Isolation and characterization of the rat liver AVP receptor using [125I][d(CH2)5'sarcosine7]AVP.
1. A vasopressin (AVP) binding protein was purified from rat liver membranes by an improved method using [125I][d(CH2)5'Sarcosine7]AVP, a selective V1 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 KD 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 [125I][d(CH2)5'Sarcosine7]AVP to the solubilized membranes was dependent on the protein concentration, and was inhibited by the unlabelled peptides [d(CH2)5'Sarcosine7]AVP, AVP, and to a lesser degree by peptides with high V2 receptor affinity, such as 1-desamino-D-AVP and [d(CH2)5'D-Ileu2-Ileu4]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 V1 receptor.
Trinder
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D
University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Australia.
Kelly
J M
JM
Fernley
R
R
Mooser
V
V
Phillips
P A
PA
Johnston
C I
CI
D000818:Animals; D000911:Antibodies, Monoclonal; D001127:Arginine Vasopressin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000302:isolation & purification*; D017483:Receptors, Vasopressin
D000911:Antibodies, Monoclonal; D010743:Phospholipids; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; C058531:argipressin, (1-beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-Sar(7)-; D001127:Arginine Vasopressin