Identification of nucleotide binding sites in V-type Na+-ATPase from Enterococcus hirae.
A and B subunits of the V-type Na+-ATPase from Enterococcus hirae were suggested to possess nucleotide binding sites (Murata, T. et al., J. Biochem., 132, 789-794 (2002)), although the B subunit did not have the consensus sequence for nucleotide binding. To further characterize the binding sites in the V-ATPase, we did the photoaffinity labeling study using 8-azido-[alpha-32P]ATP. A and B subunits were labeled with 8-azido-[alpha-32P]ATP when analysed with SDS polyacrylamide gel electrophoresis. The peptide fragment of A subunit obtained by lysyl endopeptidase digestion after labeling showed a molecular size of 9 kDa and its amino acid sequencing revealed that it corresponded to residues Arg423-Lys494. The peptide fragment from B subunit after photoaffinity labeling and lysyl endopeptidase digestion showed the size of 5 kDa and corresponded to residues Phe404-Lys443. In our structure model, these peptides were close to the adenine ring of ATP. We suggest that non-catalytic B subunit of E. hirae V-ATPase has a nucleotide binding site, similarly to eukaryotic V-ATPases and F-ATPases.
Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan.
Toshiaki
T
Hosaka
Takeshi
T
Murata
Yoshimi
Y
Kakinuma
Ichiro
I
Yamato
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D001386:Azides; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016983:Enterococcus / Q000201:enzymology*; D006868:Hydrolysis; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry
D001386:Azides; D027682:Cation Transport Proteins; D009711:Nucleotides; D010455:Peptides; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase