Universität zu Köln

Bier, Dirk ORCID: 0000-0001-9231-4303, Schulze, Annette, Holschbach, Marcus, Neumaier, Bernd and Baumann, Arnd (2020). Development and Evaluation of a Versatile Receptor-Ligand Binding Assay Using Cell Membrane Preparations Embedded in an Agarose Gel Matrix and Evaluation with the Human Adenosine A(1)Receptor. ASSAY DRUG DEV. TECHNOL., 18 (7). S. 328 - 341. NEW ROCHELLE: MARY ANN LIEBERT, INC. ISSN 1557-8127

Full text not available from this repository.

Abstract

Guanosine-5 '-triphosphate (GTP)-binding protein-coupled receptors are the target of up to 40% of prescribed medications worldwide. To evaluate the suitability of novel receptor ligands, frequently elaborate, time-consuming, and expensive receptor-ligand interaction studies have to be carried out. This work describes the development and proof of principle of a rapid, sensitive, and reliable receptor-ligand binding assay. CHO cells were stably transfected with a construct encoding the human A(1)adenosine receptor (hA(1)AR). For ligand binding assays, membranes from these cells were prepared and embedded in low melting point agarose. These immobilized samples were incubated with tritiated 8-cyclopentyl-1,3-dipropylxanthine ([H-3]DPCPX), a well-established receptor antagonist. TheK(D)andB(max)values as well as kinetic parameters (k(on)andk(off)) of receptor-ligand interaction were determined. Unspecific binding of various radiotracers to either the carrier material or the agarose gel matrix was negligible. The dissociation constant (K-D) for [H-3]DPCPX at the hA(1)AR was determined by saturation, competition binding, and kinetic experiments. These studies resulted inK(D)values of similar to 3 nM, which is in good accordance with previously published data obtained from conventional receptor-ligand binding assays. The procedure described in this study simplifies classical binding studies to a kit-like assay. The receptors retained their binding properties even when preparations were dried completely. Transport and delivery of the material are conceivable without loss of biological activity. Therefore, other laboratories can perform binding studies without special equipment or the necessity to run a cell culture laboratory and/or to dissect tissue on their own.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Bier, Dirk UNSPECIFIED orcid.org/0000-0001-9231-4303 UNSPECIFIED Schulze, Annette UNSPECIFIED UNSPECIFIED UNSPECIFIED Holschbach, Marcus UNSPECIFIED UNSPECIFIED UNSPECIFIED Neumaier, Bernd UNSPECIFIED UNSPECIFIED UNSPECIFIED Baumann, Arnd UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-325524
DOI:	10.1089/adt.2020.991
Journal or Publication Title:	ASSAY DRUG DEV. TECHNOL.
Volume:	18
Number:	7
Page Range:	S. 328 - 341
Date:	2020
Publisher:	MARY ANN LIEBERT, INC
Place of Publication:	NEW ROCHELLE
ISSN:	1557-8127
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language A(1); BRAIN; RADIOLIGAND; PHARMACOLOGY; ANTAGONIST; CONSTANT; POTENT; SLEEP Multiple languages Biochemical Research Methods; Pharmacology & Pharmacy Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/32552