Stabilization of soluble active rat liver glucagon receptor.
Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.
McVittie
L D
LD
Department of Chemistry, Indiana University.
Gurd
R S
RS
D000818:Animals; D001667:Binding, Competitive; D004355:Drug Stability; D005934:Glucagon / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008099:Liver / Q000032:analysis*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification / Q000378:metabolism*; D018027:Receptors, Glucagon; D012492:Salts / Q000494:pharmacology; D012995:Solubility
D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D012492:Salts; D006160:Guanosine Triphosphate; D005934:Glucagon