Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 121-133).

Since Meyerhof separated hexokinase in a purified form from bakers' yeast a great deal of information on the properties of this enzyme has bene obtained by numerous workers. In particular the question of the specificity of yeast hexokinase was believed to initiate hexose metabolism in the living organism, and hence the enzyme would be expected to play an important part in determining whether certain sugars could be utilized or not by yeast. Certain cases of inhibition of various metabolic processes in the living cell were found to have been caused by the specific inhibition of hexokinase. The pattern of specificity of the substrates of hexokinase falls into two different classes. Some sugars are phosphorylated in the pyranose form and others in the furanose form. The specific requirements for pyranoses have been investigated extensively but there is relatively little information in the literature on the furanose aspect of hexokinase specificity. The object of this research was to investigate the specificity of the enzyme in respect of analogues of fructose and in particular those sugars which differ only slightly from fructofuranose at carbon-1 and carbon -2. To this end, the chemical syntheses of the following compounds were undertaken: 1_amino_l_deoxy_D_fructose 1_deoxy_D_fructose 2.5_anhydro_D_mannitol 2.5_anhydro_D_sorbitol Preparations of 1_amino_1_deoxy_D_fructose and 2.5_anhydro_D_sorbitol have been published, but the other two sugars are new compounds. Part I of this thesis contains a review of the chemistry of 1_amino_1_deoxy_D_fructose, 1_deoxyketoses and 2_deoxyketoses, a report of the experimental work carried out by the author in synthesising the above mentioned compounds, and a discussion of the results obtained. Part II contains a review of the knowledge of yeast hexokinase, a report of the experimental work carried out in investigating the effects of the synthesised fructose analogues in the hexokinase reaction, and a discussion of these results in the light of other information on the specificity of the enzyme. A systematic investigation of a spectrophotometric assay method for following hexokinase activity is included in Part II. The appendix contains the data from which the graphs and tables, shown in the thesis, have been completed.

La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellules. / Mitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells.

Fusel alcohols signal nitrogen scarcity to elicit a range of responses in the yeast Saccharomyces cerevisiae. These alcohols activate pseudohyphal growth and cause rapid inhibition of translation initiation. Previous work from our lab has highlighted that the translation initiation factor eIF2B is a target for this regulation. eIF2B is the guanine nucleotide exchange factor required for recycling eIF2•GDP to eIF2•GTP. The GTP bound form of eIF2 can interact with the Methionyl initiator tRNA to form the ternary complex. Fusel alcohols target eIF2B leading to reduced ternary complex; however the mechanism by which alcohols cause this effect is currently unknown. This study aims to characterize the effects of fusel alcohols on eIF2B and identify post-translational modifications, which may be responsible for translation inhibition. Following purification of eIF2B, a number of novel phosphorylation sites have been identified using mass spectrometry. In particular, phosphorylated serine has been identified at position 131 within yeast eIF2Bδ. Phosphoantibody analysis suggests that the phosphorylation status of this residue differs following fusel alcohol treatment. Mutagenesis experiments are consistent with phosphorylation of this residue being essential for the translational inhibition seen following fusel alcohol exposure. Therefore, phosphorylation of this residue may prime eIF2B for regulation and provide a switch to sensitize the process of translation to particular conditions.

Transport of proteins to the nucleus through a nuclear envelope is controlled mostly via nuclear localization signal (NLS). Nuclear localization signal is rich in positively charged amino acids arginine and lysine. It was observed that activity of this NLS could be regulated through a phosphorylation of serine in its close proximity. Either a phosphorylation of serine or phosphomimetic changes of these "presequences" could represent an important mechanism regulating a localization of protein in cells in relation to a cellular activation. In our laboratory was identified protein - Fragile X mental retardation syndrome 1 neighbor (Fmr1nb), whose cellular localization could be driven by this posttranslational modification.

Nonmuscle myosin from bovine thymus was purified, characterized , and phosphorylated with MLCK, H4PK, and Protein Kinase C. Phosphorylation occured exclusively on the myosin regulatory light chain. Phosphorylation by MLCK and H4PK resulted in the activation of the MgATPase activity as well as filament assembly of nonmuscle myosin.

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