EC2.1.1.23); and AdoMet:cytochrome c-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.
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PMID:Comparative studies on S-adenosyl-L-methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-L-methionine as photoaffinity probe. 814 3

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.
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PMID:Enzymatic methylation of recombinant heterogeneous nuclear RNP protein A1. Dual substrate specificity for S-adenosylmethionine:histone-arginine N-methyltransferase. 828 64

Heterogeneous nuclear RNP protein A1, one of the major proteins in hnRNP particle (precursor for mRNA), is known to be posttranslationally arginine-methylated in vivo on residues 193, 205, 217 and 224 within the RGG box, the motif postulated to be an RNA binding domain. Possible effect of NG-arginine methyl-modification in the interaction of protein A1 to nucleic acid was investigated. The recombinant hnRNP protein A1 was in vitro methylated by the purified nuclear protein/histone-specific protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase) stoichiometrically and the relative binding affinity of the methylated and the unmethylated protein A1 to nucleic acid was compared: Differences in their binding properties to ssDNA-cellulose, pI values and trypsin sensitivities in the presence and absence of MS2-RNA all indicate that the binding property of hnRNP protein A1 to single-stranded nucleic acid has been significantly reduced subsequent to the methylation. These results suggest that posttranslational methyl group insertion to the arginine residue reduces protein-RNA interaction, perhaps due to interference of H-bonding between guanidino nitrogen arginine and phosphate RNA.
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PMID:Recent advances in protein methylation: enzymatic methylation of nucleic acid binding proteins. 989 55