Hormone Receptors III. PROPERTIES OF GLUCAGON-BINDING PROTEINS ISOLATED FROM LIVER PLASMA MEMBRANES

摘要: Abstract Purified rat liver plasma membranes, rich in glucagon-stimulatable adenylate cyclase activity, were extracted by the non-ionic detergent, Lubrol-PX. Glucagon-binding proteins could be identified crude and purified membrane extracts gel filtration following saturation of binding sites with 125I-glucagon before or after extraction. The Hummel-Dreyer principle was adapted to provide a quantitative microbinding assay requiring as little 0.25 µg partially glucagon-binding proteins. apparent molecular size proteins, determined on columns, function presence absence, concentration, detergent. Chromatography 8% agarose 0.5% Lubrol yielded over 1.5 x 106 weight; under these conditions much also associated micelles detergent form complex about 125,000 weight. bound either native radioactive glucagon, but not adrenocorticotropic hormone. remained soluble even reduction concentration below 0.001% ultrafiltration; 75-fold increase specific activity accompanied this step. Filtration solution absence separated two-binding one included excluded pores gel. No micellar glucagon appeared conditions. detergent-reduced further 40-fold chromatography hydroxylapatite; small amount protein, containing essentially all recovered retained adsorbent low ionic strength buffers. Relative original Lubrol-PX extract, an over-all 3000-fold realized. Binding saturable process. Partially protein exhibited, Scatchard plots, two classes sites, high affinity (Ka = 8.7 109 m-1) other quite affinity. 125I-glucagon, which complete 15 20 min at 30°, readily reversible merely application gradient hormone-binding complex. Native both competed for displaced hormone from its protein. Concentrations 0.01% greatly inhibited binding, did pH values greater than 8.5. whose weight estimated calibrated column 190,000, 125I-insulin, albeit lesser extent 125Iglucagon, 125I-adrenocorticotropic all.