Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro[14C]benzofurazan.
When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan [14C]Nbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of [14C]Nbf/mol of the F1-ATPase indicating that, of the 1.15 mol of [14C]Nbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of [14C]Nbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the [14C]Nbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the [14C]Nbf group.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001572:Benzofurans / Q000494:pharmacology*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002417:Cattle; D008239:Lysine; D008929:Mitochondria, Heart / Q000201:enzymology*; D010069:Oxadiazoles / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D014357:Trypsin
D000596:Amino Acids; D001572:Benzofurans; D002250:Carbon Radioisotopes; D010069:Oxadiazoles; D010446:Peptide Fragments; C043708:7-chloro-4-nitrobenzofuran; C011514:4-nitrobenzofurazan; D014357:Trypsin; D006180:Proton-Translocating ATPases; D008239:Lysine