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 inorgani c pyrophosphatase (H+-PPase) of pumpkin which are covalently labeled b y two fluorescent labeling reagents; N-cyclohexyl-N'-[4-(dimethyl amin o)-alpha-naphthyl] carbodiimide (NCD) and N-pyrenylmaleimide (NPM), NC D and NPM are fluorescent analogues of N,N'-dicycrohesylcarbodiimide a nd N-ethylmaleimide, respectively, and inactivate H+-PPase activity. E xcess Mg2+ protected the H+-PPase from the inactivation by these reage nts. Furthermore, we identified the cDNA clone encoding the pumpkin Hf -PPase in order to determine the position of labeled residues. The nuc leotide sequence of the cDNA clone contains a 2,304 bp open reading fr ame encoding a polypeptide with 768 amino acids. Chemical sequence ana lysis of fluorescent peptide fragments revealed that Glu(749) located in the C-terminal putative transmembrane alpha-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 al so 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 transmembra ne alpha-helix. We also Pound that the H+-PPase shows significant amin o acid sequence similarity to Kdp-ATPase A chain of E. coli, This simi larity between the two different proteins suggest that they have evolv ed from a common ancestor and may utilize a common basic mechanism for ion transport.