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JW Clack, M Juhl, C Rice, FA Witzmann; Proteome Analysis of Structural Heterogeneity of Transducin ß Subunit . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1388.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the nature of the structural heterogeneity of the beta subunit of Transducin (Tß). Methods: Partially purified Transducin was resolved using 2-D gel electrophoresis using both IPG/small-format gels and IEF tubes/large-format gels. Peptide mass fingerprinting of several different spots believed to correspond to Tß was performed. Spots were excised and proteolyzed using modified trypsin. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on the peptide mixture resulting from each spot. Results: As many as six spots with different pI«s, ranging from 5.2 to 6.1, were observed when separated using 2-D electrophoresis. MALDI Peptide mass fingerprinting determined with high probability (9-18 peptide mass matches per spot, coverage of 29-40%) that all of the spots were the same gene product, Guanine nucleotide-binding protein GI/GS/GT beta subunit 1 (P04901). This suggested that post-translational modification was responsible for the differences in pI. A subset of the Tß spots were specifically recognized by a polyclonal anti-farnesyl antibody; treatment with methyl iodide induced loss of anti-farnesyl reactivity and a large shift (≷ 1 pH unit) in pI of the spots, although some heterogeneity remained. Preliminary phosphorylation experiments using [γ-32P]ATP indicated that Tß1 is phosphorylated, perhaps to different extents. Conclusions: Tß1 displays structural heterogeneity that is probably due to post-translational modifications. Preliminary experiments suggest that Tß1 may be farnesylated and/or phosphorylated. CR: None. Support: AFSOR Grants F49620-99-1-0153 and F49620-00-1-0225 to FAW.
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