Design of angiotensin II derivatives suitable for indirect affinity techniques: potential applications to receptor studies.
The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-AII (Bio-Ahx-AII) and dinitrophenyl- aminohexanoyl-AII (Dnp-Ahx-AII). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (Kd values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala1,Phe(4N3)8)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido AII-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.
Bonnafous
J C
JC
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier.
Seyer
R
R
Tence
M
M
Marie
J
J
Kabbaj
M
M
Aumelas
A
A
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000031:analogs & derivatives*; D000818:Animals; D001710:Biotin; D004136:Dinitrobenzenes; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000032:analysis*
D000345:Affinity Labels; D004136:Dinitrobenzenes; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D001710:Biotin