Proteomic analysis of transducin beta-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 pI, 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 pI. 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.
Clack
James W
JW
Department of Biology, Indiana University - Purdue University at Indianapolis, Columbus 47203, USA. jclack@iupui.edu
Juhl
Martha
M
Rice
Carol A
CA
Li
Junyu
J
Witzmann
Frank A
FA
D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015293:Transducin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*
D015293:Transducin