Protein Synthesis by Isolated Pea Chloroplasts

Suspensions of chloroplasts, prepared as described in Fig. 1, contain between 40 and 50% intact chloroplasts, as judged by phase contrast and electron microscopy. They incorporate [ C]leucine, [ C]phenylalanine, and P S]methionine into proteins when illuminated (Fig. 1). The rate of incorporation falls rapidly to zero after about 20 min this falling rate is not accompanied by lysis of the chloroplasts. A vital component of the incubation medium is the high concentration of KCl (0.2 m). When chloroplasts are incubated in media containing sucrose as the osmoticum, protein syn- 

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Incorporation of labeled amino acids by other components of the incubation medium can be excluded. Bacterial contamination of the chloroplast preparation is minimized by using sterile glassware and media. If the preparations are solubilized in 2% Triton X-100 at the end of the incubation, less than 0.1% of the radioactivity incorporated into protein is present in the pellet spun down at 10,000 for 10 min. This indicates that incorporation is not due to bacteria, nuclei, or whole leaf cells. The large stimulation of incorporation by light and the sensitivity to inhibitors of photophosphorylation argue for chloroplast, rather than mitochondrial, protein synthesis. If stored for 1 h at O C, the activity of the crude chloroplast suspensions decreases by about 50%. Therefore, we have not attempted to purify the chloroplasts from other cellular components present in the suspension. 

We conclude from all these characteristics that protein synthesis is proceeding in intact chloroplasts only, is being driven by photophosphorylation, and is probably using messenger RNA synthesized before the chloroplasts were isolated. The question now is Does this system make discrete identifiable proteins  

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Table I. Characteristics of Protein Synthesis by Isolated Pea Chloroplasts"...

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