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Title: The molecular cell biology of a xyloglucan specific endo 1,4 β-D-glucanase from Tropaeolum majus L. cotyledons
Author(s): Stronach, Morag Shona
Issue Date: 1991
Publisher: University of Stirling
Abstract: Certain seeds store carbohydrate, not as starch, but as a polysaccharide component of thickened cotyledonary and endosperm cell walls. In nasturtium (Tropaeolum majus L.) the storage carbohydrate is xylogluoan. One of the enzymes Involved in xyloglucan mobilisation, an endo 1,4 β-D- glucanase was investigated further. The purified enzyme was found to be a single molecular species of 29,000 and to have an isoelectric point of 5.1. It was found not to be a glycoprotein and the assembled protein contained 66% β-sheet, no α-helix and 34% "other structures". A polyclonal antiserum was raised against the enzyme and used in further studies. The enzyme was localised in nasturtium cotyledons, over a germination time course, at the light microscope level using an immunofluorescent technique. The enzyme was found to localise initially at the plasma membrane before being distributed throughout the storage wall. It did not co-localise with visible structures thought to be zones of dissolution in the wall, hence it was thought that the endo 1,4 β-D-glucanase was involved in loosening the xyloglucan for attack by the other enzymes (present in the dissolution zones). The enzyme was shown to be synthesised de novo following germination, the increase in enzyme activity being a direct result of an increase in protein synthesis. The increase in activity correlates closely with the mobilisation of storage xyloglucan as viewed histochemically (and determined biochemically). The enzyme activity decreased rapidly when the substrate had been mobilised but the level of enzyme protein content does not, suggesting activity denaturation probably due to the desiccation of the seed. The protein was rapidly degraded at a later stage and this depletion is thought to be a controlled process, possibly via a developmentally regulated protease. Successful isolation of the messenger RNA from nasturtium was achieved and analysis of the imunoprecipitable protein revealed a single molecular species of M, 33,000 (as opposed to 29,000 for the pure protein). The difference in M, is almost certainly due to the presence of a signal peptide on the sequence. The production of the enzyme was found to require the presence of the embryonic axis. The effect produced by the removal of the axis could not be replaced by GA3 or 2-4-D.
Type: Thesis or Dissertation

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