• BMB Reports
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• v.52 no.3
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• pp.163-164
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• 2019

The ribosomal synthesis of proteins in the eukaryotic cytosol has always been thought to start from the unformylated N-terminal (Nt) methionine (Met). In contrast, in virtually all nascent proteins in bacteria and eukaryotic organelles, such as mitochondria and chloroplasts, Nt-formyl-methionine (fMet) is the first building block of ribosomal synthesis. Through extensive approaches, including mass spectrometric analyses of the N-termini of proteins and molecular genetic techniques with an affinity-purified antibody for Nt-formylation, we investigated whether Nt-formylated proteins could also be produced and have their own metabolic fate in the cytosol of a eukaryote, such as yeast Saccharomyces cerevisiae. We discovered that Nt-formylated proteins could be generated in the cytosol by yeast mitochondrial formyltransferase (Fmt1). These Nt-formylated proteins were massively upregulated in the stationary phase or upon starvation for specific amino acids and were crucial for the adaptation to specific stresses. The stress-activated kinase Gcn2 was strictly required for the upregulation of Nt-formylated proteins by regulating the activity of Fmt1 and its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins could be distinct N-terminal degradation signals, termed fMet/N-degrons, and that Psh1 E3 ubiquitin ligase mediated the selective destruction of Nt-formylated proteins as the recognition component of a novel eukaryotic fMet/N-end rule pathway, termed fMet/N-recognin.

• Applied Biological Chemistry
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• v.6
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• pp.107-117
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• 1965

(1) In order to study the specificity of Aspergillus soya protease to soybean protein, as well as the types of peptides formed during soybean-koji prerapation the amino acid sequence for the di & tripeptide and N-terminal amino acid residue and C-terminal amino acid residue were identified. As the results of the study, the following were obtained. Gly, Glu. Ala. Ser. Glu. Ser. Ala. Val (Cys, Glu, Ser, Ala, Arg, Try, Leu or Ileu) Asp. Phe (His, Arg, Cys, Asp, Ser, Ala, Leu or Ileu) Glu. Ala (Cys, Gly, Met) Glu. Ala (Asp, Glu,) Gly. Met (Asp, Glu, Ala, Tyr, Leu or Ileu, Lys,) Gly. Leu or Ileu (His, Asp, Glu, Gly, Ser, Lys, Thr, Phe,) Cys. Gly (Asp, Tyr,) Glu. Pro (Asp, Glu, Ser, Gly, Thr, Ala, Val, Leu or Ileu) Try. Ser (Gly, Glu, Arg, Ala, Met, Leu or Ileu,) Asp. Met (Asp, Glu, Ala, Try, Pro, Leu or Ileu,) His Thr (Ser, Gly, Tyr, Pro, Leu or Ileu,) Glu. Gly (Asp, Ala, Ser, Glu,) Leu or Ileu (2) It has revealed that Aspergillus soya protease has considerably wider range of specificity than that of chymotrypsin, pepsin and trypsin but not mold protease and Aspergillus saitoi protease. It can be said that Asp. soya protease split the bond adjacent to glutamic acid, aspartic acid, glycine, serine, alanine, cystine, tryptophan, histidine preferably acidic amino acid as C-terminal amino acid residue.

• BMB Reports
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• v.35 no.2
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• pp.228-235
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• 2002

Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAE-Sepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala-Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at $80^{\circ}C$, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at $80^{\circ}C$ for 45 min. The Km and Vmax values of the enzyme were 3.0mM and 1.7 mmol/min/mg, respectively. The B. stearothernmophilus MetAP was completely inactivated by EDTA and required $Co^{2+}$ ion(s) for activation, suggesting the metal dependence of this enzyme.

• The Korean Journal of Food And Nutrition
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• v.4 no.2
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• pp.161-166
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• 1991

The blocked N-terminus and N-terminal sequence of soybean B-amylase were aetermined by analyzing the acidic peptides derived on peptic digestion of the enzyme. The acidic peptides were separated from the digest on a Dowex 50$\times$2 column(1X5cm) and purified by reversed phase-high performance liquid chromatography(RP-HPLC). The major acidic peptide, PEP-1, was a heptapeptlde. The N-terminal 7 amino acid sequence of soybean B-amylase was deduced to be acetyl-Ala-Thf-Ser-Asp-Ser-Asn-Met- from the results of sequence analysis of PEP-1 and amino acid analysis of other acidic peptides.

• Applied Biological Chemistry
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• v.15 no.2
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• pp.111-142
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• 1972

An experimental Chung-Kook-Jang was prepared using the strain Bacillus subtilis sp. isolated by the author Samples were taken in 12 hrs interval during the fermentation and the oligopeptides were separated by the method of molecular sieving using the ion exchange resin column of Dowex-50. Only the X-16 fraction of oligopeptides was taken and the components of oligopeptides were developed in two dimensional thin layer chromatograms. The each peptide spot was eluted and each peptide was isolated. The pattern and kinds of amino acids, and N and C-terminal amino acids were studied. Fourteen different oligopeptides could be detected by the two dimensional thin layer chromatography, all of which were consisted of $4{\sim}9$ kinds of amino acids. No dipeptides and no tripeptides could be found. The N and C-terminal amino acids and the residual component amino acids of all these 14 peptides could be summarized as the follows. [P]-I. Pro (Cys Ala Asp Trp Ile Val) Glu [P]-II. Val (His Arg Glu Thr Ala Met) Asp [P]-III. Glu (Cys Lys Asp Thr Met) Ala [P]-IV. Glu(His Ser Ala) Met) [P]-V. Ile (Cys Asp Arg Gly Pro T.p Phe) His [P]-VI. Gly(Asp ser) Lys [P]-VII. Thr(Pro Tyr Phe) Asp [P]-VIII. Phe(Tyr Leu Ile) Val [P]-IX. Trp (Phelle) Thr [P]-X. Ile (Arg Leu) Phe [P]-XI. Asp (Lys His Ser Gly Glu Pro) Ala [P]-XII. Glu (Cys Asp Gly) Ser [P]-XIII. Ala (Arg Tyr) Glu [P]-XIV. Met (Glu Ala) His It appears that the protease of the Bacillus subtilis K-27 syrain has rather wider range of specificity than proteases of Aspergoillus soya, pepsin, chymotrypsin, and trypsin.

• Journal of the Korean Magnetic Resonance Society
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• v.9 no.2
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• pp.110-121
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• 2005

E.coli methionyl tRNA synthetase consist of 676 amino acids and plays a key role in initiation of protein synthesis. The native form of this enzyme is a homodimer, but the monomeric enzyme truncated approximately C-terminal 120 amino acids retains the full enzymatic activities. X-ray crystal structure of the active monomeric enzyme shows that it has two domains. The N-terminal domain is thought to be a binding site for acceptor stem of tRNA, ATP, and methionine. The C-terminal domain is mainly a-helical and makes an interaction with the anticodon of $tRNA^{Met}$. Especially it is suggested that the region of helix-loop-helix including the tryptophan residue at the position 461 may be the essential for the interaction with anticodon of $tRNA^{Met}$. In this work the structure and function of E. coli methionyl-tRNA synthetase was studied by spectroscopic method (NMR, CD, Fluorescence). The importance of tryptophan residue at the position 461 was investigated by fluorescence spectroscopy. Tryptophan 461 is expected to be an essential site for the interaction between E. coli methionyl-tRNA synthetase and E. coli $tRNA^{Met}$. Proton and heteonuclear 2-dimensional NMR spectroscopy were also used to elucidate the protein-tRNA interaction.

• KSBB Journal
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• v.9 no.2
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• pp.165-173
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• 1994

In this paper we have described the structural characterization of recombinant bovine somatotropin produced in Escherichia coli. Recombinant bovine somatotropin consists of 191 amino acid residues with a calculated molecular weight of 21,802 Da. For fragmentation of recombinant bovine somatotropin, we have used trypsin, Staphylococcus aureus V8 pretease, CNBr, and mild acid hydrolysis method. Digestion and cleavage with these proteases and chemicals yielded peptides of various size for amino acid sequence determination. The N-terminal sequence analysis was carried out up to thirty residues. Because the design of the recombinant bovine somatotropin gene for expression was such that the coding sequence begins with an initiation codon, AUG, before Ala, the first amino acid of bovine somatotropin, we could expect the initial amino acid as N-formyl Met. But the first amino acid of this protein, expressed in E. coli cells as inclusion bodies, was Ala. And the amino acid composition of RP-HPLC purified recombinant bovine somatotropin was determined and no essencial difference was observed. The amino acid sequence of the recombinant bovine somatotropin was identical to that predicted from its recombinant gene. There was no processing or replacement of amino acid residues in recombinant bovine somatotropin expressed in E. coli. The hydropathy plot of recombinant bovine somatotropin revealed a hydrophobic region at the NH2-terminus and hydrophilic region at the COOH-terminus. The E. coli expression system is thought to be valuable for the expression of recombinant bovine somatotropin because protein was processed to remove the N-terminal Met residue by methionyl-aminopeptidase autonomously.

• Molecules and Cells
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• v.45 no.3
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• pp.158-167
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• 2022

Ubiquitin (Ub) is post-translationally modified by Ub itself or Ub-like proteins, phosphorylation, and acetylation, among others, which elicits a variety of Ub topologies and cellular functions. However, N-terminal (Nt) modifications of Ub remain unknown, except the linear head-to-tail ubiquitylation via Nt-Met. Here, using the yeast Saccharomyces cerevisiae and an Nt-arginylated Ub-specific antibody, we found that the detectable level of Ub undergoes Nt-Met excision, Nt-deamination, and Nt-arginylation. The resulting Nt-arginylated Ub and its conjugated proteins are upregulated in the stationary-growth phase or by oxidative stress. We further proved the existence of Nt-arginylated Ub in vivo and identified Nt-arginylated Ub-protein conjugates using stable isotope labeling by amino acids in cell culture (SILAC)-based tandem mass spectrometry. In silico structural modeling of Nt-arginylated Ub predicted that Nt-Arg flexibly protrudes from the surface of the Ub, thereby most likely providing a docking site for the factors that recognize it. Collectively, these results reveal unprecedented Nt-arginylated Ub and the pathway by which it is produced, which greatly expands the known complexity of the Ub code.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.17-17
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• 2003

tRNA (m¹ G37) methyltransferase (TrmD) catalyze s the trans for of a methyl group from S-adenosyl-L-methionine (AdoMet) to G/sup 37/ within a subset of bacterial tRNA species, which have a residue G at 36th position. The modified guanosine is adjacent to and 3' of the anticodon and is essential for the maintenance of the correct reading frame during translation. We have determined the first crystal structure of TrmD from Haemophilus influenzae, as a binary complex with either AdoMet or S-adenosyl-L-homocysteine (AdoHcy), as a ternary complex with AdoHcy/phosphate, and as an apo form. The structure indicates that TrmD functions as a dimer (Figure 1). It also suggests the binding mode of G/sup 36/G/sup 37/ in the active site of TrmD and catalytic mechanism. The N-terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C-terminal domain shows a structural similarity to DNA binding domain of trp or tot repressor. We propose a plausible model for the TrmD₂-tRNA₂ complex, which provides insights into recognition of the general tRNA structure by TrmD (Figure 2).

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.218-223
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• 1998

Casein was hydrolyzed by alcalase to produce iron binding peptide (IBP). IBP was effectively separated from casein hydrolysates by immobilized $Fe^{3+}$ affinity chromatography and further purified by reverse phase chromatography. $25,\;50\;and\;100\;{\mu}g/mL$ of IBP solubilized $4.2,\;5.7\;and\;7.1\;{\mu}g$ of ferric at duodenum condition $(pH\;6,\;37^{\circ}C)$, respectively. According to the result of MALDI analysis, molecular weight of IBP was determined to 2,175 dalton. IBP was mainly composed of proline (24.5 mol%), lysine (15.7 mol%), and glutamine or glutamic acid (14.9 mol%) and its N-terminal sequence was Met-Ala-Pro-Lys-His. According to the information obtained from molecular weight, amino acids composition and N-terminal sequence of IBP, it was evident that IBP was from f102-119 of ${\beta}-casein$.