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Detection of phosphoinositide‐binding proteins in Tetrahymena vorax using liposomes as an affinity binding matrix
Author(s) -
Yarbrough Thomas Michael,
Ryals Phillip E
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.939.12
Subject(s) - microsome , phosphatidylethanolamine , biochemistry , chemistry , liposome , phospholipid , chinese hamster ovary cell , tetrahymena , phosphatidylinositol , biology , phosphatidylcholine , enzyme , membrane , receptor , kinase
Tetrahymena vorax , a unicellular eukaryotic organism, was surveyed for the presence of proteins that bind to inositol‐containing phospholipids. Cultures of T. vorax were homogenized at logarithmic growth phase, and microsomal fractions isolated by ultracentrifugation. All fractions were assayed for the microsomal marker cytochrome c reductase. Fractions enriched in microsomes were carbonate extracted and dialyzed. Microsomal protein samples were incubated with control liposomes containing biotin‐tagged phosphatidylethanolamine and lacking any phosphoinositides, and experimental liposomes contained the same lipids and an inositol phospholipid. After the liposomes were allowed to bind to streptavidin‐agarose beads, protein was electrophoresed and stained with SYPRO‐Ruby. Preliminary data show at least two bands (44 and 49.5 kDa) in the experimental samples that have greater staining intensity than the corresponding control bands, suggesting the presence of phosphoinositide‐binding proteins in the microsomal samples. Further investigation of phosphoinositide‐binding proteins is underway using protein from macrostomal and microstomal cell types of Tetrahymena patula .