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Title: The development of methods for the purification of inositol monophosphatase from plants
Author: Afsar, S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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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 Mr 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 Ins3P, was a weak competitive inhibitor. Partially purified soybean IMPase was insensitive to Li+, with marked inhibition at 250mM Li+. Other inhibitors tested were: a methylene bisphosphonic acid derivative, NaF, HgC12 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available