Proteomic analysis of transducin β-subunit structural heterogeneity

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 beta-subunit of transducin (T-beta) 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 pl, 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) beta-subunit 1 (GNB1; T-beta1). This suggested that post-translational modification was responsible for the differences in pl. Phosphorylation experiments showed that at least one T-beta1 spot was phosphorylated in vitro with [gamma(-32)P]ATP by an endogenous kinase. Treatment of T-beta with alkaline phosphatase caused a large change in the spot pattern of T-beta, suggesting that phosphorylated T-beta is a substrate for alkaline phosphatase. We conclude that T-beta1 constitutes over 99% of the T(beta)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.