Mechanism of Glycolate Transport in Spinach Leaf Chloroplasts

Abstract

The incorporation of ¹⁴CO₂ into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to ¹⁴CO₂ (NaH¹⁴CO₃, 1 millimolar) in the light was determined as a function of O₂ concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K_m (O₂) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic ¹⁴CO₂ fixation under an O₂ atmosphere. Glycolate transport into and out of chloroplasts was studied using the silicon oil centrifugation method. Both uptake and loss of glycolate were found to be rapid, with small temperature dependencies between 0°C and 25°C (Q₁₀ = 1.1). The reaction rate as a function of the concentration of glycolate up to 30 millimolar was linear in both directions. The effect of external pH on the reaction rate in both directions was also examined. Glycolate penetrates rapidly, even at pH 8, showing a surprisingly high permeation of the glycolate anion. This rate was about 30 micromoles per milligram Chl per hour at 0°C, and the initial concentration of glycolate of 10 millimolar with a pH range of 7 to 8. The observed rate is comparable to the reported value for glycolate synthesis in chloroplasts under photorespiratory conditions. The uptake of glycolate into chloroplasts was accelerated below pH 7, while the rate of excretion was considerably lowered. It is, thus, suggested that undissociated glycolic acid penetrates the chloroplast envelopes more rapidly than does the anion.