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Abstract

1. ¹²⁵I-labelled secretin bound rapidly and specifically to membranes from cat pancreas. Binding of labelled hormone was competitively inhibited by unlabelled secretin in the same range of concentrations that stimulated pancreatic adenylate cyclase in these membranes. The dissociation constant of the membrane binding sites for unlabelled secretin as evaluated by these displacement experiments was 4.1-10^-9 M and the number of binding sites 1.0 pmol per mg of membrane protein. 2. Studies using different concentrations of [¹²⁵I]secretin (at a constant ratio of labelled to unlabelled hormone) revealed a similar value of 4-4-10^-9 M for the dissociation constant. 3. Both the association and dissociation rate constants of [¹²⁵I]secretin binding were temperature sensitive; the dissociation rate constant increased more rapidly with increase in temperature. The ratio k_-1/k₊₁ (at 22 degrees C) gave a dissociation constant of 3.7-10^-9M which agrees closely with the figure obtained from equilibrium data. These data indicate that ¹²⁵I-labelled secretin and unlabelled secretin bind to the same binding site on pancreatic membranes, with high affinity. 4. Unlabelled secretin stimulated pancreatic adenylate cyclase with an apparent K_m of 8.4-10^-9 M, while [¹²⁵I]secretin apparently did not stimulate the adenylate cyclase. Together with the binding data this might suggest that different portions of the secretin molecule are responsible for binding and adenylate cyclase activation. 5. Studies on the specificity of [¹²⁵I]secretin binding carried out with various peptide hormones (glucagon, human gastrin, pancreozymin and caerulein) which are all inefficient in stimulating pancreatic fluid secretin, showed that these hormones have no influence on the binding of [¹²⁵I]secretin. In contrast, vasoactive intestinal polypeptide, which stimulates pancreatic fluid and bicarbonate secretion, showed a competitive inhibition of secretin binding to the plasma membrane preparation.