Abstract
Abstract Illumination of chloroplasts in the presence of sulfate, ADP, and Mg2+ causes 35 to 50% inhibition of a subsequent phosphorylation reaction; ADP cannot be replaced by other nucleotides. Compounds that normally inhibit photophosphorylation prevent the appearance of the sulfate-induced inhibition. No evidence was found for phosphorylation site specificity of the sulfate inhibition. In chloroplasts inhibited by preliminary illumination with sulfate, the light-induced pH rise and the P/e2 ratio in a phosphorylating Hill reaction are inhibited as much as photophosphorylation is. The pH rise can be restored by dicyclohexylcarbodiimide. Both the trypsin- and dithiothreitol-activated Ca2+-dependent ATPase activities associated with the chloroplast coupling factor (CF1) are decreased in sulfate-inhibited chloroplasts. Removal of CF1 from the membranes followed by recombination with CF1 from noninhibited chloroplasts restores the pH rise completely and phosphorylation to a large extent. From these and other experimental results it is concluded that sulfate, in the presence of ADP and Mg2+, interacts with CF1 on chloroplast membranes under conditions of coupled electron flow so as to cause a modification of CF1 structure.
Highlights
In chloroplasts inhibited by preliminary illumination with sulfate, the light-induced pH rise and the P/e2 ratio in a phosphorylating Hill reaction are inhibited as much as photophosphorylation is
Reagents-Phenazine methosulfate, trypsin, dithiothreitol, and phlorizin were purchased from Nutritional Biochemicals, methylviologen from British Drug Houses, and N, N’-dicyclohexylcarbodiimide from Sigma
We have found that less phosphate is required to prevent the irreversible inhibition from occurring than is needed to compete with sulfate directly in a phosphorylation reaction
Summary
Chloroplast Preparation-Greenhouse-grown spinach leaves were homogenized in a Waring Blendor for 15 set with a medium containing 0.8 M sucrose-20 m&r Tricine-NaOH (pH 7.8)-10mM NaCl-5 mM ascorbate. Chloroplast Preparation-Greenhouse-grown spinach leaves were homogenized in a Waring Blendor for 15 set with a medium containing 0.8 M sucrose-20 m&r Tricine-NaOH (pH 7.8)-10. After filtering through Miracloth (Chicopee Mills, Inc., New York), the homogenate was centrifuged at 1500 x g for 30 set and chloroplasts were isolated from the supernatant fluid by centrifugation at 1500 x g for 7 min. The chloroplasts were washed once in the grinding medium and resuspended in sucrose-Tricine-NaOH-NaCl. Chlorophyll was determined by the method of Arnon [10]. Sulfate Inhibition-In a final volume of 2.0 ml, chloroplasts (200 pg of chlorophyll) were illuminated at 23” in a reaction mixture containing 50 InM Tricine-NaOH (pH 8.5), 5 maI MgCl,, 2.5 mM ADP, 10 mrvr K2S04, and 50 PM pyocyanine. “Pi was purchased from New England Nuclear
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