Ammonia Assimilation and Metabolite Transport in Isolated Chloroplasts. I. Kinetic Measurement of 2-Oxoglutarate and Malate Uptake Via the 2-Oxoglutarate Translocator in Oat and Spinach Chloroplasts

Abstract

The stable double-layer silicone centrifugation system was used to determine the kinetic properties of the 2-oxoglutarate (2-OG) translocator in isolated oat and spinach chloroplasts. The uptake of [¹⁴C]2-OG and [¹⁴C]malate via the 2-OG translocator were measured in the presence of 20 mM glutamate in chloroplasts preloaded with unlabelled 2-OG. The characteristics of the general dicarboxylate (Dct) translocator were also determined using chloroplasts preloaded with glutamate. The V_max values obtained for transport activity via the 2-OG translocator in oat and spinach chloroplasts exceeded 150 μmol mg^-1 Chl h^-1 and for the Dct translocator less than 100 μmol mg^-1 Chl h^-1. The K½ (malate) values of the 2-OG and Dct translocators also showed large differences in the two species. In spinach chloroplasts they were 2.7 and 0.6 mM for the 2-OG and Dct translocators respectively whereas, in oat chloroplasts the corresponding values were 2.7 and 1.4 mM. This suggests that, in spinach, malate would be transported into the chloroplasts preferentially via the Dct translocator, thus providing a kinetic basis for the 'push and pull' mechanism proposed for dicarboxylate transport during photorespiratory NH₃ recycling in the 2-translocator model.