Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Abstract

Uptake of D,L-glycerate into the chloroplast stroma was studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3-5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2.degree. C and 8.5 at 20.degree. C, but at all pH values, the rate of uptake was higher at 20.degree. C than at 2.degree. C. Uptake was concentration dependent, saturating above 8 mM glycerate. At 2.degree. C, the Km was 0.3 mM and the Vmax was 13 .mu.mol/mg of chlorophyll/h. At 20.degree. C initial rates of glycerate uptake were > 40 .mu.mol/mg of Chl/h. Although there was some metabolism of glycerate by chloroplasts in the light, even at 2.degree. C, the actual concentration of glycerate in the stroma, determined by chromatography, was 2-4 times that in the incubation medium. NH4Cl stimulated glycerate uptake and decreased its metabolism by the chloroplasts resulting in stromal concentrations of glycerate up to 6 times higher than in the medium. A number of weak acids inhibited glycerate uptake, lactate and glyoxylate being the most effective. The inhibition was not competitive with respect to glycerate. Glycerate uptake was also inhibited by p-chloromercuriphenyl sulfonate. Substrates of the dicarboxylate transporter did not inhibit glycerate uptake. Of the 4 substrates of the phosphate transporter tested, 3-phosphoglycerate and dihydroxyacetone phosphate inhibited glycerate uptake but Pi and glyceraldehyde-3-phosphate had little effect. The inhibition by 3-phosphoglycerate was competitive with respect to glycerate, with a Ki of 0.2 mM, suggesting that 3-phosphoglycerate binds to the same site as glycerate. Glycerate only inhibited 3-phosphoglycerate uptake slightly and the inhibition was not competitive, indicating that glycerate does not bind to the phosphate transporter. Glycerate did not counter exchange with 32P in chloroplasts which had been preloaded with 32Pi, confirming that glycerate did not enter via the phosphate transporter. Evidently, the chloroplast envelope contains a specific glycerate transporter, which differs from the previously reported transporters in that it is stimulated by light.