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The Role of GTP-binding Proteins in Exocytosis

Lillie, Thomas Henry Wallace; (1992) The Role of GTP-binding Proteins in Exocytosis. UNSPECIFIED thesis (Ph.D), UCL (University College London). Green open access

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Streptolysin-0 permeabilised rat mast cells release their granule contents by exocytosis in response to micromolar levels of Ca2+ and GTP-γ-S, the dual-effectors. Provision of ATP shifts the sensitivity of secretion to these dual-effectors to lower concentrations. ATP can also maintain the responsiveness of permeabilised cells to the dual effectors, restore responsiveness in permeabilised cells and delay the onset of exocytosis. Other non-adenine nucleoside triphosphates can also increase the sensitivity to the dual-effectors. This is an indirect effect, involving the phosphorylation of endogenous ADP to generate ATP, a reaction catalysed by nucleoside diphosphate kinase. When rat mast cells are permeabilised in iso-osmotic solutions composed of zwitterions, secretion occurs in response to either Ca2+ or GTP-γ-S when ATP is also provided. The response to GTP-γ-S does not require ATP (although its presence does enhance the response) or Ca2+, but is dependent on Mg2+. The response to Ca2+ is totally dependent on the presence of ATP or other nucleoside triphosphates. It can be inhibited by GDP, and it would appear ATP is being used as the substrate for generation of GTP from endogenous GDP. This being the case, secretion appears to be dependent on provision of guanine nucleotide and, by implication, a GTP-binding protein. The presence of Ca2+ is not necessary, although when present it modulates the response to guanine nucleotides. Studying the kinetics of secretion reveals that Ca2+ and GTP-γ-S stimulated secretion is modulated by Mg2+. This modulation bears similarity to the effects of Mg2+ on the Gs regulated adenylyl cyclase system. The results are interpreted in terms of the interaction of Mg with GTP-binding proteins. Using in-situ oxidisation and reduction of radioactive guanine nucleotides it is possible to label GTP-binding proteins inside Streptolysin-0 permeabilised cells. I have labelled GTP-binding proteins in rat hepatocytes, and am able to detect changes in labelling of G 5 in response to stimulation with glucagon. It is hoped that this method will allow identification of GTP-binding proteins involved in exocytosis in rat mast cells.

Type: Thesis (UNSPECIFIED)
Qualification: Ph.D
Title: The Role of GTP-binding Proteins in Exocytosis
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest
Keywords: Pure sciences; Mast cells
URI: https://discovery.ucl.ac.uk/id/eprint/10111930
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