The biological tissues used were cauliflower and soybean cells capable of synthesizing their own inositol. The existing assays for monitoring IMPase activity were investigated, and a major achievement of the project was the development of a dual-label radiochemical assay which measured both IMPase and 'other phosphatase' activities simultaneously. A number of methods for the purification of IMPase were investigated and developed. Adopting ammonium sulphate precipitation as the first step, soybean IMPase was found to precipitate in the 30% to 50% saturation fraction and a purification of 2.4 fold obtained. Ion exchange chromatography, using Q-Sepharose Fast Flow, resulted in a purification of 12 fold. Various types of immobilized ligand and salts were tested to improve separation by hydrophobic interaction chromatography. A purification of 13 fold was achieved. Purification using preparative iso-electric focusing by the Rotofor cell proved to be the most successful; a purification of 115 fold was obtained. Gel filtration chromatography was also examined as a possible step in the purification strategy as well as preparative native acrylamide gel electrophoresis. The <I>M</I><SUB>r</SUB> of soybean IMPase was found to be 60 kDa and the pI 5.3 Soybean IMPase was shown to be less thermostable than its mammalian counter-part and heat treatment proved not to be an effective technique to use in the purification of IMPase. Inhibitor studies on IMPase revealed that 6-deoxy-6-phosphonoglucose, an isosteric analogue of Ins3<I>P, </I>was a weak competitive inhibitor. Partially purified soybean IMPase was insensitive to Li<SUP>+</SUP>, with marked inhibition at 250mM Li<SUP>+</SUP>. Other inhibitors tested were: a methylene bisphosphonic acid derivative, NaF, HgC1<SUB>2</SUB> and β-mercaptoethanol. Overall, considerable progress has been made in achieving the aims of this project. Different techniques which exploit different characteristics of the protein have been successfully applied. As a result, purification of soybean IMPase may be expected when the techniques are assembled in the optimal order.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595369 |
| Date | January 1997 |
| Creators | Afsar, S. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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