HPLC analysis of methylated amino acids: Methylated amino acids on HPLC

Park, Kwang Sook; Hong, Sungyoul; Lee, Hyang Woo; Kim, Sangduk; Paik, Woon Ki

doi:10.1007/bf02857700

Cited by 6 publications

(2 citation statements)

References 16 publications

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“…The enzymically methyl-14C-labelled myelin membrane was first extracted with 0.2 M-H2S04 as described above and hydrolysed in 6 M-HCI at 110°C for 24 h. Amino acid analysis of the hydrolysate was performed on a Waters h.p.l.c. apparatus as described previously (Park et al, 1986), by using the o-phthaldialdehyde derivative-formation method. Fractions (1.0 min) were collected and counted for radioactivity with ACS II scintillator.…”

Section: Amino Acid Analysis Of Methyl-'4c-labelled Proteinmentioning

confidence: 99%

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Enzymic methylation of myelin basic protein in myelin

Ghosh

Rawal

Syed

et al. 1991

Biochemical Journal

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Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition.

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Section: Amino Acid Analysis Of Methyl-'4c-labelled Proteinmentioning

confidence: 99%

“…eluted between 14 and 16 min. Some 3000 of MMeArg (15 min) and 600 of DiMeArg (16 min) were estimated on the basis of the retention time of authentic methylated compounds (Park et al, 1986).…”

Section: Identification Of the Methyl-'4c-labelled Myelin Protein Andmentioning

confidence: 99%

Enzymic methylation of myelin basic protein in myelin

Ghosh

Rawal

Syed

et al. 1991

Biochemical Journal

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In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

et al. 1992

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We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) from Euglena gracilis [J. Biol. Chem., 260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as epsilon-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as the in vitro substrate. Presently, histone H1 has been purified from Euglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. The Euglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higher Rf value than the other histones H1 in acid/urea gel electrophoresis. When the Euglena histone H1 was [methyl-3H]-labeled in vitro by a homologous enzyme (one of the two Euglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C18 columns on HPLC, we demonstrated that Euglena histone H1 contains approximately 9 mol% of epsilon-N-methyllysines (1.40, 1.66, and 5.62 mol% for epsilon-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of epsilon-N-methyllysines in histone H1.

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Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

1993

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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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HPLC analysis of methylated amino acids: Methylated amino acids on HPLC

Cited by 6 publications

References 16 publications

Enzymic methylation of myelin basic protein in myelin

Enzymic methylation of myelin basic protein in myelin

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

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