Premium
ISOLATION AND CHARACTERIZATION OF THE RAT LIVER AVP RECEPTOR USING [ 125 I][d(CH 2 ) 5′ SARCOSINE 7 ]AVP 1
Author(s) -
Trinder Deborah,
Kelly Janice M.,
Fernley Ross,
Mooser Vincent,
Phillips Paddy A.,
Johnston Colin I.
Publication year - 1992
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.1992.tb00447.x
Subject(s) - sarcosine , chaps , chemistry , fast protein liquid chromatography , vasopressin , receptor , size exclusion chromatography , affinity chromatography , chromatography , arginine , membrane , biochemistry , amino acid , biology , high performance liquid chromatography , endocrinology , glycine , enzyme
SUMMARY 1. A vasopressin (AVP) binding protein was purified from rat liver membranes by an improved method using [ 125 I][d(CH 2 ) 5′ Sarcosine 7 ]AVP, a selective Vi AVP radioligand and a combination of CHAPS solubilization, gel filtration, lectin affinity and FPLC ion exchange chromatography. 2. The purified protein exhibited a maximum binding activity of 2480 pmol/ mg protein with a K D of 4.5 nmol/ L, which corresponds to a purification of approximately 26 700‐fold. The molecular weight of this protein was 70 000 Da. 3. The binding of [ 125 I][d(CH 2 ) 5′ Sarcosine 7 ]AVP to the solubilized membranes was dependent on the protein concentration, and was inhibited by the unlabelled peptides [d(CH 2 ) 5′ Sarcosine 7 ]AVP, AVP, and to a lesser degree by peptides with high V 2 receptor affinity, such as 1‐desamino‐D‐AVP and [d(CH 2 ) 5′ D‐Ileu 2 ‐Ileu 4 ]AVP. 4. In addition, an AVP anti‐idiotypic monoclonal antibody bound to both the partially purified and purified lectin affinity AVP binding protein in a concentration‐dependent manner. These results indicate that the purified protein displays similar characteristics to the liver membrane‐bound AVP V 1 receptor.