Characterization and use of crude alpha-subunit preparations for quantitative immunoblotting of G proteins.
G proteins are heterotrimeric membrane-associated proteins that couple a large number of receptors to a variety of effector systems within the cell. Characterization of G proteins expressed in a particular cell type represents an important first step in defining the potential candidates to which a receptor might couple. A difficulty often encountered using G protein antisera from various commercial and private sources is relating the intensity of bands on a Western blot to the relative amount of G protein present in a membrane preparation. This problem is especially noteworthy when comparing across G protein subtypes due to differences in titer, affinity, and specificity among various antisera. Conventional approaches to obtaining G protein standards of sufficient purity to address these issues in a quantitative manner are time-consuming and difficult, but the procedures outlined herein demonstrate a method for using DEAE fractions from Escherichia coli expressing individual alpha-subunits. The key features of the present approach are to estimate saturable GTP gamma S binding in each alpha-subunit preparation and calculate the moles of alpha-subunit present in the respective preparations based on the known stoichiometry of GTP gamma S binding (1:1). The extent of correspondence between GTP gamma S binding and immunoreactivity is then determined by trypsin protection assays, which estimate the proportion of immunodetectable G protein which can bind GTP gamma S. After characterization in this manner, DEAE fractions from bacteria transformed with the respective cDNA for Gi alpha-1, G1 alpha-2, and G1 alpha-3 were used to construct standard curves on Western blots and estimate endogenous G protein concentrations in cell lines (CHO and HeLa) and across species (rat and mouse) in isolated adipocyte preparations. Plasma membranes from CHO cells contained Gi alpha-2 (4.8 +/- 0.3 pmol/mg protein) and Gi alpha-3 (0.6 +/- 0.1 pmol/mg protein), but not Gi alpha-1, while HeLa cell membranes contained Gi alpha-1 (0.11 +/- 0.01 pmol/mg protein) and Gi alpha-3 (1.3 +/- 0.1 pmol/mg protein), but not Gi alpha-2. In contrast, rat and mouse adipocyte membranes contained Gi alpha-1 (48 +/- 2 vs 36 +/- 2 pmol/mg protein), Gi alpha-2 (77 +/- 1.5 vs 25 +/- 1.4 pmol/mg protein), and Gi alpha-3 (26 +/- 1.2 vs 15 +/- 1 pmol/mg protein). The method described herein provides an innovative solution to the technically difficult problem of obtaining pure standards for the assay of G protein alpha-subunits and does so using simple biochemical and immunological techniques.
Gettys
T W
TW
Department of Medicine, Medical University of South Carolina, Charleston 29425.
Sheriff-Carter
K
K
Moomaw
J
J
Taylor
I L
IL
Raymond
J R
JR
D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008297:Male; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis*; D051381:Rats; D012015:Reference Standards; D014357:Trypsin
D010446:Peptide Fragments; D014357:Trypsin; D019204:GTP-Binding Proteins