SOLUBILIZATION AND MOLECULAR CHARACTERIZATION OF ACTIVE PANCREASTATINRECEPTORS FROM RAT-LIVER MEMBRANES

Pancreastatin receptors were solubilized from rat liver membranes with the nonionic detergent Triton X-100. Binding of a iodinated analog of rat pancreastatin ([I-125-Tyr(0)]pancreastatin) to the soluble fracti on was time dependent, saturable, and reversible. Scatchard analysis o f binding under equilibrium conditions indicated that the soluble extr acts contained a single class of pancreastatin-binding sites, with a b inding capacity of 14 fmol/mg protein and a K-d of 0.3 nM. As observed with membrane-bound receptors, binding of [I-125]pancreastatin to sol uble extracts was inhibited by guanine nucleotides with the following rank order of potency: guanyl-5'-yl-imidodiphosphate > GTP > GDP > GIM P, indicating that the soluble receptors are functionally linked to G proteins. Molecular analysis of the soluble pancreastatin receptor by covalent cross-linking to [I-125]pancreastatin using disuccinimidyl su berate and further identification on SDS-PAGE indicated a single band of 85,000 M(r). Gel filtration of soluble extracts on Sephacryl S-300 revealed two molecular components viith binding abilities (M(r), 80,00 0 and 170,000). The higher molecular mass component was more sensitive to guanine nucleotides, and covalent cross-linking of both components to [I-125]pancreastatin and further SDS-PAGE analysis revealed again a single band of 85,000 M(r), suggesting an association of the recepto r with a G protein. Moreover, direct evidence that a G(q) was present in the same chromatographic fraction was obtained by specific immunode tection. The soluble receptor is a glycoprotein that can be specifical ly bound to the wheatgerm agglutinin lectin. We conclude that we solub ilized active pancreastatin receptors from rat liver membranes, and th ese results support the conclusion that the liver pancreastatin recept or consists of a 80,000 M(r) glycoprotein associated with G proteins.