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The glycolate/glycerate transporter of spinach (Spinacia oleracea L.) chloroplast inner envelope membranes was solubilized by treatment of the membranes with sodium cholate. Mixtures of the cholate extracts and soy asolectin were subjected to gel filtration to remove the detergent. The reconstituted vesicles were frozen, thawed, and sonicated in a buffer that contained 10 millimolar d-glycerate and, usually, [(3)H]sucrose as an internal space indicator. The dilution of the vesicles into a medium that contained 0.4 millimolar [(14)C]d-glycerate resulted in a rapid accumulation of labeled glycerate, followed by a much slower loss of [(14)C]d-glycerate from the vesicles. This behavior is characteristic of counterflow. The accumulation of [(14)C]d-glycerate was strongly inhibited by HgCl(2), which blocks glycolate/glycerate transport in intact chloroplasts. In the absence of proton ionophores, the extent of [(14)C]glycolate accumulation under similar conditions was much greater than that of [(14)C]d-glycerate. External glycolate inhibited d-glycerate counterflow and external d-glycerate inhibited glycolate counterflow. The external pH dependence of the efflux of [(14)C]d-glycerate accumulated in vesicles by counterflow and its inhibition by external l-mandelate are characteristics displayed by glycolate transport in intact chloroplasts. Partial purification of the transporter was achieved by glycerol gradient centrifugation. The solubilized glycolate and glycerate counterflow activities, assayed by reconstitution into vesicles, were found to sediment similarly.

Experiments in which [1-(14)C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C(3) plants.

A method has been devised for loading chloroplast inner envelope vesicles prepared from pea (Pisum sativum L. var Progress No. 9) or spinach (Spinacia oleracea L.) with 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), a membrane impermeant, fluorescent pH indicator. Two known proton-linked transport activities of the inner envelope, glycolate/H(+) co-transport and phosphate/phosphoglycerate exchange have been shown to cause quenching of the internal pyranine fluorescence. This represents the first demonstration that these vesicles are sealed and competent for transport measurements. The technique, as it now stands, is essentially qualitative. It does, however, offer advantages over transport measurements with intact chloroplasts, for example compatibility with rapid mixing techniques and accessibility of the transport proteins to antibodies.

The transport of glycolate and d-glycerate, the substrate and end product of the photorespiratory carbon pathway, respectively, by isolated intact pea (Pisum sativum) chloroplasts has been compared. d-Glycerate uptake was inhibited by the 2-hydroxymonocarboxylates, glycolate, glyoxylate, and d-lactate. Phosphate and phosphoglycerate and triose phosphates were without effect when the assays were carried out for two seconds. Glycolate was found to be a competitive inhibitor of d-glycerate uptake and the presence of glycolate in the chloroplast stroma strongly enhanced d-glycerate uptake from the medium. For optimal rates of d-glycerate-dependent O(2) evolution by pea chloroplasts, phosphate, ADP or ATP, and a mediator of cyclic electron flow had to be added. The inhibition of d-glycerate-dependent O(2) evolution by triose phosphates and 2-hydroxymonocarboxylates was tested. The inhibition of d-glycerate-dependent O(2) evolution by these metabolites did not correlate with their effects on glycerate transport. Thus, metabolism of d-glycerate, rather than its transport, limits the rate of glycerate-dependent oxygen evolution. The ramifications of d-glycerate metabolism on the interpretation of d-glycerate uptake data obtained with prolonged incubations are discussed. We conclude that d-glycerate and glycolate transport are mediated by the same transport system.