Sequence Analysis of Cloned cDNA and Proteolytic Fragments for Nitrate Reductase from Spinacia oleracea L.

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概要

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• Proteolytic fragments were obtained by limited proteolysis of 120 kDa nitrate reductase from Spinacia oleracea L. using trypsin and Staphylococcus aureus V8 protease. Determination of NH_2-terminal sequences in 9 to 14 Edman degradation steps allowed the exact localization of the fragments within the amino-acid sequence of spinach nitrate reductase was deduced from the nucleotide sequence of cDNA clone pSPNR117 which was initially identified by hybridization to squash nitrate reductase cDNA clone [Crawford, N. M., Campbell W. H. and Davis R. W. (1986) Proc. Natl. Acad. Sci. USA 83: 8073] and anti spinach nitrate reductase polyclonal antibodies. This clone has a 2324 base insert, and the amino acid sequence deduced from its open reading frame, which contains 640 residues. The predicted sizes 42.5 and 30 kDa were in reasonable agreement with previous determination of the apparent molecular sizes of the FAD-cytchrome b_<557>-binding, and FAD-binding fragments, respectively. Arginine residue was the cleavage site for trypsin and glutamic acid was for S. aureus V8 protease. The amino acid residues within the linker regions which connect the functional domains, could be cleaved with trypsin or S. aureus V8 protease may be well conserved in the amino acid sequences deduced from the nitrate reductase cDNA sequences. A sequence identity of 61.2-80.1% was found in the amino acid sequences deduced from the cDNA sequences as obtained by spinach and other higher plant nitrate reductases. However, the amino acid sequences surrounding the proteolytic cleavage sites of nitrate reductase had poor homology.

• 日本植物生理学会の論文

著者

• Nakagawa Hiroki

  Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University

• KIYOTA Seiichiro

  Variable Star Observers League in Japan (VSOLJ)

• Nakagawa Hiroki

  Department Of Agricultural Chemistry Faculty Of Agriculture Saga University

• Nakagawa Hiroki

  Department Of Agricultural Chemistry Faculty Of Horticulture Chiba University:institute Of Agrobiolo

• Shiraishi Naomasa

  Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University

• Kubo Yoshihiro

  Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University

• Takeba Go

  Laboratory of Applied Biology, Faculty of Life Science, Kyoto Prefectural University

• Takeba Go

  Laboralory Of Applied Biology Faculty Of Living Science Kyoto Prefectural University

• Shiraishi Naomasa

  Department Of Agricultural Chemistry Faculty Of Horticulture Chiba University

• SAKANO KATSUHIRO

  Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University,

• Sakano Katsuhiro

  Laboratory Of Biochemistry Faculty Of Agriculture Nagoya University

• Sakano Katsuhiro

  Laboratory Of Nitrogen Metabolism Department Of Applied Physiology National

• Kiyota Seiichiro

  Laboratory of Nitrogen Metabolism, Department of Applied Physiology, National

• Kiyota S

  Vsolj

• Kiyota S

  National Inst. Agrobiological Resources Tsukuba Jpn

• Kubo Yoshihiro

  Department Of Agricultural Chemistry Faculty Of Horticulture Chiba University

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