Structural Studies of the Vacuolar H^+-Pyrophosphatase : Sequence Analysis and Identification of the Residues Modified by Fluorescent Cyclohexylcarbodiimide and Maleimide
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概要
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We determined the amino acid residues of the H^+-translocating inorganic pyrophosphatase (H^+-PPase) of pumpkin which are covalently labeled by two fluorescent labeling reagents; N-cyclohexyl-N'-[4-(dimethyl amino)-α-naphthyl] carbodiimide (NCD) and N-pyrenylmaleimide (NPM). NCD and NPM are fluorescent analogues of N, N'-dicycrohexylcarbodiimide and N-ethylmaleimide, respectively, and inactivate H^+-PPase activity. Excess Mg^<2+> protected the H^+-PPase from the inactivation by these reagents. Furthermore, we identified the cDNA clone encoding the pumpkin H^+-PPase in order to determine the position of labeled residues. The nucleotide sequence of the cDNA clone contains a 2, 304 bp open reading frame encoding a polypeptide with 768 amino acids. Chemical sequence analysis of fluorescent peptide fragments revealed that Glu^<749> located in the C-terminal putative transmembrane α-helix was a NCD-labeled residue, and Cys^<632> was a NPM-labeled residue located in a putative cytosolic domain. The amino acid sequence of the region that includes Glu^<749> is highly conserved in H^+-PPases from other plants and it also shows some sequence similarity with the region of the carbodiimide-reactive Glu (or Asp) of F_0F_1-ATPase c-subunit. The reactive glutamic acids in these proteins are located at the last C-terminal transmembrane α-helix. We also found that the H^+-PPase shows significant amino acid sequence similarity to Kdp-ATPase A chain of E.Coli. This similarity between the two different proteins suggest that they have evolved from a common ancestor and may utilize a common basic mechanism for ion transport.
- 日本植物生理学会の論文
著者
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竹安 邦夫
京大生命科学研究科
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竹安 邦夫
京都大学大学院生命科学研究科分子情報解析学分野
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TAKEYASU Kunio
Department of Medical Biochemistry and Neurobiotechnology Center
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Maruyama Chio
Research Laboratory for Resources Utilization, Tokyo Institute of Technology
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Tanaka Yoshiyuki
National Institute of Agrobiological Resources
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Yoshida Masasuke
Research Laboratory for Resources Utilization, Tokyo Institute of Technology
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Sato Masa
Department of Natural Environmental Sciences, Faculty of Integrated Human Studies, Kyoto University
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Sato M
Kyoto Univ. Kyoto Jpn
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Yoshida M
Kinki Univ. Nara Jpn
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Tanaka Y
National Inst. Agrobiological Resources Ibaragi Jpn
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Maruyama Chio
Research Laboratory For Resources Utilization Tokyo Institute Of Technology
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Yoshida Masasuke
Research Laboratory For Resources Utilization Tokyo Institute Of Technology
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Yoshida Masasuke
Research Lab. Resources Util. Tokyo Inst. Tech.
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Tanaka Yoshiyuki
National Inst.of Agrobiol.res.
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Sato Masa
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology
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