Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts of Euglena gracilis

Abstract

1. A procedure is described for the isolation of intact polyribosomes from the cytoplasm, chloroplasts and mitochondria of Euglena gracilis. 2. All three polyribosomal preparations incorporated labelled amino acids in a system in vitro. The cytoplasmic system was inhibited by chcloheximide but not by chloramphenicol. Both the chloroplast and the mitochondrial systems, however, were inhibited by chloramphenicol but not by cycloheximide. It is shown that mitochondrial polyribosomes, like the polyribosomes from cytoplasm and chloroplasts, can participate directly in protein synthesis without supplementary mRNA being added to the synthesizing system, as in previously reported instances. 3. Sedimentation coefficients were measured for the ribosomes, ribosomal subunits, and rRNA of the cytoplasm, chloroplasts and mitochondria. 4. The G+C content was 55% for cytoplasmic rRNA, 50% for chloroplast rRNA, and 29% for mitochondrial rRNA. 5. The cytoplasmic ribosomal subunits contained a ribonuclease activity that was inhibited by heparin.

Hybridization studies with RNA and DNA isolated from Euglena gracilis chloroplasts and mitochondria

“…These latter authors inferred that the 14 S, 11 S RNA though of ribosomal origin could be degradation products of the 23 S, 16 S RNA. This assumption is supported by the fact, that the base composition reported for both RNA preparations [6,18] match within the limits of analytical error.…”

“…Mitochondria were isolated from freshly harvested cells (batches of 150 g, wet weight) by grinding with a mortar, pestle and glass beads [6]. The mitochondria were purified by differential centrifugation and flotation in a Renografin (Squibb and Sons, Inc., Princeton) step gradient [15].…”

“…Both types of organelles contain their own set of duplex DNA [1][2][3] which replicate at a different pace than the nuclear DNA [4,5] does. These organelles also are equipped with functional 70 S ribosomes containing 23 S, 16 S rRNA [6].…”

Ribulose 1,5-Diphosphate Carboxylase Synthesis in Euglena

“…These inhibitors also prevent protein synthesis on the 70S type ribosomes isolated from either Euglena chloroplasts or mitochondria but they do not affect protein synthesis on the larger cytoplasmic ribosomes (2). In contrast, cycloheximide specifically blocks protein synthesis by cytoplasmic ribosomes (2).…”

“…These inhibitors also prevent protein synthesis on the 70S type ribosomes isolated from either Euglena chloroplasts or mitochondria but they do not affect protein synthesis on the larger cytoplasmic ribosomes (2). In contrast, cycloheximide specifically blocks protein synthesis by cytoplasmic ribosomes (2). When chloramphenicol at a concentration of 1 mg/ml is added to the suspending medium of Euglena cells, it effectively blocks protein synthesis on 68S chloroplast ribosomes; RuDPCase synthesis during the regreening of dark grown cells was completely inhibited (5,7).…”