Proteomic analysis of transducin β-subunit structural heterogeneity

James W. Clack, Martha Juhl, Carol A. Rice, Junyu Li, Frank A. Witzmann

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

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 (Tβ) 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/, 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 GI/GS/GT β-subunit 1 (GNB1; Tβ1). This suggested that post-translational modification was responsible for the differences in p/. Phosphorylation experiments showed that at least one Tβ1 spot was phosphorylated in vitro with [γ-32P]ATP by an endogenous kinase. Treatment of Tβ with alkaline phosphatase caused a large change in the spot pattern of Tβ, suggesting that phosphorylated Tβ is a substrate for alkaline phosphatase. We conclude that Tβ1 constitutes over 99% of the Tβexpressed 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.

Original languageEnglish (US)
Pages (from-to)3493-3499
Number of pages7
JournalELECTROPHORESIS
Volume24
Issue number19-20
StatePublished - Oct 1 2003

Keywords

  • G-Proteins
  • Photoreceptors
  • Post-translational modification
  • Proteomics
  • Signal transduction

ASJC Scopus subject areas

  • Clinical Biochemistry

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  • Cite this

    Clack, J. W., Juhl, M., Rice, C. A., Li, J., & Witzmann, F. A. (2003). Proteomic analysis of transducin β-subunit structural heterogeneity. ELECTROPHORESIS, 24(19-20), 3493-3499.