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atch was tested with at least four increasing protein concentrations, and only the straight
lines presenting slopes similar to that obtained with the standard hormone (10-100 pg/tube)
were considered as positive. Specific activity, as it represents the quantity of CGRP-like
molecules (ng) per µg of protein, was calculated. Receptor binding ability of each purified
fraction (ED50) was also determined and expressed as the quantity of protein (mg) that
induced a 50% inhibition of the initial binding to rat liver membranes. The experiment was
performed in triplicate.
2.3.1.9 Gastrin and CGRP radioimmunoassay (RIA)
The presence of gastrin-like molecules in the crude extracts was determined by gastrin-
radioimmunoassay. Rabbit antiserum, synthetic 125 I as tracer, and synthetic gastrin 1 as
standard were used (GASK-PR, CIS Bio International). Results were expressed as pg of
bioactive molecules per mg of dry weight. ED50 was also determined and expressed as the
quantity of protein (mg) that induced a 50% inhibition of the initial binding of CCK to its
specific antibody.
The quantity of immunoreactive CGRP-like molecules presented in the fractions collected
after molecular sieving was measured following a previously described assay for human
CGRP (Fouchereau-Peron et al. 1990): in brief, an anti-CGRP antiserum at a final dilution of
1/150,000 was incubated with serial dilutions of synthetic human CGRP or fractions of cod
hydrolysates collected after molecular sieving (18 h at 30°C). Then, 125 I labelled human
CGRP was added and the incubation continued during 24 hours at 4°C. Bound and free
hormone were separated by charcoal-dextran precipitation. Results were expressed as pg of
CGRP-like molecules per ml.
Control (specific antibody omitted) tubes were incubated in each assay. The detection limit
for radioimmunoassay was 10 pg of immunoreactive peptide per tube.
2.3.1.10 Liver membrane preparation
Liver membranes were prepared using male Wistar rats according to the method of Neville
until step 11 (Neville 1968). Proteins were quantified by the method of Lowry using BSA as
standard (Lowry et al. 1951).
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