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Highly purified intact chloroplasts from mesophyll protoplasts of the 4 carbon pathway plant digitaria sanguinalis inhibition of phospho glycerate reduction by ortho phosphate and by phosphoenol pyruvate



Highly purified intact chloroplasts from mesophyll protoplasts of the 4 carbon pathway plant digitaria sanguinalis inhibition of phospho glycerate reduction by ortho phosphate and by phosphoenol pyruvate



Physiologia Plantarum 57(3): 330-338



Purified mesophyll protoplasts from the C4 plant D. sanguinalis were used to prepare intact mesophyll chloroplasts with low cytoplasmic contamination. The procedure involved breakage of protoplasts, differential centrifugation, partition in a dextran-polyethylene glycol 2-phase system and Percoll density gradient centrifugation. The final chloroplast preparation contained .apprx. 80% intact chloroplasts with a phosphoenolpyruvate carboxylase contamination of 0.2-1% 9 of the original protoplast activity, corresponding to 1-6 .mu.mol 14CO2 fixed/mg Chl .cntdot. h. The purified chloroplasts showed substrate-dependent O2 evolution in the range of 40-150 .mu.mol substrate reduced/mg Chl .cntdot. h, with phosphoglycerate or oxaloacetate as substrate. Both reactions were stimulated 1.5-fold by pyruvate and further by addition of the other substrate. These measurements indicated that phosphoglycerate reduction was limited by substrate transport across the chloroplast envelope. Without added substrate, the chloroplasts consumed O2 via pseudo-cyclic electron transport in the light. This reaction was also stimulated by pyruvate. Phosphoglycerate-dependent O2 evolution was inhibited by Pi and by phosphoenolpyruvate to about the same extent with purified chloroplasts, but only by Pi with protoplast extracts. This suggests that phosphoglycerate, Pi and phosphoenolpyruvate share a common carrier, similar to the Pi-translocator in C3 chloroplasts, and that the lack of inhibition obtained with phosphoenolpyruvate and unpurified chloroplasts is artefactual, possibly due to oxaloacetate formation from added phosphoenolpyruvate and concomitant stimulation of O2 evolution by oxaloacetate reduction. Apparently, phosphoenolpyruvate is transported with a Km similar to that of Pi in C4 mesophyll chloroplasts.

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