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Title: Evaluation of pyruvate kinase as a potential phylogenetic marker : studies on the pyruvate kinase of the archaebacterium Thermoplasma acidophilum
Author: Potter, Simon
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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In this study the glycolytic enzyme pyruvate kinase was chosen as a potential phylogenetic marker. It was considered to be particularly appropriate for two reasons; first, it fulfils the criteria outlined above and secondly, there is a wealth of structural information for the enzyme from various sources available in the sequence databases. The pyruvate kinase chosen for study was that of the the thermoacidophilic archaebacterium, Thermoplasma acidophilum, because whilst there is a great deal of information in the databases on pyruvate kinases isolated from organisms within the eucaryal and bacterial domains, no sequence information previously existed for an archaebacterial equivalent. The aim of this study, therefore, was first to characterise the enzyme to ensure that the constancy of function requirement was fulfilled, and secondly to obtain sufficient primary structure information to facilitate a realistic evaluation of the potential of pyruvate kinase as a phylogenetic marker. The pyruvate kinase was purified to homogeneity using a series of chromatographic steps, and then characterised with respect to its physical and kinetic properties. The enzyme has a native Mr of 250 K, and a subunit Mr of 60 K. It exhibits typical Km values towards its substrates PEP and ADP, and is allosterically regulated by AMP. It is one of the most thermostable pyruvate kinases yet isolated, being active at up to 90oC. Initial sequencing attempts were frustrated by the chemical blockage of the N-terminus of the enzyme, and hence it was cleaved both chemically and proteolytically into peptide fragments, which were then sequenced by automated Edman degradation. The sequences of these internal peptides were then used in conjunction with a codon usage table derived from the citrate synthase gene of T. acidophilum to design a number of oligonucleotide probes. These probes were then a) fluorescently labelled and hybridised to Southern blots of restriction digests of T. acidophilum genomic DNA, and b) used as primers for the polymerase chain reaction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available