Structural Characteristics of the Disulfide-reduced Ovotransferrin N-Lobe Analyzed by Protein Fragmentation
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概要
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The isolated N-lobe (1-332) and C-lobe (342-686) of ovotransferrin were each found to form an opaque gel by incubation with 70 mM 2-mercaptoethanol at 37℃ for 24 h, suggesting that a protein conformational change due to extensive disulfide reduction was the initial mechanism prior to subsequent intermolecular protein aggregation forming gel networks. To investigate the conformational state of an extensively disultide-reduced protein, the structural characteristics of a two-disulfide protein form that had been shown to be produced by extensive disulfide reduction of the N-lobe [H. Yamashita, T. Nakatsuka, and M. Hirose, J Biol. Chem., 270, 29806-29812 (1995)] were analyzed, as a model protein, by the protein fragmentation approach. When the two-disulfide form was proteolyzed with chymotrypsin, a protease-resistant 8-kDa fragment was produced. This fragment was purified by ion-exchange column chromatography, and chemical analyses and matrix-assisted laser desorption ionization time-of-flight mass spectrometry clearly showed that the 8-kDa fragment comprised Ala1-Tyr72 of ovotransferrin. The fragment was found by amino acid analysis to retain the two intra-chain disulfide bonds intact. Far-UV CD spectrum analyses revealed that the 8-kDa fragment retained a native-like folded conformation. These data are consistent with the view that the N-terminal degment, Ala1-Tyr72, assumed a local native-like conformation in the two-disulfide form of the ovotransferrin N-lobe.
- 社団法人日本農芸化学会の論文
- 1997-04-23
著者
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Hirose M
The Division Of Applied Life Sciences The Graduate School Of Agriculture Kyoto University
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Hirose Masaaki
Graduate School Of Agriculture Kyoto Univ.
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Mizutani K
Maruzen Pharmaceuticals Co.
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Hirose Masaaki
The Research Institute For Food Science Kyoto Univesity
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Hirose Masaaki
Division Of Applied Life Sciences The Graduate School Of Agriculture Kyoto University
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MIZUTANI Kimihiko
The Research Institute for Food Science, Kyoto University
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YAMASHITA Honami
The Research Institute for Food Science, Kyoto University
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OE Hideo
The Research Institute for Food Science, Kyoto University
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Hirose Masaaki
The Research Institute For Food Science Kyoto University
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Yamashita H
Biotechnology Research Center Toyama Prefectural University
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Hayakawa Y
Institute Of Low Temperature Science Hokkaido University
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Oe Hideo
The Research Institute For Food Science Kyoto University:(present Address)shiga Junior College Of Cu
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Yamashita H
Dep. Of Nutritional Sci. Fac. Of Health And Welfare Sci. Okayama Prefectural Univ.
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Yamashita Honami
The Research Institute For Food Science Kyoto University
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Mizutani Kimihiko
Division Of Applied Life Science Graduate School Of Agriculture Kyoto University
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