Thompson, L. Susan (1987) Cloning of the enolase gene from Escherichia coli and characterization of a multimeric enolase from sulfolobus acidocaldarius. Masters thesis, Memorial University of Newfoundland.
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Glycolytic enzymes involved in three-carbon metabolism are found in representatives of every phylogenetic lineage. As these enzymes evolve slowly and their amino acid sequences are highly conserved, these proteins and their genes should be important tools for studying long term evolutionary relationships. Enolase was the glycolytic enzyme chosen for this thesis. A hybrid recombinant plasmid, pLC10-47, is known to complement an Escherichia coli enolase mutant (Thomson et al., (1979), J. Bacteriol. 137: 502-506). As a start to comparing the structure of the enolase gene from E. coli with its eukaryotic counterparts, the location of the E. coli enolase gene on pLClO-47 was determined. This was achieved by a process of restriction endonuclease digestion of pLC10-47, subcloning the fragments into pUC 12 and transforming E. coli with recombinant plasmids. Plasmid DNA was isolated from the transformants and examined for the presence of DNA from pLC10-47. Cell extracts of the transformants were assayed for enolase activity. The E. coli enolase gene was localized to the region around the unique Eco RI site of pLC10-47. As part of a study designed to compare the enolases from representatives of the three primary kingdoms, the enolase from Sulfolobus acidocaldarius was partially purified and characterized. S. acidocaldarius is a sulphur metabolizing archaebacterium which grows at temperatures of 70-75°C and at a pH of 2-3. The starting material for the enzyme purification was a post-ribosomal cytoplasmic extract kindly provided by Dr. A. Matheson, U. of Victoria, B.C. Enolase was purified by a combination of ion exchange chromatography on DEAE-cellulose (DE52) and gel filtration on Sephacryl S-300. The enzyme was characterized with respect to its native and subunit molecular weights. Parameters such as the Km for 2-phosphoglycerate, pH optimum, magnesium dependence and heat stability were determined and compared with those of enolases from eubacteria and eukaryotes. Enolases from S. acidocaldarius and members of the eubacterial genus Thermus are unusual in that they are thermostable and octamers whereas enolases from other organisms are dimers.
|Item Type:||Thesis (Masters)|
|Additional Information:||Bibliography: leaves 120-125.|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Enolase; Archaebacteria|
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