Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to the same allosteric site, however, MgADP activates and PEP inhibits the binding of fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can differentiate between the two ligands and cause the appropriate response is important for the understanding of the basis of allosteric regulation. We studied the interactions between an active site and an allosteric site (heterotropic interactions) within the protein, and found that each of the four unique heterotropic interactions is unique and the magnitudes of the coupling free energies for MgADP activation sum up to 100% that of wildtype EcPFK without homotropic cooperativity in F6P binding. We took on the kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which are important for the allosteric responses to both MgADP and PEP. Interestingly, Lys214 is only important in PEP inhibition and not MgADP activation. We can also differentially disrupt the MgADP heterotropic interactions with the introduction of G184C within the protein. These results suggest that there are different pathways for allosteric communication within the enzyme: different paths for MgADP activation and PEP inhibition, and different paths for each heterotropic interaction with Gly184 being important for the 33Å MgADP heterotropic interaction.

The biochemical basis for the regulation of sucrose accumulation is not fully understood. The present study was thus aimed at investigating aspects of 'coarse' (enzyme activity) and 'fine' (metabolite) control of glycolytic enzyme activity in relation to carbon partitioning in the developing stalk (internodes 3 to 10), and between varieties with significant differences in sucrose content. Particular emphasis was placed on studying pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), since this enzyme has been implicated in sucrose metabolism in other plant species. Within the developing stalk, internodal maturation was associated with a redirection carbon from the insoluble matter and total respiration (C02 production and biosynthesis) to sucrose storage. Between varieties, with significant variation in sucrose content, there was an inverse relationship between hexose monophosphate partitioning into respiration and sucrose. The reduction in carbon flux to respiration was not associated with a decline in the extractable specific activity of PK, PFK and PFP. There was also no alteration in the regulation of PK, PFK and FBPase by change in the mass action ratios. Hexose monophosphate concentration declined approximately two to three-fold from internodes 3 to 9 and Fru-6-P concentration was within the lower Km or 80.5 range (Fru-6-P) of PFP and PFK, respectively (as reported from the literature) . Within the developing stalk, substrate limitation might have contributed to the decline in carbon partitioning to respiration. In sugarcane, the levels of PFP activity were controlled in part by PFP protein expression. 8ugarcane PFP polypeptide(s) are resolved as a single protein with a molecular mass of approximately 72 kO. PFP catalysed a reaction close to equilibrium in all intemodes investigated, and the concentration of Fru-2,6-P2 was shown to be in excess of the requirement to stimulate PFP activity. Carbon flux from the triose-P to hexose monophosphate pool was apparent in sugarcane, suggesting that PFP activity was functional in vivo. The developmental profile of specific PFP activity was not positively correlated to the increasing rate of sucrose accumulation in the top ten internodes of the developing stalk. Between different sugarcane varieties, specific PFP activity was shown to be inversely correlated to sucrose content. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

La Polyarthrite Rhumatoïde est caractérisée par une synovite à l'origine de lésions progressives ostéo-articulaires induites par les formes réactives de l'oxygène (ROS) produites par la NADPH oxydase des polynucléaires neutrophiles (PMN). La NADPH oxydase des phagocytes, est formée d'un centre catalytique membranaire, le cytochrome b558, sur lequel vient s'associer des protéines cytosoliques régulatrices (p67phox, p47phox, p40phox et Rac1/2). Nous avons étudié la spécificité de l'interaction entre la (6-phosphofructokinase 2) et de la 6PGDH (6-phosphogluconate déshydrogénase) et la NADPH oxydase des PMN. D'autre part, nous avons caractérisé les domaines de l'hétérocomplexe S100A8/A9 impliqués dans l'activation de la NADPH oxydase phagocytaire. Par ailleurs, une étude de la signature protéique dans le liquide synovial a été menée afin de rechercher l'empreinte de l'activation du PMN dans la PR.Après stimulation par le PMA, la 6PGDH et la PFK2 co-imunoprécipitent avec les facteurs cytosoliques p67phox, p47phox and p40phox. Les expériences de microscopie confocale suggèrent une co-localisation de ces deux enzymes du métabolisme du glucose avec la NADPH oxydase, dans des micro-domaines membranaires : les radeaux lipidiques. La 6PGDH est impliquée dans l'activation de la NADPH oxydase phagocytaire en élevant la concentration du NADPH cytosolique mais également en augmentant l'affinité de cette enzyme pour son substrat, le NADPH. PFK2 est l'enzyme majeure de la régulation de la glycolyse, voie est essentielle pour la production d'ATP du PMN. L'utilisation du complexe S100A8/A9 et de protéines chimères de fusion nous a permis de révéler que la partie C-terminale de S100A8 est impliquée dans la liaison avec le cytochrome b558 et l'activation de la NADPH oxydase phagocytaire. In vivo, le profil protéique du liquide articulaire de PR a révélé l'empreinte de l'activation du PMN dans cette pathologie avec une surexpression des protéines S100A8 et S100A9. Une production ectopique de S100A8/A9 par les synoviocytes de type fibroblastique a été mise en évidence.En conclusion, la 6PGDH, la PFK2 et l'hétérodimère S100A8/A9 sont de nouveaux partenaires d'activation de la NADPH oxydase des phagocytes. Dans la PR, l'activation des PMNs conduit à la sécrétion de S100A8/A9 qui semblent constituer à la fois des biomarqueurs pertinents, mais également des cibles thérapeutiques potentielles.

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 166-179). Also issued on the Internet.

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