Journal Article

Structural Studies of the Vacuolar H<sup>+</sup>-Pyrophosphatase: Sequence Analysis and Identification of the Residues Modified by Fluorescent Cyclohexylcarbodiimide and Maleimide

Chio Maruyama, Yoshiyuki Tanaka, Kunio Takeyasu, Masasuke Yoshida and Masa H. Sato

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 39, issue 10, pages 1045-1053
Published in print October 1998 | ISSN: 0032-0781
Published online October 1998 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/oxfordjournals.pcp.a029301
Structural Studies of the Vacuolar H+-Pyrophosphatase: Sequence Analysis and Identification of the Residues Modified by Fluorescent Cyclohexylcarbodiimide and Maleimide

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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 Mg2+ 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 Glu749 located in the C-terminal putative transmem-brane a-helix was a NCD-labeled residue, and Cys632 was a NPM-labeled residue located in a putative cytosolic domain. The amino acid sequence of the region that includes Glu749 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 F0F1-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.

Keywords: Chemical modification; H+-PPase; Kdp ATPase; Pumpkin; Vacuole

Journal Article.  0 words. 

Subjects: Plant Sciences and Forestry ; Biochemistry ; Molecular and Cell Biology

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