Biotinyl endothelin-1 binding to endothelin receptor and its applications.
The endothelin (ET) system consists of two membrane receptor types A and B and three 21-mer isopeptides endothelin-1, endothelin-2, and endothelin-3 as ligands. This system is involved in many physiological processes such as vasomodulation, neurotransmission, embryonic development, renal function, and regulation of cell proliferation. In many pathophysiological conditions involving endothelin system, the endothelin antagonism could be a possible clinical treatment. Designing of an antagonist involves the characterization of the binding of the test compounds to the endothelin receptors. This is being carried out using radioactive ligand. A simpler and quicker method will be of great advantage. This study reports a non-radioactive method for establishing the IC50 concentrations of the ligand. This method uses biotinylated-endothelin-1 and streptavidin conjugated with horseradish peroxidase. Hydroxyl apatite gel is used for separating the bound and unbound biotin-tagged endothelin-1. This method is applicable to detergent solubilized receptors and purified recombinant receptors. The endothelin receptor type A expressed in Pichia pastoris system has been used in this study. We show that this method is applicable in Western blot analysis of endothelin-1 and its receptor complex. This can be used to localize the receptor molecules as well.
Saravanan
K
K
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
Paramasivam
M
M
Dey
S
S
Singh
T P
TP
Srinivasan
A
A
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D001710:Biotin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D017886:Durapatite / Q000737:chemistry / Q000378:metabolism; D019332:Endothelin-1 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D005544:Forecasting; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D010843:Pichia / Q000187:drug effects / Q000235:genetics; D044022:Receptor, Endothelin A / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D019332:Endothelin-1; D008024:Ligands; D044022:Receptor, Endothelin A; C058865:Strep-avidin conjugated horseradish peroxidase; D001710:Biotin; D017886:Durapatite; D006735:Horseradish Peroxidase