This work has investigated the permeability of amyloplasts isolated from developing wheat endosperm to a range of metabolites that could be taken lip by the amyloplasts for starch synthesis and carbohydrate oxidation. A rapid method for the isolation and purification of amyloplasts from the endosperm of spring wheat, Triticum aestivum L.cv Axona, has been developed. Amyloplasts were mechanically released from cells of plasmolysed endosperm tissue and purified by low speed centrifugation through 2% Nycodenz onto an agar cushion. Amyloplasts prepared by this method were routinely 55-65% intact and the recovery was greater than 20% as determined by the plastid marker enzyme, alkaline inorganic pyrophosphatase (APPase). Contamination by the cytosol determined by UDP-glucose pyrophosphorylase and alcohol dehydrogenase was less than 1% whilst the contamination attributable to mitochondria, the endomembrane system and microbodies was 0.2%, 0.5% and 0% respectively. Amyloplast integrity was dependent upon the sorbitol concentration of the extraction buffer and at the optimal concentration of O.8M sorbitol, intactness was maintained for up to 2 hours. Proteolytic protection experiments demonstrated that amyloplast marker enzymes were protected from digestion with trypsin. Preparations incubated with a range of [U_14C] labelled substrates, in the presence and absence of ATP, were able to support starch synthesis at physiological rates similar to rates obtained in whole endosperm when supplied with 5 mM GlclP and 1 mM ATP or 5 mM ADP-glucose. The Km for GlclP was calculated as 0.46 mM. The ability of amyloplast preparations to support starch synthesis from GlclP and ATP in the presence of known translocator inhibitor compounds was examined. DIDS, P5P and CAT, inhibitors of the phosphate and adenylate trans locators, reduced the amount label incorporation from [U_14C] GlclP, supplied in the presence of ATP, into starch by up to 80%. Stimulation" of the OPPP by the addition of substrates for the glutamate synthase reaction, 2-oxoglutarate and glutamine in the presence of either Gle 1P or Glc6P and ATP resulted in an asymmetric distribution of carbon utilisation between starch synthesis and carbohydrate oxidation. 14C from supplied [U_I4C] GlclP and [U_14C] Glc6P was measured in synthesized starch and evolved CO2. Whilst GlclP was clearly the preferred substrate for starch synthesis, Glc6P was readily oxidised when 2-oxoglutarate and glutamine were supplied to isolated plastid preparations. Metabolite levels inside the amyloplast were quantified under different conditions of starch synthesis and carbohydrate oxidation. Under conditions in which the OPPP was not stimulated, GlclP demonstrated no interconversion to Glc6P, suggesting that the enzyme which interconverts the two compounds is not catalysing an equilibrium reaction and may be the regulatory branchpoint between the pathways of starch biosynthesis and carbohydrate oxidation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:488035 |
| Date | January 1996 |
| Creators | Blissett, Kerry Joy |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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