Chloroplast F1 ATPase has more than three nucleotide binding sites, and 2-azido-ADP or 2-azido-ATP at both catalytic and noncatalytic sites labels the beta subunit.
The photolabeling of chloroplast F1 ATPase, following exposure to Mg2+ and 2-azido-ATP and separation from medium nucleotides, results in derivatization of two separate peptide regions of the beta subunit. Up to 3 mol of the analogue can be incorporated per mole of CF1, with covalent binding of one moiety or two moieties per beta subunit that can be either AMP, ADP, or ATP derivatives. These results, the demonstration of noncovalent tight binding of at least four [3H]adenine nucleotides to the enzyme and the presence of three beta subunits per enzyme, point to six potential adenine nucleotide binding sites per molecule. The tightly bound 2-azido nucleotides on CF1, found after exposure of the heat-activated and EDTA-treated enzyme to Mg2+ and 2-azido-ATP, differ in their ease of replacement during subsequent hydrolysis of ATP. Some of the bound nucleotides are not readily replaced during catalytic turnover and covalently label one peptide region of the beta subunit. They are on noncatalytic sites. Other tightly bound nucleotides are readily replaced during catalytic turnover and label another peptide region of the beta subunit. They are at catalytic sites. No alpha-subunit labeling is detected upon photolysis of the bound 2-azido nucleotides. However, one or both of the sites could be at an alpha-beta-subunit interface with the 2-azido region close to the beta subunit, or both binding sites may be largely or entirely on the beta subunit.
D000244:Adenosine Diphosphate / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism; D001345:Autoradiography; D001386:Azides / Q000008:administration & dosage / Q000378:metabolism*; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006358:Hot Temperature; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D014316:Tritium
D001386:Azides; D009711:Nucleotides; D014316:Tritium; D000244:Adenosine Diphosphate; C022295:2-azidoadenosine 3',5'-diphosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases