Regulation of the supply of oxaloacetate for mitochondrial metabolism via phosphoenolpyruvate carboxylase in barley leaf protoplasts. II. Effects of metabolites on PEPC activity at different activation states of the protein.

The regulation of the supply of oxaloacetate (OAA) for mitochondrial metabolism via phosphoenolpyruvate carboxylase (PEPC) by metabolites is studied in barley (Hordeum vulgare L.) leaf protoplasts in light or darkness as well as under photorespiratory or non-photorespiratory conditions. Measurements on PEPC activity were performed on samples quickly frozen in liquid nitrogen to break the cell and stop metabolism and thus preserve the in vivo activation state. Glycine, serine, pyruvate, acetyl-CoA, glycolate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and ADP had no significant effect on PEPC activity. Malate, aspartate and glutamate were strong inhibitors of PEPC activity decreasing the activity more in light versus darkness. However, at the physiological cytosolic concentration of these metabolites under the respective conditions, inhibition of PEPC activity was about the same with the exception of aspartate which inhibits more under non-photorespiratory than under photorespiratory conditions. 2-Oxoglutarate and glyoxylate decreased PEPC activity by 20 to 40% in the range of its physiological cytosolic concentration. Inhibition by physiological cytosolic concentrations of glutamine was limited. Glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate, dihydroxyacetonphosphate and P(i) stimulated PEPC activity significantly in their physiological cytosolic concentration range. Physiological cytosolic concentrations of glucose 6-phosphate and fructose 6-phosphate activated PEPC activity to about the same extent under all conditions applied, while 3-phosphoglycerate and dihydroxyacetonphosphate stimulating stronger under non-photorespiratory versus photorespiratory conditions. Moreover, dihydroxyacetonphosphate stimulated PEPC activity more in light versus darkness under non-photorespiratory conditions. P(i) activation of PEPC activity decreases in light versus darkness under non-photorespiratory conditions. Stimulation of PEPC activity by citrate in its physiological concentration range is limited. Glucose 1-phosphate and AMP activated PEPC activity only at concentrations higher than their physiological levels in the cytosol. Determinations of PEPC activity in the presence of different malate/glucose 6-phosphate ratios revealed that glucose 6-phosphate totally relieved the inhibitory effect of malate. The regulatory properties of PEPC activity will be discussed in relation to its functions in C3 plants.