Large-Scale Purification of Beta-Adrenergic Receptors from Mammalian Cells in Culture

Abstract

S49 Mouse lymphoma wild-type cells were grown in spinner cultures of 40 liters to a density of approximately 3 million cells/ml. Growth of cells to high density (2–3 million cells/nil) required that the cell suspensions be bubbled with oxygen. Cells from 40 liter cultures were collected by centrifugation and disrupted by nitrogen cavitation. Highly purified membranes (0.35 g membrane protein) that were rich in beta-adrenergic receptor (0.4 – 0.7 pmol receptor/mg membrane protein) were prepared by differential centrifugation and then solubilized with the plant glycoside, digitonin (1.5% digitonin at 3 mg of membrane protein/ml). Beta-adrenergic receptors were isolated and purified by sequential affinity chromatography, ion-exchange chromatography, and steric exclusion high-pressure liquid chromatography. The extract was subjected to affinity chromatography on a dcrivatized Scpharose-4B CL column to which the high-affinity, beta-adrenergic antagonist (-)alprcnolol had been immobilized. Following extensive washing, the receptor bound to this matrix was eluted using a 0 – 100 micromolar linear gradient of (-)alprenolol. The receptor eluted as a sharp peak at 30 micromolar ligand and displayed a specific activity of 280 pmol receptor/mg of protein. Ion-exchange chromatography on DEAE-Sephacel increased the specific activity to 950 pmol/mg of protein. The final step in the purification, steric-exclusion high-pressure liquid chromatography on two TSK-3000 and one TSK-2000 columns, tandem linked, resulted in a beta-adrenergic receptor preparation with a specific activity of 6700 pmol/mg of protein (15, 900-fold purification). Autoradiography of the radioiodinated pure receptor, the receptor photolabcled with [¹²⁵l]iodoazidobenzylpindolol or silver-staining of chemical amounts of protein revealed that the M_r of the pure receptor is 66, 000 upon polyacrylamide gel electrophoresis in sodium dodccyl sulfate under reducing conditions. The receptor is a bcta₂-subtype adrenergic receptor.