[35S]-GTPγS binding experiments
[35S]-GTPγS experiments were performed on crude
membrane fractions of MOR-expressing HEK293 cells to determine
mu-agonist-induced G-protein activation (as reflected by the exchange
rate of GDP for GTP) at different pH values (6.5-7.4). GTP was replaced
by a high concentration of [35S]-GTPγS in the
assay solvent, and the accumulation of
[35S]-GTPγS-bound G proteins in the membrane was
measured. To this end, membranes were homogenized and dissolved in HEM
G-protein buffer at pH 7.4 or 6.5, including freshly added 0.1 % (w/v)
BSA and 1 mM dithiotreitol (DTT). In analogy to (Ludwig et al., 2003),
50 µg of membrane fractions in duplicates were incubated with GDP (30
µM) and [35S]-GTPγS (0.05 nM) for 90 min at
30oC to determine dose-response curves and
EC50 values. Unspecific
[35S]-GTPγS binding in the presence of
non-radioactive GTPγS (10 µM) was subtracted to yield specific binding.
Bound and free ligands were separated by rapid filtration under vacuum
through Whatman GF/B glass fibre filters soaked in water followed by 6
washes with Tris Buffer. Bound radioactivity was determined by liquid
scintillation spectrophotometry for 35S after
overnight extraction of the filters in scintillation fluidoptiphase HISAFE3 . Concentrations of radioactive compound were
calculated based on the half-life of 35S (87.4 days).
Experiments were randomized to compensate for position effects in the
filter apparatus or unequal sample processing times. Data processing and
analysis were blinded for pH values (6.5 or 7.4) with the help of a
colleague.