Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits.
The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.
Bragg
P D
PD
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Hou
C
C
D009327:4-Chloro-7-nitrobenzofurazan; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D001386:Azides; D001426:Bacterial Proteins / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D004024:Dicyclohexylcarbodiimide; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008024:Ligands; D008565:Membrane Proteins / Q000502:physiology / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D008024:Ligands; D008565:Membrane Proteins; D004024:Dicyclohexylcarbodiimide; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan