The preparation, solubilisation and binding characteristics of a β2-adrenoceptor isolated from transfected Chinese hamster cells

Abstract

beta 2-Adrenoceptor agonists (beta-agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta-agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta 2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg-1 of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta-agonists, with IC50 values ranging from 2.1 +/- 0.76 x 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta 2-adrenoceptors are a convenient source of active receptor material. Solubilised beta 2-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells

“…At present, few of the radio-receptor assays have already been developed for multiresidue detection of β-agonists using natural membrane-bound β 2 -AR prepared from bovine teat muscles [ 34 ], or recombinant β 2 -AR expressed in Chinese hamster ovary cells [ 35 ], E . coli [ 36 ], and NCB20-D1 cells [ 37 ].…”

Solubilisation and binding characteristics of a recombinant β2-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of β-agonists and antagonists residues in food-producing animals

“…The ␤ 2 -adrenergic receptor has already been functionally expressed in a variety of different organisms including insect and mammalian cells [17] or Escherichia coli [18,19]. Radio-receptor assays (RRA) have already been described for multianalyte detection of ␤-agonist residues using natural membrane bound ␤ 2 -adrenergic receptors prepared from bovine teat muscles [20], or recombinant ␤ 2 -adrenoreceptors expressed in membranes of a transfected Chinese hamster ovary (CHO) cell line [21] or expressed in human embryonic kidney cells [22]. In the past, we developed a multi-residue screening method for the detection of ␤ 2 -adrenergic residues in urine samples using a radioactive tracer and a genetically modified E. coli expressing the human ␤ 2 -adrenergic receptor in their inner membrane [23].…”

Identification of an unknown β‐agonist in feed by liquid chromatography/bioassay/quadrupole time‐of‐flight tandem mass spectrometry with accurate mass measurement

“…This is particularly relevant in screening methods for compounds for which no detailed information is present with regard to the identity. Meenagh et al 9 developed a competition binding assay for b-agonists based on solubilised human b2-adrenoreceptors isolated from transfected Chinese hamster ovary cells and the tritiated antagonist dihydroalprenolol as radioactivity label. Such an assay is able to detect novel derivatives, which exhibit the same pharmacological action as b-agonists but may have modified chemical structures.…”