The function of chloroplast ribosomes was investigated by analysing the products of in vitro protein synthesis by isolated pea chloroplasts. Since previous attempts at identifying newly synthesised proteins in isolated chloroplasts had either been unsuccessful or inconclusive, careful attention was paid both to the choice of chloroplast preparation and to the analytical techniques used to identify labelled proteins. A rapid method of chloroplast isolation was used which gave a chloroplast preparation which contained about 50% intact chloroplasts and showed high rates of amino acid incorporation into protein. It was felt that the proteins synthesised by these chloroplasts would accurately reflect the nature and pattern of protein synthesis which occurs in chloroplasts in vivo. High rates of incorporation would also aid identification of newly-synthesised proteins. Amino acid incorporation was shown to be sensitive to selective inhibitors of 70S-type ribosomes, but was not affected by inhibitors of 80S-type ribosomes. In addition, incorporation was shown to by insensitive to ribonuclease, suggesting that protein synthesis was taking place in intact chloroplasts. When the labelled chloroplasts were fractionated by differential centrifugation, approximately 25% of this incorporation was present in a 150 000 x g chloroplast supernatant fraction. Further analysis was confined to this supernatant fraction since only released, and therefore completed, polypeptides should be present in this fraction, thus aiding identification. The 150 000x g supernatant fraction was analysed on polyacrylamide gels in the presence and absence of a denaturant, sodium dodecyl sulphate, and by gel chromatography on Sephadex G100 in a sodium dodecyl sulphate-containing buffer. Only one polypeptide was found to be labelled by all these procedures. This polypeptide was identified as the large subunit of Fraction 1 protein, a major protein constituent of the chloroplast. Identity of the in vitro product present in the soluble phase of the chloroplast with the large subunit of Fraction I protein was established by comparing a two-dimensional tryptic peptide map of its [S35] methionine-labelled peptides with a tryptic peptide map of the large subunit of Fraction I protein labelled in vivo with [S35] methionine. It may therefore be concluded that only one of the many proteins present in the soluble phase of the chloroplast, namely the large subunit of Fraction 1 protein, is synthesised on chloroplast ribosomes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595027 |
| Date | January 1974 |
| Creators | Blair, George Eric |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/74384/ |
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