Identification of the initially NBD-labeled essential tyrosine residue in bovine heart MF1-ATPase.
Bovine heart MF1-ATPase was labeled with limiting amounts of [14C]NBD-C1[( 14C]4-chloro-7-nitro-2,1,3-benzoxadiazole) and the resulting radioactive label on the essential Tyr was stabilized by reduction with zinc in the presence of multidentate ligand EDTA and redox mediator 4,4'-dipyridyl. Subsequent treatment of the labeled protein with cyanogen bromide and separation of the reaction mixture by ion-exchange chromatography yielded essentially only one radioactive polypeptide. Further cleavage of this polypeptide with TPCK-trypsin, lactonization of the terminal homoserine residue and reaction with derivatized polystyrene resin gave a shorter peptide attached to the solid support which contained all the radioactivity. Edman degradation showed that the amino acid sequence of this peptide was Glu . Gly . Asn . Asp . Leu . Tyr . His . Glu . Met, which corresponds to residues 192-200 in the beta subunit of bovine heart MF1-ATPase as determined by Runswick and Walker (1983). Since this specifically labeled Tyr-197 is separated by only one amino acid residue from the essential Glu-199 which was labeled specifically with dicyclohexylcarbodiimide by Yoshida et al. (1982) it seems most likely that both Tyr-197 and Glu-199 play direct roles in the catalytic hydrolysis and synthesis of ATP.
D009327:4-Chloro-7-nitrobenzofurazan; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D009206:Myocardium / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000032:analysis*; D013053:Spectrophotometry; D014443:Tyrosine / Q000032:analysis*
D014443:Tyrosine; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D003488:Cyanogen Bromide