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dc.contributor.authorLi, Liangen_US
dc.date.accessioned2007-05-18T20:03:13Z
dc.date.available2007-05-18T20:03:13Z
dc.date.issued2000-05-01T00:00:00Zen_US
dc.identifier.urihttp://hdl.handle.net/1993/1909
dc.description.abstractPhospholipase 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.extent9292268 bytes
dc.format.extent184 bytes
dc.format.mimetypeapplication/pdf
dc.format.mimetypetext/plain
dc.language.isoengen_US
dc.rightsinfo:eu-repo/semantics/openAccess
dc.titleRegulation of phospholipase D in submandibular glandsen_US
dc.typeinfo:eu-repo/semantics/doctoralThesis
dc.typedoctoral thesisen_US
dc.degree.disciplineOral Biologyen_US
dc.degree.levelDoctor of Philosophy (Ph.D.)en_US


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