Isolation and Enzymic Characterization of Euglena Proplastids

Abstract

Organelles were isolated from dark-grown E. gracilis Klebs by sucrose density gradient centrifugation. Plastids, identified by triosephosphate isomerase and NADP glyoxylate reductase were present at an equilibrium density of 1.24 g/ccm3 clearly separated from mitochondria at an equilibrium density of 1.22 g/ccm3. Assay for choline phosphotransferase and glucose-6-phosphatase showed that endoplasmic reticulum membranes were present at a density of 1.12 g/ccm3. The plastid fraction contained phosphofructokinase, pyruvate kinase, triosephosphate isomerase and aldolase indicating the operation of a glycolytic pathway. During regreening pyruvate kinase and phosphofructokinase in the developing proplastid decreased, neither enzyme being present in the mature chloroplast. However, plastids were present in the photosynthetic cell as shown by a peak of glycolysis enzymes at an equilibrium density of 1.24 g/ccm3. The integrity of isolated plastids was demonstrated by their capacity for protein synthesis. Plastids isolated from dark-grown cells rapidly incorporated [35S]methionine into protein with an absolute dependence on added ATP. The large subunit of ribulose diphosphate carboxylase was the major polypeptide synthesized by these isolated plastids.