Studies on the molecular organisation and properties of the first three enzymes of pyrimidine biosynthesis, carbamoyl phosphate synthetase (CPTase), aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase), in various organisms have been reviewed. The molecular organisation of these three enzymes has been investigated in wheat using gel filtration chromatography. CPSase activity could not be detected in gel filtered extracts and in crude extracts from wheat seedlings was shown to be highly labile. ATCase and DHOase activity was detected and the molecular weights of these enzymes were estimated to be 1.03 x 105 ( 1.4 x 10^4) and 8.6 x 10^4 (6 x 103), respectively. At no time during these investigations were high molecular weight species (consistent with the presence of a multifunctional complex containing these enzymes) detected. During the course of these investigations, a protease was detected which was shown to co-migrate with ATCase and DHOase activities. This protease was shown to be insensitive to the serine protease inhibitor PMSF, but was partially inactivated by iodoacetamide, consistent with the protease being a member of the cysteinyl protease family. Inclusion of iodoacetamide during chromatography also failed to reveal high molecular weight species of these enzymes. Antisera were raised against purified wheat ATCase and were characterised by their ability to inactivate the enzyme. These antisera were then used to probe western blots of crude extracts from wheat seedlings and screen a wheat cDNA expression library to ATCase sequences. Western blotting failed to show any immunoreactive species in extracts prepared under conditions which suppressed protease activity (SDS, -mercaptoethanol), although a low molecular weight (approximately 3.7 x 10^4) ATCase could be detected in samples obtained after gel filtration chromatography. Antisera also showed very little cross-reactivity with the ATCase from E.coli a result consistent with studies on the enzyme from B. subtilis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:314743 |
| Date | January 1992 |
| Creators | Bartlett, Terence James |
| Publisher | University of Greenwich |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://gala.gre.ac.uk/6106/ |
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