Asymmetry of the alpha subunit of the chloroplast ATP synthase as probed by the binding of Lucifer Yellow vinyl sulfone.
The catalytic portion of the chloroplast ATP synthase (CF1) is structurally asymmetric. Asymmetry of the otherwise symmetrical alpha3beta3 heterohexamer is induced by the presence of tightly bound nucleotides and interactions with the single-copy, smaller subunits. Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulfonic acid) rapidly and covalently binds to lysine 378 on one alpha subunit [Nalin, C. M., Snyder, B., and McCarty, R. E., (1985) Biochemistry 24, 2318-2324] [Shapiro, A. B. (1991) Ph.D. Thesis, Cornell University, Ithaca, NY). The asymmetrical binding of Lucifer Yellow to CF1 provides a method to investigate the cause of asymmetry in the alpha subunits. The reaction of CF1 with Lucifer Yellow was monitored by total fluorescence of bound Lucifer Yellow as well as by quantitative determination of Lucifer Yellow bound to the tryptic peptide that contains lysine 378 of the alpha subunit. The total binding of Lucifer Yellow to CF1 was not affected by the presence of tightly bound nucleotides or nucleotide in the medium. Neither the total binding of Lucifer Yellow to CF1 nor the reaction of alpha-lysine 378 with Lucifer Yellow was changed by the removal of the epsilon subunit, the delta subunit, or both subunits. The extent of incorporation of Lucifer Yellow into lysine 378 of the alpha subunit in (alphabeta)n was about three times that of Lucifer Yellow incorporation into CF1. Reconstitution of (alphabeta)n with gamma restored the binding of one Lucifer Yellow per alpha3beta3gamma. Therefore, the interactions between gamma and the alphabeta heterohexamer are important in conferring asymmetry to the alpha subunits of CF1.
Lowe
K M
KM
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
McCarty
R E
RE
D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D007546:Isoquinolines / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D013450:Sulfones / Q000378:metabolism; D014357:Trypsin
D005456:Fluorescent Dyes; D007546:Isoquinolines; D009711:Nucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013450:Sulfones; C009873:divinyl sulfone; C017475:lucifer yellow; D014357:Trypsin; D006180:Proton-Translocating ATPases