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.
Highlights
Duction have been shown previously to regulate the hydrolysis in the dark, even though they catalyze very high
Theseresults suggest thatthey subunit of CFI plays an important role in regulatioonf ATP synthesis and hydrolysis
The sulfhydryl compound was removed by washing and the thylakoidswere incubatedwith[3H]N-ethylmaleimideinthe dark.The[3H]N-ethylmaleimidewasincorporatedalmost aexclusively into they subunit, indicatingthat a disulfide bond in this subunit wascleaved
Summary
The y component that migrates faster is probably the Effect of Illumination in the Presence of Dithiothreitol on the Modification of Sulfhydryl Croups of CF,-Since illumination of thylakoids in the presenceof dithiothreitol induces a Ca2+-dependent ATPase activity in CF, extractefdrom the thylakoids with EDTA [4], this treatmentclearly alters CF,. Since tahcecessible thiol (S4)on the y subunit is blocked by pretreatment with nonradioactive N-ethylmaleimide [35], this incorporation of radioactive maleimide into the y subunit is probably the result of limited cleavage of a disulfide This conclusion is consistent with the observation that CFI extracted from thylakoids incubated with dithiothreitol in the dark hsaosme ATPase activity [4]. CFI was modified with [3H]N-ethylmaleimidein thylakoids in the dark followingdithiothreitol treatment in the dark or light (described in Table I), isolated, pretreated with dithiothreitol, no addition, or oiodosobenzoate, and electrophoresed (described in Fig. 1).The relative amounts of protein in the y l and y2 bands were estimated by densitometric scanning.
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