Date of Award
Doctor of Philosophy
Zoology and Genetics
Sheldon S. Shen
This work was the first to provide direct evidence for the presence of a PLCbeta isoform within the sea urchin egg, named suPLCbeta. The coding sequence was identified and cloned from the eggs of Lytechinus pictus and when translated, determined to have the greatest degree of homology and identity with the mammalian PLCbeta4 isoforms. This alone is a unique finding in that the presence of PLCbeta4 has only been found in the retina and specific neuronal tissues. The presence of the protein within the egg was verified using antibodies that were generated specifically against suPLCbeta, which indicate that the majority of the enzyme is localized in the non-soluble fraction of the cell, presumably associated with the plasma membrane. This distribution is found both in the unfertilized eggs as well as one minute post-fertilization. In order to further characterize the enzymatic activity of suPLCbeta, recombinant proteins were expressed and purified from Sf9 cells as GST-fusion proteins. Initial experiments were carried out with the fusion protein bound to Glutathione-Sepharose beads using the level of hydrolytic activity of the PLC as a measure of activity. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is dependent on the levels of free calcium that are present. Removal of the GST tag via Thrombin-mediated proteolysis not only freed the recombinant suPLCbeta from the Sepharose beads, but also resulted in a truncated form of the protein that was missing the first 93 amino terminal amino acid residues. When assayed for its enzymatic activity, this truncated form still possessed its calcium-dependent hydrolytic activity that was regulated by Gbetagamma.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Andre Peter Joseph Kulisz
Kulisz, Andre Peter Joseph, "The identification, cloning, and characterization of a Phospholipase C-beta isoform from the sea urchin egg " (2004). Retrospective Theses and Dissertations. 1105.