Isolation of plasma membrane compartments from rat brain cortex; detection of agonist-stimulated G protein activity.
BACKGROUND
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.


METHODS
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.
Bourova
Lenka
L
Laboratory of Membrane Receptors, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. bourova@biomed.cas.cz
Stohr
Jiri
J
Lisy
Vaclav
V
Rudajev
Vladimir
V
Novotny
Jiri
J
Svoboda
Petr
P