Different Effects of Carboxy-Terminal Deletion in the Adrenodoxin Molecule on Cytochrome c and Acetylated Cytochrome c Reductions
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概要
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In immunoblotting analysis using rabbit antibody to bovine adrenodoxin, the total proteins of the bovine adrenal cortex gave two bands, suggesting the presence of two forms of adrenodoxin in vivo : full-length and carboxy-terminal deleted adrenodoxins. To examine the effect of the carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrendoxin were constructed. The wild type [Ad(2-128)] and carboxy-terminal deleted [Ad(2-114) and Ad(2-112)] recombinant adrenodoxins expressed in Escherichia coli were purified to give a single band on SDS-PAGE. They showed an A_<414>/A_<276> value of 0.92. In an NADPH-cytochrome c reduction assay, the K_m values for cytochrome c in the reconstituted system with Ad(2-128), Ad(2-114) and Ad(2-112) were 39,235 and 618 nM, respectively. The V_<max> values were 638,700 and 898 mol/min/mol flavin, respectively. In an NADPH-acetylated cytochrome c reduction assay, the maximum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maximum activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values were 4-times higher than that of Ad(2-128). In the presence of 100 mM NaCl, the K_m values for acetylated cytochrome c in the system reconstituted with Ad(2-128), Ad(2-114) and Ad(2-112) were 220,33 and 22 μM, respectively. The V_<max> values were 352,305 and 382 mol/min/mol flavin, respectively. These results indicate that the effects of the carboxy-terminal deletion of adrendoxin on NADPH-cytochrome c and acetylated cytochrome c reductions are different; the carboxy-terminal region (residues 113-128) of adrendoxin largely contributes to the binding with cytochrome c but disturbs the binding with acetylated cytochrome c.
- 公益社団法人日本薬学会の論文
- 1996-11-15
著者
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Yasukochi Takanori
Department Of Microbiology Faculty Of Pharmaceutical Sciences Kyushu University
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Horiuchi Tadao
Department Of Biochemistry School Of Medicine Keio University
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SAGARA Yasuhiro
Department of Medical Biology, Kochi Medical School
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HARA Takayuki
Department of Biochemistry, Graduate School of Health and Nutrition Sciences, Nakamura Gakuen Univer
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ARIYASU Yasuyuki
Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University
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KAJIYAMA Akiko
Department of Biochemistry, Graduate School of Health and Nutrition Sciences, Nakamura Gakuen Univer
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Hara Takayuki
Department Of Biochemistry Graduate School Of Health And Nutrition Sciences Nakamura Gakuen Universi
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Kajiyama Akiko
Department Of Biochemistry Graduate School Of Health And Nutrition Sciences Nakamura Gakuen Universi
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Horiuchi Tadao
Department Of Bioengineering Faculty Of Engineering Soka University
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Horiuchi T
Department Of Bioengineering Faculty Of Engineering Soka University
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Ariyasu Yasuyuki
Department Of Microbiology Faculty Of Pharmaceutical Sciences Kyushu University
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Sagara Yasuhiro
Department Of Medical Biology Kochi Medical School
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