Thesis (Ph. D.) -- California Institute of Technology, 2010. / Title from home page (viewed 03/31/2010). Advisor and committee chair names found in the thesis' metadata record in the digital repository. Includes bibliographical references.

Habilitationsschrift--Marburg. / At head of title: Aus dem Physiologisch-Chemischen Institut der Universität Marburg. Includes bibliographical references.

No description available.

Phosphoglycerate mutase catalyses the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic/gluconeogenic pathways. There are two main types of phosphoglycerate mutase: 2,3-bisphosphoglycerate dependent and 2,3-bisphosphoglycerate independent. The enzyme from Saccharomyces cerevisiae has been extensively studied: the high resolution crystal structure of this tetrameric enzyme, subunit Mr 27,000, is known (Winn et al., 1981), the amino acid sequence has been determined (Fothergill and Harkins, 1982) and the gene encoding the enzyme has been isolated and sequenced (White and Fothergill-Gilmore, 1988). Phosphoglycerate mutase from the fission yeast, Schizosaccharomyces pombe, has been purified and partially characterised (Price et al., 1985; Johnson and Price, 1987). It is monomeric, of Mr 23,000, and the sequences of a number of peptides produced by digestion of this enzyme have been determined (Fothergill and Dunbar. unpublished). Alignment of these sequenced peptides with the sequence of S.cerevisiae phosphoglycerate mutase shows 40% identity and the conservation of a number of residues which are known to be essential to the activity of the S. cerevisia enzyme e. g. His-8, Arg-7, Ser-11. Thr-20 and Arg-59. Attempts were made to isolate and sequence the gene encoding S. pombe phosphoglycerate mutase. The S.cerevisiae phosphoglycerate mutase gene failed to detect gene sequence homologies in the S.pombe genome. An oligonucleotide, designed against part of the S.pombe phosphoglycerate mutase sequence (a stretch which was not homologous to the S. cerevisiae sequence) also failed to detect sequence homologies in the S.pombe genome. Thus under the conditions used, neither the S. cerevisiae gene nor the degenerate oligonucleotide appeared to be a suitable molecular probe to screen the S.pombe cDNA expression library in λgt11 (which was synthesised by V. Simanis). A polyclonal antibody against S.pombe phosphoglycerate mutase was prepared and used to screen the S. pombe cDNA expression library. A number of small identical clones were isolated and sequenced. the cDNA inserts encoded 69 residues and part of this sequence was similar to part of the sequence of phosphoglycerate mutase from other sources. Part of the sequence was also similar to a stretch of fructose-2,6-bisphosphatase sequence (fructose-2,6-bisphosphatase appears to be divergently related to phosphoglycerate mutase, Pilkis et al., 1987). A purification scheme for phosphoglycerate mutase from the prokaryote, Streptomyces coelicolor, has been devised. The N-terminal sequence of this enzyme was determined and confirmed that the gene isolated and sequenced by Peter White, encoded phosphoglycerate mutase from S. coelicolor. The enzyme was shown to be a tetramer with a subunit Mr of 29,000. S. coelicolor phosphoglycerate mutase was also shown to be partially 2,3-bisphosphoglycerate dependent.

Some of the most profound changes elicited by cell growth stimuli influence dramatic rewiring of metabolism. Intriguingly, rapidly dividing cells with aberrant growth factor signalling, such as cancer cells, tend to rely on glycolysis to generate an adequate supply of building blocks required for cell proliferation and invasion. In this study, we observed that in response to stimulation with insulin-like growth factor 1 (IGF-1), MCF-7 breast adenocarcinoma cells show increased levels of the key glycolysis proteins pyruvate kinase M2 and lactate dehydrogenase A. We then developed targeted multiple reaction monitoring (MRM) mass spectrometry assays to conduct quantitative analysis of glycolysis proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), the latter implicated in pyruvate kinase splicing and many other aspects of cell proliferation. Application of the glycolysis MRM assay to examine IGF-1 stimulated MCF-7 cells revealed increased levels of all sequential proteins from phosphoglycerate mutase 1 to lactate dehydrogenase A in the glycolysis pathway. An extension of this study to cell lines of varying invasiveness, suggest a relation between glycolysis and metastasis. The clinical applicability of glycolysis MRM assay was also shown by its successful application to lung cancer biopsy analysis. Success with the targeted analysis of glycolysis proteins led to a similar approach for the hnRNP family. Our results showed evidence that a poorly characterized hnRNP (A/B) may be regulated by the c-Myc transcription factor but does not evidently influence pyruvate kinase splicing. Our approach using MRM to examine small subsets of proteins downstream of cellular growth signals is relatively novel. Our results demonstrate the potential for such targeted MS strategies because of their high selectivity and multiplexing capabilities. Further, the findings from our analyses provide novel insights into the downstream changes elicited by growth signals such as IGF-1 and c-Myc.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The allosteric phosphofructokinase from <i>E. coli</i> crystallized in the absence of ligands. X-ray diffraction data were collected to 2.4 aa, AA resolution and the structure was solved by the method of molecular replacement, using the model of the liganded R-state of the enzyme. The atomic model was refined by rigid body refinement and by the least-squares method of Hendrickson and Konnert. The final structure is compared to the high resolution model of the liganded, active form of the enzyme and to the low resolution structure of the inhibited phosphofructokinase from B. stearothermophilus. It is found that the quaternary structure of the unliganded model is more similar to the liganded, active form than to the inhibited T-state of B. stearothermophilus pfk. There are however considerable differences in the tertiary and quaternary structures, apparently resulting from ligand binding. These changes are not localised to the binding sites. The overall change, though to result mainly from the binding of the activators, is consistent with the closing of the active site observed in the structure of the active state. The ways in which the ligand binding could bring about these changes are considered. The possible effect of the changes on the enzyme activity are discussed. It is possible that the structure represents an inactive T-state, different from that in the presence of inhibitors. Whatever the exact interpretation it is clear that the structure shows considerable flexibility suggesting that the two-state allosteric model is an oversimplification.

No description available.

No description available.