Simultaneous solubilization of high-affinity receptors for VIP and glucagon and of a low-affinity binding protein for VIP, shown to be identical to calmodulin.

Anion-exchange chromatography of solubilized pig liver cell membranes on DEAE-Sepharose gave a fraction with high affinity binding proteins for VIP and glucagon distinct from each other. Scatchard analysis indicated the presence of one binding site for VIP (Kd 1.5 +/- 0.6 nM and Bmax 1.3 +/- 0.4 pmol/mg). The order of potency for VIP-related peptides to inhibit [125I]VIP binding was: VIP > peptide histidine isoleucine amide (PHI) > rat growth hormone releasing factor (rGRF) > secretin. GTP-gamma-S inhibited [125I]VIP binding and reduced the affinity of VIP binding sites to 6.5 nM. In the same isolated fraction, [125I]glucagon binding was displaced by glucagon preferentially to oxyntomodulin, and GTP did not affect this [125I]glucagon binding. Scatchard analysis indicated the presence of one binding site for glucagon (Kd 0.08 +/- 0.03 nM and Bmax 0.31 +/- 0.01 pmol/mg). A low-affinity VIP binding protein (IC50 0.7 microM) was detected in a fraction eluting later and exhibited a peptide specificity: rGRF > VIP > VIP(10-28) > secretin > PHI. This rGRF-preferring protein (18 kDa) was purified and had a partial amino-acid sequence identical to that of calmodulin. Its [125I]VIP binding was competitively inhibited by VIP and calmidazolium in a manner similar to that for pig brain calmodulin. Thus we have co-solubilized VIP and glucagon high affinity receptors from pig liver cell membranes and separated them from VIP-binding calmodulin.