The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells.
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.
Meenagh
S A
SA
Department of Agriculture and Rural Development for Northern Ireland, Veterinary Sciences Division, Stoney Road, Belfast, N. Ireland, UK BT4 3SD.
Elliott
C T
CT
Buick
R K
RK
Izeboud
C A
CA
Witkamp
R F
RF
D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2