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Proteomic analysis of transducin β‐subunit structural heterogeneity
Author(s) -
Clack James W.,
Juhl Martha,
Rice Carol A.,
Li Junyu,
Witzmann Frank A.
Publication year - 2003
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305520
Subject(s) - transducin , peptide , protein subunit , trypsin , phosphorylation , phosphatase , chemistry , gel electrophoresis , microbiology and biotechnology , peptide mass fingerprinting , biochemistry , biology , enzyme , proteomics , g protein , signal transduction , gene
Partially purified transducin was resolved using two‐dimensional gel electrophoresis (2‐DE). Peptide mass fingerprinting of several different spots believed to correspond to the 37 kDa β‐subunit of transducin (T β ) 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. As many as six spots with different p I , ranging from 5.2 to 6.1, were observed when separated using 2‐DE. MALDI peptide mass fingerprinting determined with high probability that all of the spots were the same gene product, guanine nucleotide‐binding protein G I /G S /G T β‐subunit 1 (GNB1; T β1 ). This suggested that post‐translational modification was responsible for the differences in p I . Phosphorylation experiments showed that at least one T β1 spot was phosphorylated in vitro with [γ‐ 32 P]ATP by an endogenous kinase. Treatment of T β with alkaline phosphatase caused a large change in the spot pattern of T β , suggesting that phosphorylated T β is a substrate for alkaline phosphatase. We conclude that T β1 constitutes over 99% of the T β expressed in bovine rod outer segments and displays structural heterogeneity that is due to post‐translational modification. We also conclude that some, but not all, of the heterogeneity observed is due to phosphorylation of Tb1.