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Full-Text Articles in Biochemistry
Investigation Of The Saccharomyces Cerevisiae Gpi Transamidase: Insights Into Its Activity And Subunit-Subunit Interactions, Travis Ness
Wayne State University Dissertations
Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, posttranslational
modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces
cerevisiae GPI-T is composed of five membrane-bound subunits: Gaa1, Gpi8, Gpi16,
Gpi17, and Gab1. Structural and functional studies have been hindered by the
complexity of this enzyme. Conditions to purify the Gpi8:Gaa1:Gpi16 GPI-T heterotrimer
from yeast have been reported, but an understanding of the subunit functions,
interactions, and stoichiometry remain unclear. Furthermore, a reliable, quantitative, in
vitro assay for this important post-translational modification has remained elusive for
nearly three decades.
Our laboratory has developed an in vitro peptide cleavage assay that correlates
changes …
Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka
Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka
Wayne State University Dissertations
DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE
By
SANDAMALI AMARASINGHA EKANAYAKA
December 2013
Advisor: Dr. Tamara L. Hendrickson
Major: Biochemistry
Degree: Doctor of Philosophy
The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for …