Effect of Chloramphenicol on Amino Acid Incorporation by Chloroplasts and Comparison with the Effect of Chloramphenicol on Chloroplast Development in Vivo

Amino acid incorporation into protein by chloroplasts and chloroplast ribosomes is more sensitive to inhibition by chloramphenicol than amino acid incorporation into protein by cytoplasmic ribosomes (3, 4). Chloramphenicol also inhibits the formation of some chloroplast enzymes without affecting cell division in Euglena (11) or leaf development in bean (8, 9). Chloramphenicol also selectively increases the proportion of 70 S ribosomes present as polysomes (6). Chloramphenicol is bound to chloroplast 70 S ribosomes but not to 80 S cytoplasm ribosomes of Euglena (1). These observations have led to the conclusion that chloramphenicol inhibits protein synthesis by chloroplasts, but not protein synthesis by the cytoplasm, as was suggested by Stutz and Noll (13). We report here that the effect of varying chloramphenicol concentration on inhibition of amino acid incorporation into protein by bean chloroplasts in vitro is the same as the effect of varying chloramphenicol concentration on inhibition of chlorophyll accumulation and on inhibition of development of photosynthetic activity during greening of etiolated leaves. Our observations also suggest that chloramphenicol acts in vivo by inhibiting plastid protein synthesis. Chloroplasts were prepared from leaves of Phaseolus vulgaris var. Black Valentine which had been grown in darkness for 6.0 days and then exposed to light for 0.5 days (10). Leaves were homogenized for 15 sec in a Waring Blender with 120 ml of Honda medium (5) for each 30 g of leaves. The slurry was filtered through a pad of cheesecloth and glass wool and refiltered through 4 thicknesses of facial tissue. The filtrate was centrifuged at 3500g for 3 min, and the pellet was resuspended in 10 ml of Honda medium for each 60 g of leaves. Incorporation of '4C leucine (250 mc/mmole) into protein was carried out as described elsewhere (10, 12). The amount of radioactivity incorporated into protein was determined according to Mans and Novelli (7). Amino acid incorporation into protein is inhibited by concentrations of chloramphenicol greater than 10 ,ug/ml (Fig. 1). The highest concentration of chloramphenicol that was used, 800 Mg/ml, inhibited amino acid incorporation into protein 72%. Chloramphenicol inhibits development of photosynthetic activity when the antibiotic is present during the greening of etio-