Importance of Disulfide Bridge Formation on Folding of Phospholipase D from Streptomyces antibioticus
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概要
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The effects of redox conditions on the folding of phospholipase D(PLD) of Streptomyces antibioticus were investigated. Although the enzyme was very stable even in the presence of 1.0M guanidinehydrochrolide(Gdn-HCl), the coexistence of dithiothreitol(DTT) and Gdn-HCl inactivated the enzyme completely. The inactivated enzyme recovered its activity by dialysis in which DTT was removed prior to Gdn-HCl, whereas its activity was not recovered when Gdn-HCl was removed prior to DTT. In vitro protein synthesis was used for further analyses of the folding process. Active PLD was synthesized in the absence of DTT. The activity increased as the protein synthesis proceeded. In contrast, inactive PLD was synthesized in the presence of DTT. The inactive PLD could not be effectively activated by simple removal of the reductant, while incubation with Gdn-HCl and subsequent removal of DTT followed by that of Gdn-HCl was a much more effective method for the synthesis of active enzymes. From these results, it is suggested that: (i) PLD contains disulfide bridge(s), which is(are) necessary for maintaining its tertiary structure, (ii) correct formation of the disulfide bridge(s) is a critical step in the early stage of the (re)folding process, and(iii) the disulfide bridge(s) further facilitate the folding process, resulting in the synthesis of the active enzymes with the correct structure.
- 社団法人日本生物工学会の論文
- 2000-05-25
著者
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Nakano Hideo
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya Universit
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KAWARASAKI YASUAKI
Laboratory of Molecular Biotechnology, Division of Molecular and Cellular Mechanisms, graduate Schoo
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YAMANE TSUNEO
Laboratory of Molecular Biotechnology, Division of Molecular and Cellular Mechanisms, graduate Schoo
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Iwasaki Yugo
Laboratory of Molecular Biotechnology, Department of Bioengineering Sciences, Graduate School of Bio
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Iwasaki Y
Lab. Of Molecular Biotechnology Dep. Of Bioengineering Sciences Graduate School Of Bioagricultural S
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Iwasaki Yugo
Laboratory Of Molecular Biotechnology Department Of Bioengineering Sciences Graduate School Of Bioag
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Iwasaki Yugo
Laboratory Of Molecular Biotechnology Graduate School Of Bio- And Agro-sciences Nagoya University
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Iwasaki Yugo
Department Of Applied Biological Sciences Faculty Of Agriculture Nagoya University
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Yamane T
Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University:
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Yamane Tsuneo
Laboraotory Of Bioreaction Engineering Department Of Food Science And Technology Faculty Of Agricult
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Yamane Tsuneo
Laboratory Of Bioreaction Engineering Department Of Food Science And Technology School Of Agricultur
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Yamane T
Laboratory Of Molecular Biotechnology Graduate School Of Biological & Agricultural Sciences Nago
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Yamane T
Laboratory Of Bioreaction Engineering Department Of Food Science And Technology School Of Agricultur
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Kawarasaki Yasuaki
Laboratory Of Molecular Biotechnology Division Of Molecular And Cellular Mechanisms Graduate School
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Nakano Hideo
Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University
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Yabuki Takashi
Protein Research Group Riken Genomic Sciences Center
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NISHIYAMA TATSUAKI
Laboratory of Molecular Biotechnology Graduate School of Biological and Agricultural Sciences, Nagoy
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Yamane Tsuneo
Dept. Of Food Science And Technology Faculty Of Agriculture Nagoya University
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Nishiyama Tatsuaki
Laboratory Of Molecular Biotechnology Graduate School Of Biological And Agricultural Sciences Nagoya
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Yamane Tsuneo
Laboratory Of Molecular Biotechnology Graduate School Of Bio- And Agro-sciences Nagoya University
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Nakano Hideo
Laboratory Of Molecular Biotechnology Department Of Bioengineering Sciences Graduate School Of Bioag
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