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Highly purified intact chloroplasts from mesophyll protoplasts of the C 4 plant Digitaria sanguinalis . Inhibition of phosphoglycerate reduction by orthophosphate and by phosphoenolpyruvate
Author(s) -
Hallberg Mats,
Larsson Christer
Publication year - 1983
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1983.tb02296.x
Subject(s) - digitaria sanguinalis , chloroplast , protoplast , phosphoenolpyruvate carboxykinase , botany , biology , tricine , digitaria , biochemistry , enzyme , weed , gene
Purified mesophyll protoplasts from the C 4 plant Digitaria 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 two‐phase system, and Percoll density gradient centrifugation. The final chloroplast preparation contained about 80% intact chloroplasts with a phosphoenolpyruvate carboxylase contamination of 0.2–1% of the original protoplast activity, corresponding to 1–6 μmol 14 CO 2 fixed/mg Chl h. The purified chloroplasts showed substrate‐dependent oxygen evolution in the range of 40–150 μmol substrate reduced/mg Chl 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 oxygen via pseudo‐cyclic electron transport in the light. Also this reaction was stimulated by pyruvate. Phosphoglycerate‐dependent oxygen evolution was inhibited by P i and by phosphoenolpyruvate to about the same extent with purified chloroplasts, but only by P i with protoplast extracts. This suggests that phosphoglycerate, P i and phosphoenolpyruvate share a common carrier, similar to the P i ‐translocator in C 3 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 oxygen evolution by oxaloacetate reduction. Furthermore, the results suggest that phosphoenolpyruvate is transported with a K m similar to that of P i in C 4 mesophyll chloroplasts.