Broken Chloroplasts. To prepare lysed chloroplasts, the intact plastids were "osmotically shocked" in 50 mM Hepes-HCI (pH 7.6). After being kept at 0 C for 5-10 min, the suspension was centrifuged at 5,100g for 5 min. The pellet was suspended in 40 ml 50 mM Hepes-HCI (pH 7.6) and recentrifuged at 5,100g for 5 min. The supernatant was discarded, and the resulting pellet consisting of broken plastids (lamellar membranes) was resuspended in 2.0 ml grind medium.

Photoassimilation of 'CO2 by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praeakum was investigated. The main watersoluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction.Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. There have been only a few reports of CO2 assimilation by isolated chloroplasts from CAM plants. Levi and Gibbs (19) reported assimilation by mechanically isolated chloroplasts from Kalanchoe daigremontiana. Nishida and Sanada (21) Plant Material. Sedum praealtum D. C., supplied by Edwards and Spalding, was propagated with stem cuttings in 15-cm pots containing a vermiculite-soil (1:1) mixture. The plants were kept in a greenhouse, but natural light was supplemented with banks of fluorescent lights in the winter months. The plants were watered when dry, approximately every 2 to 3 days. They were fertilized with one-quarter strength Hoagland solution once a week. Leaf material was not removed from the plants until 2 months after the cuttings became established. Young, but fully expanded leaves were used for chloroplast isolation. Protoplast yields diminished significantly when the established plants were older than about 8 months.Protoplast Isolation. Protoplast isolation was modeled after the procedure developed by Spalding and Edwards (29), except several changes were made in the procedure which increased the yield. Isolation was begun near the end of the solar day. The leaves were thinly sliced and placed in cold 0.3 M sorbitol. The slices were washed once, very gently vacuum-infiltrated for a few seconds, and washed once again with 0.3 M sorbitol. The slices (40g) were then transferred to 65 ml cooled digestion medium on a bed of ice. The digestion medium contained 0.3 M sorbitol, 0.9% (w/v) cellulase, 0.45% Macerozyme, and 4% BSA at pH 5.6. The next morning, the digestion mixture was manually swirled, passed through a 210-,pm nylon net, and centrifuged for 10 to 15 min at 20g in a swinging bucket rotor. The pellets were resuspended in 40 ml 30%o (w/v)