• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.209-209
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• 1996

Muscarinic acetylcholine receptors contain two highly conserved tyrosine residues which are located within or at the extracellular border of the second transmembrane domain. These tyrosine residues are located at positions 82 and 85 of the sequence of the ml subtype of muscarinic receptors. In this wok, we studied the involvement of these two residues in ligand binding to and agonist-induced activation this receptor subtype. our data suggest an important role for these two tyrosines in these processes, with a more prominent role for the tyrosine residue located at position 82 than that located at position 85. Evidence is also provided that while the aromatic moiety of these tyrosine residues is important for antagonist binding, both this moiety and the tyrosine phenolic hydroxyl group are involved in agonist binding and receptor activation.

• Archives of Pharmacal Research
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• v.24 no.5
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• pp.446-454
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• 2001

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-car-boxymethylated peptide derivative, which was further hydrohysed by endoproteinase-Asp-N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase-Asp-N andthe mass spectra data, a peptide fragment turned out to correspond to a peptide, DALCCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

• Archives of Pharmacal Research
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• v.18 no.6
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• pp.371-375
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• 1995

Erythropoietin (EPO), a glycoprotein hormone, regulates the proliferation and differentiation of ereythroid progenitor cells. Many attempts have been made to identify the functionally important amino acids of the hormone. One of those early studies has found that heavy redioiodination of EPO caused the loss of its biological activity, suggesting some important role of one of the four tyrosine residues (Goldwasser, 1981). Thus, in this study, we have generated and tested four $Tyr{\dashrightarrow}Phe$ substitution mutants to clarify the possible role of the tyrosine residue(s) in the hormone's Tyrosine residue(s) in the hormone's biological activity. When the mutant and wild type EPO cDBAs were transfected into COS-7 cells and the biological activities of the muteins were assayed using the primary murine erythroid spleen cells, no mutation tested was found to affect the biological activity of the hormone. Thus we conclude that, contrary to the previous observation, none of the four tyrosine in eryghropoietin is critically involved in the binding of the hormone to its receptor.

• BMB Reports
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• v.45 no.5
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• pp.317-322
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• 2012

2-deoxy-D-glucose(2DG)-caused endoplasmic reticulum (ER) stress inhibits protein phosphorylation at tyrosine residues. However, the accurate regulatory mechanisms, which determine the inflammatory response of chondrocytes to ER stress via protein tyrosine phosphorylation, have not been systematically evaluated. Thus, in this study, we examined whether protein phosphorylation at tyrosine residues can modulate the expression and glycosylation of COX-2, which is reduced by 2DG-induced ER stress. We observed that protein tyrosine phosphatase (PTP) inhibitors, sodium orthovanadate (SOV), and phenylarsine oxide (PAO) significantly decreased expression of ER stress inducible proteins, glucose-regulated protein 94 (GRP94), and CCAAT/ enhancer-binding-protein- related gene (GADD153), which was induced by 2DG. In addition, we demonstrated that SOV and PAO noticeably restored the expression and glycosylation of COX-2 after treatment with 2DG. These results suggest that protein phosphorylation of tyrosine residues plays an important role in the regulation of expression and glycosylation during 2DG-induced ER stress in rabbit articular chondrocytes.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.302-307
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• 2011

Native chitin deacetylase of Aspergillus nidulans was purified to apparent homogeneity by a combination of phenyl-Sepharose and Q-Sepharose column chromatography. In order to analyze the amino acid residues involved in the enzyme activity, the enzyme was chemically modified with chemical agent, which selectively reacted with the specific amino acid residue on the protein. When the enzyme was chemically modified with diethylpyrocarbonate, which specifically reacted with histidine residues on the protein, the activity was eliminated. The chitin deacetylase, chemically modified with 100 ${\mu}M$ modifier at the residue of arginine or tyrosine, has shown to have decreased activities. It was shown that the modification at aspartic acid or glutamic acid did not affect the enzyme activity to a greater extent, which would not implicate that acid amino residues were directly involved in catalytic reaction and would affect on the global structures of the proteins. This results demonstrated that histidine and tyrosine residues of enzyme would participate in an important function of the chitin deacetylase activity.

• Journal of the Korean Chemical Society
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• v.14 no.2
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• pp.155-160
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• 1970

The kinetics of the pepsin-catalyzed hydrolysis of N-carbobenzoxy-L-glutamyl-L-tyrosine at pH 3.5 and $37^{\circ}C$ were determined by a spectrophotometric technique. The pepsin used was further purified on a Sephadex G-75 column. The kinetics data were Km = l.7 ${\times}10^{-3}M,\;-{\Delta}F^{\circ}$ = 3.99Kcal/mole, and $k^3=\;2.1{\times}10^{-2}\;sec^{-1}$. An analysis of the above data and other investigators' data obtained from some dipeptides led to the following conclusions. (1) Phenylalanyl residues in a synthetic peptide are bound to pepsin more strongly than glutamyl or tyrosyl residues, supporting the theory that a part of the binding region of the active center is hydrophobic. (2) Dipeptides are bound to pepsin principally through their side chains and the binding involves both side-chain residues. (3) The nature of amino acids in dipeptides $R_2-R_1,\;affect\;the\;k_3$ values.

• BMB Reports
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• v.34 no.1
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• pp.33-38
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• 2001

The human PTK6 (also known as Brk) polypeptide, which is deduced from its full-length cDNA, represents a non-receptor protein tyrosine kinase (PTK). It contains SH3, SH2, and tyrosine kinase catalytic domains that are closely related to Src family members. We generated an antihuman PTK6 antibody by immunizing rabbits with a PTK6-specific oligopeptide conjugated to BSA, which corresponds to 11 amino acid residues near the C-terminus. An immunoblot analysis with the antibody detected an expected 52-kDa band in various mammalian transformed cell lines. Immunoprecipitation and immunoblot analyses demonstrated that PTK6 is phosphorylated on the tyrosine residues) and interacts with approximately a 23-kDa tyrosine-phosphorylated polypeptide (most likely a substrate of PTK6) in breast carcinoma T-47D cells. An immunofluorescence analysis demonstrated that PTK6 is localized throughout the cytoplasm of T-47D cells. These results support a possible role for PTK6 in the intracellular signal transduction through tyrosine phosphorylation.

• Journal of Life Science
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• v.9 no.2
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• pp.44-48
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• 1999

The ionization of tyrosine residue is known to be involved in the stabilization of transition-state in catalysis of astacin based upon the astacin-transition state analog structure. Two tyrosine residues, Tyr-216 and Tyr-219, are conserved in all MMPs related with astacin family, We replaced Tyr-216 and Tyr-219 into phenylalanine, respectively and the zinc binding properties, kinetic parameters, and pH dependence of each mutant are determined in order to examine the role of tyrosine residue in matrilysin catalysis. Both mutants contain two zinc atoms per mol of enzyme, indicating that either tyrosime does not affect the zinc binding property of the enzyme. Y216F and Y219F mutants are highly active and the kcat/Km values are only decreased 1.1-1.5-fold compared to the wild-type enzyme. The decrease in the activity of the mutants is essentially due to the increase in Km value. The pH dependencies of the kcat/Km values for both mutants are similar to the corresponding dependencies obtained with the wild type enzyme. The pKa values at the alkaline side of both mutants are not changed. These kinetic and pH dependence results indicate that the ionization of active site tyrosine residue of matrilysin is not reflected in the kinetics of peptide hydrolysin as catalyzed by astacin.

• Bulletin of the Korean Chemical Society
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• v.25 no.5
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• pp.711-714
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• 2004

Peroxynitrite enters erythrocytes through band 3 anion exchanger and oxidizes cytosolic proteins therein. As a protein associated with band 3, carbonic anhydrase II may suffer from peroxynitrite-induced oxidative damages. Esterase activity of carbonic anhydrase II decreased as the concentration of peroxynitrite increased. Neither hydrogen peroxide nor hypochlorite affected the enzyme activity. Inactivation of the enzyme was in parallel with the release of zinc ion, which is a component of the enzyme's active site. SDS-PAGE of peroxynitrite-treated samples showed no indication of fragmentation but non-denaturing PAGE exhibited new bands with lower positive charges. Western analysis demonstrated that nitration of tyrosine residues increased with the peroxynitrite concentration but the sites of nitration could not be determined. Instead MALDI-TOF analysis identified tryptophan-245 as a site of nitration. Such modification of tryptophan residues is responsible for the decrease in tryptophan fluorescence. These results demonstrate that peroxynitrite nitrates tyrosine and tryptophan residues of carbonic anhydrase II without causing fragmentation or dimerization. The peroxynitrite-induced inactivation of the enzyme is primarily due to the release of zinc ion in the enzyme's active site.

• Journal of Microbiology
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• v.35 no.4
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• pp.300-308
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• 1997

The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.