Structural studies of the vacuolar H(+)-pyrophosphatase: sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide.
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)-alpha-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 transmembrane alpha-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 alpha-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.
Maruyama
C
C
Department of Natural Environmental Sciences, Faculty of Integrated Human Studies, Kyoto University, Sakyo-ku Kyoto, Japan.
Tanaka
Y
Y
Takeyasu
K
K
Yoshida
M
M
Sato
M H
MH
D000595:Amino Acid Sequence; D002234:Carbodiimides / Q000378:metabolism; D003001:Cloning, Molecular; D019662:Cucurbitaceae / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D008301:Maleimides / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000201:enzymology
D002234:Carbodiimides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008301:Maleimides; D010446:Peptide Fragments; C003944:N-(3-pyrene)maleimide; C038088:N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase