Show simple item record Li, Liang en_US 2007-05-18T20:03:13Z 2007-05-18T20:03:13Z 2000-05-01T00:00:00Z en_US
dc.description.abstract Phospholipase D (PLD) is a centrally important enzyme in signal transduction, since its hydrolysis of membrane phospholipids initiates a series of reactions that release several intracellular regulators, including phosphatidic acid, diacylglycerol and arachidonic acid. Studies were carried out on the regulation of PLD and its physiological significance, in enzymatically-dispersed acinar cells of rat submandibular glands. In permeabilized submandibular cells, the guanine nucleotide, GTP_S, activated PLD by a mechanism that involved both a heterotrimeric GTP-binding protein and the small GTPase, Arf. Purified recombinant Arf activated PLD, and endogenous Arf translocated from the cytosolic to the particulate fraction after GTP_S stimulation. Activation of PLD by the muscarinic agonist, carbachol, was significantly inhibited by the Arf-specific Mocker brefeldin A. A receptor-coupled Arf-PLD pathway is proposed, which is largely independent of the PIP2/PLC signal transduction system. Aluminum fluoride (AlF4-), an established G-protein activator, was found to inhibit GTP_S stimulation of PLD in permeabilized cells. This phenomenon was confirmed and characterized in detail in a carefully controlled cell-free PLD assay system. AlF4 - inhibited both guanine nucleotide- and oleate-sensitive PLD in doseand time-dependent ways. Several lines of evidence indicated that AlF 4- inhibited PLD activation by a mechanism that was independent of G-proteins, the cAMP signal transduction pathway or regulation of cellular phosphatases. Aluminum fluoride also potently inhibited PLD activity in submandibular Golgi membrane-enriched fractions, suggesting an additional possible regulatory role in intracellular membrane fusion/vesicle transport. In subsequent studies, purified plant PLD from cabbage was used to confirm the AlF4- inhibitory effect in another PLD superfamily member, and to investigate the possible mechanism of this inhibition. AlF4-, but not F- or Al3+ alone, directly inhibited purified PLD activity. Inhibition occurred in both PIP2-dependent and independent PLD assays. Since AlF4- is a structural analog of phosphate, the hypothesis of a phosphate-mimicking mechanism was tested. Two additional phosphate analogs, berrylium fluoride and orthovanadate, as well as phosphate itself, inhibited PLD in a dose-dependent way. Kinetic studies suggested that cabbage PLD follows the Hill kinetic model, with a possible functional oligomerization and a high degree of cooperativity among multiple substratebinding sites. Kinetic studies also indicated that AlF4- inhibits cabbage PLD activity by direct interaction with a phosphate-binding site, through a competitive mode of inhibition in which the formation of a PLD-substrate phosphatidyl-linked intermediate is blocked. en_US
dc.format.extent 9292268 bytes
dc.format.extent 184 bytes
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dc.language en en_US
dc.language.iso en_US
dc.rights info:eu-repo/semantics/openAccess
dc.title Regulation of phospholipase D in submandibular glands en_US
dc.type info:eu-repo/semantics/doctoralThesis Oral Biology en_US Doctor of Philosophy (Ph.D.) en_US

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