• Bulletin of the Korean Chemical Society
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• 제26권10호
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• pp.1611-1613
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• 2005

• Archives of Pharmacal Research
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• 제26권12호
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• pp.1047-1054
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• 2003

Molecular chaperones have a crucial role in the folding of nascent polypeptides in endoplasmic reticulum. Some of them are known to be sensitive to the modification by electrophilic metabolites of organic pro-toxicants. In order to identify chaperone proteins sensitive to alkyators, ER extract was subjected to alkylation by 4-acetamido-4 -maleimidyl-stilbene-2,2 -disulfonate (AMS), and subsequent SDS-PAGE analyses. Protein spots, with molecular mass of 160, 100, 57 and 36 kDa, were found to be sensitive to AMS alkylation, and one abundant chaperon protein was identified to be protein disulfide isomerase (PDI) in comparison with the purified PDI. To see the reactivity of PDI with cysteine alkylators, the reduced form ($PDI_{red}$) of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at $38^{\circ}C$ at pH 6.3, and the remaining activity was determined by the insulin reduction assay. Iodoacetamide or N-ethylmaleimide at 0.1 mM remarkably inactivated $PDI_{red}$ with N-ethylmaleimide being more potent than iodoacetamide. A partial inactivation of $PDI_{oxid}$ was expressed by iodoacetamide, but not N-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone ($IC_{50}, 15 \mu$ M) was the most potent, followed by 4-hydroxy-2-nonenal and 1,4-naphthoquinone. In contrast, 1,2-naphthoquinone, devoid of a remarkable inactivation action, was effective to cause the oxidative conversion of $PDI_{red}$ to $PDI_{oxid}$. Thus, the action of Michael acceptor compounds differed greatly depending on their structure. Based on these, it is proposed that POI, one of chaperone proteins in ER, could be susceptible to endogenous or xenobiotic Michael acceptor compounds in vivo system.

• Archives of Pharmacal Research
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• 제24권5호
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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.

• 대한화학회지
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• 제39권7호
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• pp.564-571
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• 1995

NaOH 수용액에서 과산화수소로 2-thiobiuret를 산화성 고리화 반응시켜 5-amion-2H-1, 2, 4-thiadiazoline-3-one을 합성하였다. 이 화합물에 대한 토오토머화 현상을 IR, $^1H$ NMR, $^13C$ NMR 등의 분광학적인 방법으로 고찰하고 분자괘도함수에 의한 이론적 계산 결과와 비교 검토하였다. 이 결과 5-Amion-2H-1, 2, 4-thiadiazoline-3-one는 락팀형보다는 락탐형으로 존재한다. 알킬화 반응을 여러 염기로 DMF 용매와 이상용매계 $H_2O$-THF에서 시도한 결과 N-2 위치에서 알킬화 반응이 일어남을 알 수 있었다. 알킬화 반응은 할로켄화 알칸을 $Li_2CO_3$/DMF의 조건하에서 반응시켰을 때 가장 수율이 좋았다. N-2 위치에서 알킬화 반응 생성물의 확인은 IR, $^1H$ NMR과 $^13C$ NMR 등의 분광학적인 방법과 5-methyl-2-thiobiuret를 thiobiuret와 같이 산화성 고리화 반응을 통하여 5-amino-2-methyl-1,2,4-thiadiazolidine-3-one 표준 시료를 합성하여 알킬화 반응 생성물과 비교 확인하였다.