Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase.
The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.
Viale
A M
AM
Vallejos
R H
RH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001120:Arginine / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D046911:Macromolecular Substances; D008666:Metalloendopeptidases; D010658:Phenylglyoxal / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D001120:Arginine; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; D006180:Proton-Translocating ATPases; D010658:Phenylglyoxal; D003488:Cyanogen Bromide