Role of the gamma subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol.

TitleRole of the gamma subunit of chloroplast coupling factor 1 in the light-dependent activation of photophosphorylation and ATPase activity by dithiothreitol.
Publication TypeJournal Article
Year of Publication1984
AuthorsKetcham, SR, Davenport, JW, Warncke, K, McCarty, RE
JournalJ Biol Chem
Volume259
Issue11
Pagination7286-93
Date Published1984 Jun 10
ISSN0021-9258
KeywordsDithiothreitol, Electrophoresis, Polyacrylamide Gel, Enzyme Activation, Ethylmaleimide, Iodobenzoates, Macromolecular Substances, Photic Stimulation, Photophosphorylation, Plants, Proton-Translocating ATPases
Abstract

In leaves and intact chloroplasts, oxidation and reduction have been shown previously to regulate the ATPase activity of thylakoids. Illumination of spinach chloroplast thylakoids in the presence of dithiothreitol, which activates the ability of thylakoids to catalyze sustained ATP hydrolysis in the dark, causes increased incorporation of N-ethylmaleimide into the gamma subunit of coupling factor 1 (CF1). A disulfide bond in the gamma subunit is reduced during activation. The residues involved in this disulfide bond are the same as those in the disulfide linkage reduced during dithiothreitol activation of soluble CF1. The disulfide and dithiol forms of the gamma subunit may be separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. N-Ethylmaleimide is preferentially incorporated in the dark into the reduced form of the gamma subunit of CF1 in thylakoids previously exposed to dithiothreitol. Only a subpopulation of the CF1 in thylakoids is susceptible to dithiothreitol reduction and subsequent reaction with N-ethylmaleimide in the dark. Alkylation of the thiol groups exposed by reduction of the disulfide bond protects ATPase activity from inhibition by oxidants. At a given value of the transmembrane pH differential, photophosphorylation rates in dithiothreitol-activated thylakoids can be as much as seven to eight times those of nonactivated controls. N-Ethylmaleimide treatment of activated thylakoids in the dark prevents the loss of the stimulation of ATP synthesis on storage of the thylakoids. Photophosphorylation by intact chloroplasts lysed in assay mixtures is also activated in comparison to that by washed thylakoids. At a low ADP concentration, the rate of photophosphorylation approaches saturation as delta pH increases. These results suggest that the gamma subunit of CF1 plays an important role in regulation of ATP synthesis and hydrolysis.

Alternate JournalJ. Biol. Chem.
Citation Key3245
PubMed ID6233282
Grant List5T32-GM07273 / GM / NIGMS NIH HHS / United States