Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase.
The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.
Cunningham
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KM
Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
McCarty
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RE
D000225:Adenine / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D003624:Darkness; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007546:Isoquinolines / Q000378:metabolism*; D008027:Light; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D018892:Proton-Motive Force / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions; D018724:Spinacia oleracea / Q000166:cytology / Q000201:enzymology; D013450:Sulfones / Q000378:metabolism*; D020524:Thylakoids / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
D005456:Fluorescent Dyes; D007546:Isoquinolines; D009711:Nucleotides; D010456:Peptides, Cyclic; D012996:Solutions; D013450:Sulfones; D014475:Uncoupling Agents; C009873:divinyl sulfone; D000255:Adenosine Triphosphate; C017475:lucifer yellow; D006180:Proton-Translocating ATPases; C003384:tentoxin; D000225:Adenine