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Molecular and enzymatic characterization of mammalian phospholipase C zeta (PLCϚ)

Nomikos, Michail 2005. Molecular and enzymatic characterization of mammalian phospholipase C zeta (PLCϚ). PhD Thesis, Cardiff University.

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In mammalian oocytes, the fertilising sperm evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation and embryonic development. Although the exact mechanism leading to the initiation of Ca2+ oscillations is still unclear, accumulating evidence suggests that sperm-specific phospholipase C zeta (PLC zeta) is delivered from the fertilising sperm into the ooplasm, triggering the Ca2+ oscillations through the inositol 1,4,5-trisphosphate (InsP3) pathway. PLC zeta is the smallest known mammalian PLC isoform comprising of two EF hand, a C2 and the X and Y catalytic core domains. In this study we examined the biochemical properties of recombinant bacterially expressed mouse PLC zeta (m PLC zeta) using the well-characterised rat PLC zeta1 (rPLC zeta1) as control. Using a PtdInsP2 hydrolysis assay we showed that both isoforms had a similar Km for PtdIns(4,5)P2 and that PLC zeta had a much higher Ca2+ sensitivity, which would predict it to be active at resting Ca2+ concentrations in eggs. PLC zeta bound with high affinity to PtdIns(3,5)P2 and PtdIns(4,5)P2 even though it lacks a PH domain from its sequence, which targets PLC zeta1 to PtdIns(4,5)P2. A series of domain deletion constructs of PLC zeta were used to demonstrate the role of the EF hands on the Ca2+ sensitivity of PLC zeta and the role of C2 domain and XY linker on its binding to PtdInsP2. Luminescent PLC constructs were generated to examine their potential to elicit Ca2+ oscillations, quantifying their expression levels in mouse eggs. Anti-human PLC zeta (h PLC zeta) monoclonal antibodies were produced and their ability to block the in vitro hydrolysing activity of recombinant h PLC zeta was tested.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: R Medicine > R Medicine (General)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 31 Jan 2020 03:38

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