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. MATERIAL/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.

Laboratory of Membrane Receptors Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

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