Modification of domains of alpha and beta subunits of F1-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms, by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP)
Abstract
Photoaffinity labeling by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed alpha and beta subunits of F1-ATPase from the thermophilic bacterium PS3 (TF1) is described. BzATP binds to both isolated alpha and beta subunits, to complexed beta subunit but not to complexed alpha subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [gamma-32P]BzATP-labeled alpha subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422-Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably alpha Q422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed beta subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments beta G10-M64, beta E75-M233, and beta D390-M469 were labeled with the isolated beta subunit. With complexed beta subunit the label was found only in CNBr fragments: beta E75-M233 and beta G339-M389. The locations where the covalently bound BzATP w...Continue Reading
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