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.
Andersson
M
M
Department of Biochemistry II, Karolinska Institute, Stockholm, Sweden.
Carlquist
M
M
Maletti
M
M
Marie
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002793:Cholic Acids; D002848:Chromatography, DEAE-Cellulose; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005934:Glucagon / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008969:Molecular Sequence Data; D011964:Receptors, Gastrointestinal Hormone / Q000187:drug effects / Q000378:metabolism*; D018027:Receptors, Glucagon; D018005:Receptors, Vasoactive Intestinal Peptide; D012995:Solubility; D013552:Swine; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D002147:Calmodulin; D002793:Cholic Acids; D003902:Detergents; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005934:Glucagon; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate