Plastids have been isolated from pea (Pisum sativum L.) roots with a high degree of purity and intactness. In these plastids, the activity of enzymes involved in carbohydrate metabolism have been analyzed and corrected for cytosolic contamination. The results show that fructose-1,6-bisphosphatase, NAD-glyceraldehyde phosphate dehydrogenase, and phosphoglyceromutase are not present in pea root plastids. Transport measurements revealed that inorganic phosphate, dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and glucose-6-phosphate (Glc6p) are transported across the envelope in a counterexchange mode. Transport of glucose-1-phosphate was definitely excluded. The oxidation of Glc6P by intact plastids resulted almost exclusively in the formation of DHAP. The parallel measurement of DHAP formation and NO2- consumption during Glc6P-supported nitrite reduction yielded a ratio of NO2-reduced/DHAP formed of 1.6, which is relatively close to the theoretical value of 2.0. These results show that the oxidation of Glc6P, involving the uptake of Glc6P and the release of DHAP, and the reduction of NO2- are very tightly coupled to each other.
The kinetic properties of the adenosine 5'-diphosphate/adenosine 5'-triphosphate (ADP/ATP) translocator from pea (Pisum sativum l.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea 1.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. l h e K,(ATP) value of the pea root ADP/ATP translocator was 10 p~ and that for ADP was 46 p~. Corresponding values of the spinach ADP/ATP translocator were 25 p~ and 28 PM, respectively. Comparable results were obtained for the reconstituted ATP transport activities. l h e transport was highly specific for ATP and ADP. Adenosine 5'-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake.With pea root plastids and spinach chloroplasts, K,,, values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.
Evidence is provided that amyloplasts from pea roots contain a translocator which transports, in a counter exchange mode, phosphate, glucose 6-phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate. The translocator has a low affinity for 2-phosphoglycerate and glucose 1-phosphate. Metabolite transport was measured by silicone oil filtering centrifugation either directly by uptake of radioactive labelled compounds or indirectly by back exchange.
Plastidial envelope membranes were isolated from tomato (Lycopersicon esculentum) leaves and green and red tomato fruits by isopycnic discontinuous sucrose density gradient centrifugation. Solubilized envelope membrane proteins were reconstituted into liposomes. Transport measurements revealed that the phosphate translocator from tomato leaves transports inorganic phosphate, 3-phosphoglycerate and triosephosphates. The phosphate translocators of green and red fruit plastids catalyze, in addition to the transport of these substrates, also the transport of glucosedphosphate, glucose-1-phosphate and phosphoenolp yruvate .
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