Isoproterenol-induced subcellular redistribution of G-protein beta subunits in S49 lymphoma cells demonstrated by a novel competitive ELISA.
A novel competitive ELISA has been developed for the determination of levels of the beta subunit of guanine-nucleotide-binding protein (G-protein) using antipeptide antibodies directed against the amino terminus of the beta subunit. Because beta subunits form highly hydrophobic.heterodimeric complexes with gamma subunits of G-proteins, specific assay conditions were required. Optimal concentrations of antibodies, detergents, Mg2+ as well as ionic strength were determined. In addition, we found that an effective binding of the used antibodies to the beta subunit was ensured only after denaturation of the beta gamma complexes. Subsequently, this ELISA was used for quantitation of the beta subunit in subcellular fractions of S49 lymphoma cells during isoproterenol-mediated desensitization of beta-adrenergic controlled transmembrane signalling system. A 10 min as well as 60 min treatment of the cells with isoproterenol (1 nmol/ml) resulted in a significant shift of G-protein beta subunits (presumably as beta gamma complexes) from the plasma membrane fractions to low-density microsomal fractions. No significant change was detected after the hormone action in the distribution of plasma membrane constitutive enzymes. In conclusion, the developed ELISA helped us to reveal that beta-adrenergic stimulation can induce redistribution of the beta gamma dimer from plasma membranes to low-density microsomes.
Novotny
J
J
Wallenberg Laboratory for Cardiovascular Research, Gothenburg University, Sweden.
Kvapil
P
P
Bokoch
G M
GM
Ransnäs
L A
LA
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002462:Cell Membrane / Q000737:chemistry; D003902:Detergents; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019204:GTP-Binding Proteins / Q000032:analysis*; D007545:Isoproterenol / Q000494:pharmacology*; D008223:Lymphoma / Q000737:chemistry* / Q000648:ultrastructure; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000737:chemistry; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D000318:Adrenergic beta-Agonists; D003902:Detergents; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019204:GTP-Binding Proteins; D008274:Magnesium; D007545:Isoproterenol