The Journal of Biological Chemiewry
نویسنده
چکیده
Subchloroplast particles were prepared by incubation with digitonin and differential centrifugation. Such particles show a high rate of cyclic photophosphorylation but have little or no capacity to form the reversible, light-dependent pH gradient. They also have no activity in the formation of the nonphosphorylated high energy intermediate, in acid-base phosphorylation, and in light-induced absorbance change. ATP formation in these particles is insensitive to amines, nigericin plus KCl, and low concentrations of Triton X-100. It is inhibited by atebrin, car-bony1 cyanide p-trifluoromethoxyphenylhydrazone, and citrate. It is concluded from the above and from other experimental results that the light-induced formation of a pII differential in chloroplasts is secondary to and in equilibrium with a postulated high energy intermediate. A mode of action for various uncouplers in photophosphorylation in chloroplasts is suggested. Isolation of ChZoropZustsChloroplasts were prepared from lettuce (Lactuca sativa var. romaine). Leaves (125 g) were homogenized in a 220-volt Waring Blendor for 15 set at 120 volts in 140 ml of medium containing 0.4 M sucrose, 0.01 M NaCI, 0.01 M Tricine (pH S.O), 0.04 M ascorbate, and 1 mg per ml of human serum albumin. The beneficial effect of albumin was reported recently in detail (5). The homogenate was filtered through gauze and centrifuged at low speed. The chloroplast pellet was then collected by centrifuging the homogenate for 7 min at 1,500 x g. To disrupt the chloroplasts, each pellet, which was obtamed from 30 ml of homogenate, was resuspended in 30 ml of 5 mM Tricine, pH 8.5. The swollen chloroplasts were precipitated by centrifugation for 5 min at 20,000 X g. This pellet was dispersed in the original medium, but without ascorbate and albumin, at a chlorophyll concentration between 1 and 2 mg per ml.
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تاریخ انتشار 2002