Document Detail


Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting.
MedLine Citation:
PMID:  1832297     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)
Authors:
E F Estrada; V Barra; C E Caorsi; S Troncoso; N Ruiz-Opazo; C B González
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biochemistry     Volume:  30     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1991 Sep 
Date Detail:
Created Date:  1991-10-11     Completed Date:  1991-10-11     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  8611-6     Citation Subset:  IM    
Affiliation:
Instituto de Fisiología, Facultad de Medicina, Universidad Austral de Chile, Valdivia.
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MeSH Terms
Descriptor/Qualifier:
Affinity Labels*
Animals
Binding, Competitive
Collodion
Cross-Linking Reagents*
Electrophoresis, Polyacrylamide Gel
Liver / chemistry
Lypressin / metabolism*
Membrane Proteins / isolation & purification
Molecular Weight
Rats
Receptors, Angiotensin / analysis*
Receptors, Vasopressin
Swine
Chemical
Reg. No./Substance:
0/Affinity Labels; 0/Cross-Linking Reagents; 0/Membrane Proteins; 0/Receptors, Angiotensin; 0/Receptors, Vasopressin; 50-57-7/Lypressin; 9004-70-0/Collodion

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