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KMID : 0043320030260121047
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Archives of Pharmacal Research
2003 Volume.26 No. 12 p.1047 ~ p.1054
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Identification of Alkylation-Sensitive Target Chaperone Proteins and Their Reactivity with Natural Products Containing Michael Acceptor
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Liu Xi-Wen
Sok Dai-Eun
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Abstract
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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 () of PDI was incubated with various alkylators containing Michael acceptor structure for 30 min at 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 with N-ethylmaleimide being more potent than iodoacetamide. A partial inactivation of was expressed by iodoacetamide, but not N-ethylmaleimide (NEM) at pH 6.3. Of Michael acceptor compounds tested, 1,4-benzoquinone ( 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 to . 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.
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KEYWORD
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PDI, Chaperone, Michael acceptor, Inactivation, Oxidative conversion, Quinone, 4-Hydroxy-2-nonenal
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