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Title: The enzymological characterization of the inositol phosphatase synaptojanin
Author: Knott, Jessica Mary May
ISNI:       0000 0004 2689 5738
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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Synaptojanin is unique in that it is the only mammalian inositol phosphatase to contain more than one catalytic domain; a CX5R Suppressor of Actin (Sac) domain and an Inositol Polyphosphate Phosphatase Catalytic (IPPc) domain. The enzyme has been shown to play a crucial role in synaptic vesicle recycling and its functioning has been implicated in the onset of Alzheimer’s disease. Many domains and sub-units in bi- and multi-functional enzymes are found to operate in a co-dependent manner. In this work, the possibility that the functioning of the Sac and IPPc domains in Synaptojanin are co-dependent was investigated. The kinetic parameters of the IPPc domain were assessed in a double Sac/IPPc phosphatase and compared to that in both a double phosphatase where the Sac activity had been rendered inactive and a single IPPc phosphatase. It was found that the VMax activity of the IPPc domain towards its lipid substrate PI(4,5)P2 is significantly lower when the Sac domain is not present or functional. Likewise, it was found that the VMax activity of the Sac domain towards PI(4)P is reduced when the IPPc domain is removed or its activity rendered inactive. Interfacial recognition and substrate channelling were investigated as mechanisms to explain the domain dependency. However, they were found not to contribute to the observed differences in VMax. Instead, it seems likely that protein-lipid interactions induce the dependency. In summary, this thesis presents the first evidence that the catalytic domains in Synaptojanin act in a co-operative manner and probes the mechanism by which the domains interact.
Supervisor: Parker, Kim ; Ces, Oscar Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral