Isolation of plasma membrane compartments from rat brain cortex; detection of agonist-stimulated G protein activity
Lenka Bourova, Jiri Stohr, Vaclav Lisy, Vladimir Rudajev, Jiri Novotny, Petr Svoboda
Med Sci Monit 2009; 15(4): BR111-122
Heterotrimeric guanine nucleotide-binding proteins (G proteins) play an essential role in linking cell-surface receptors to effector proteins at the plasma membrane. The functional activities of G proteins in various plasma membrane compartments remain to be elucidated.
Material and Method: Plasma membranes from rat cerebral cortex were isolated on Percoll and fractionated by sucrose-density gradient. Fractions were screened for plasma membrane markers and signaling molecules. G-protein activity was determined by agonist-stimulated gamma-32P-GTPase or 35S-GTPgammaS binding. The largest content of markers was found at the 35% to 40% (w/v) sucrose interface. This fraction was defined as the bulk of the plasma membrane. The low-density plasma membrane fraction was localized in 15% to 20% (w/v) sucrose.
Results: Both bulk and low-density plasma membrane fractions were characterized by high levels of nonspecific, low-affinity GTPase activity and basal, high-affinity GTPase activity. Baclofen-stimulated GTPase activity was twice as high in the bulk fraction as in the low-density fraction. The effect of other G protein-coupled receptor agonists was not significant. 35S-GTPgammaS saturation-binding experiments measured with increasing concentrations of GDP revealed high-affinity sites that were clearly distinguishable from basal binding and responded to agonists in the following order of efficacy: baclofen >(DADLE) >(DAMGO) >U-69593.
Conclusions: The method presented here describes a straightforward method for the isolation of clearly defined plasma membrane preparations from rat brain cortex. Quantitative assessment of G-protein activity, particularly the high basal activity, differs from that reported in membrane fractions from HEK 293 cells.
Keywords: Humans, Guanosine 5'-O-(3-Thiotriphosphate) - metabolism, GTP-Binding Proteins - metabolism, GTP Phosphohydrolases - metabolism, Enzyme Activation, Electrophoresis, Polyacrylamide Gel, Cell Membrane - metabolism, Cerebral Cortex - metabolism, Cell Line, Blotting, Western, Baclofen - pharmacology, Animals, Adenylate Cyclase - metabolism, Male, Rats, Rats, Wistar