Isolation of Intact Chloroplasts of Euglena gracilis by Isopycnic Sedimentation in Gradients of Silica

Abstract

A technique is described for the isolation of structurally intact and partially active chloroplasts from photoheterotrophically grown Euglena gracilis. The separation of intact chloroplasts from stripped chloroplast membranes and other subcellular particles was achieved by sedimentation in continuous, isosmotic density gradients of Ludox AM, a silica sol.The final preparations contained an average of 93% intact chloroplasts and corresponded to approximately 10% of the the chlorophyll of the original cell suspension and 20 to 30% of the chlorophyll layered on the gradients.The chloroplasts obtained were intact by the criteria of ultrastructure, their content of ribulose diphosphate carboxylase, and their activity in a modified Hill reaction assay (U. Heber and K. A. Santarius. 1970. Z. Naturforsch. 25b: 718-727). In addition, the isolated chloroplasts were capable of incorporating amino acids into protein in the light.