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标题：Conformational Change of Glyceraldehyde-3-phosphate Dehydrogenase Induced by Acetylleucine Chloromethyl Ketone is Followed by Unique Enzymatic Degradation
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作者：Mitsune Yamaguchi ; Yukihiro Tsuchiya ; Kayoko Hishinuma 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2003
卷号：26
期号：12
页码：1648-1651
DOI：10.1248/bpb.26.1648
出版社：The Pharmaceutical Society of Japan
摘要：We have previously reported that acetylleucine chloromethyl ketone (ALCK), an inhibitor of acylpeptidehydrolase, induces the inhibition and degradation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the U937 cell extract. In the present study, the process of ALCK-induced GAPDH degradation was investigated. A kinetic study revealed that GAPDH was irreversibly inhibited by ALCK. ALCK treatment induced a change in the signal intensity of GAPDH in the near-UV region of the circular dichroism (CD) spectrum, and the fluorescence intensity of GAPDH at 330 nm increased to about 10% when excited at 280 nm, suggesting that a significant conformational change of GAPDH was induced by ALCK. When the U937 cell extract was incubated with ALCK and the products were separated by SDS-polyacrylamide gel electrophoresis (PAGE), a 23-kDa fragment from GAPDH was detected by Western blotting using anti-GAPDH serum. When ALCK-treated GAPDH was incubated with protease fractions from the U937 cell extract, a 17-kDa fragment was also detected. Sequence analysis showed that the N-terminal amino acid sequence of the 23-kDa fragment was GKVKVG and that of 17-kDa fragment was RDGRGAL. Therefore, ALCK-modified GAPDH is deduced to be digested at the peptide bond Trp₁₉₅–Arg₁₉₆. The protease activity liberating a 23-kDa fragment from ALCK-treated GAPDH was effective under the basic condition. Results suggested that ALCK binds to GAPDH to modulate the conformation of enzyme, which is susceptible to chymotrypsin-like protease activity.
关键词：acetylleucine chloromethyl ketone (ALCK);glyceraldehyde-3-phosphate dehydrogenase;U937 cell extract;chymotrypsin-like protease