Preparation of Chloroplasts. Chloroplasts were isolated from leaves of spinach (Spinacia oleracea L., var. Virginia Blight Resistant) grown in a growth chamber at 21 C with a 10-hr day (2100 ft-c) and 16 C night. The light-dark cycle in the chamber was adjusted so that the plants received about 30 min of illumination before chloroplast isolation. Chloroplasts were prepared essentially as described by Jensen (12) with some modifications. The rinsed leaves, about 10 g after deribbing, were chopped gently at 4 C with razor blades in 45 ml of a grinding medium containing 330 mm sorbitol, 50 mm MES, 2 mM Na2EDTA, 1 mM MgCI2, I mm MnCI2, 0.5 mm K2HPO, (entire solution adjusted with NaOH to pH 6.5 at 4 C) plus 5 mm freshly prepared isoascorbate. The slurry was squeezed through two layers of Miracloth and centrifuged for 50 sec at 2000g. The chloroplast pellet was resuspended and stored in the dark at 4 C in the sorbitol-salt solution described above except that 50 mm HEPES (pH 6.7) replaced MES and the isoascorbate concentration was reduced to 2.5 mm. Chl was determined by the method of Arnon (1), and the chloroplast preparations contained 110 to 650 ,ug Chl/ml.Photosynthetic "C02 Assimilation. Photosynthetic CO2 fixation by the isolated chloroplasts was determined by H'4CO3-incorporation at 25 C and 3700 ft-c (74 nanoeinsteins/cm2 sec, 400-700 nm) under various He/02 atmospheres. Unless noted otherwise, the reaction vessels contained chloroplasts (5-32 tig of Chl), resuspension medium adjusted to pH 7.8, 5 mm freshly prepared Na4P207, +3 mm glycidate, and 1 mm NaH 4CO3 (2)(3)(4) Ci/mole) in a final volume of I ml. Vessels containing all the reaction medium components except chloroplasts and "4C-bicarbonate were sealed and repeatedly evacuated and refilled with the appropriate He/02 mixture and chilled, after which the chloroplasts were introduced with a 20-gauge syringe needle.Following a 4-min preillumination without shaking in the photosynthetic Warburg bath, the reactions were initiated by injecting NaH14CO3 and terminated at varying times by injecting 0.1 ml of 6 N acetic acid. Contents of the flasks were thoroughly mixed, aliquots dried at 25 C under a stream of N2, and dpm 237 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from