Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes

Abstract

We have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH2)2SS(CH2)2CO-[Ala1,Phe(4N3)8]AII, which contains a cleavable disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (Kd approximately 1 nM), proved to be suitable for indirect affinity chromatography of rat liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT1A receptor

“…To circumvent problems with traditional puri®cation strategies (Capponi & Catt, 1980;Marie et al, 1990) we introduced a six histidine a nity tag into the N-terminus of a recombinant AT 1A receptor. The histidine tag allows a rapid puri®cation of proteins by means of Ni 2+ -NTA a nity chromatography under both denaturing (Hochuli et al, 1988) and native conditions (Janknecht et al, 1991).…”

Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin‐labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles

“…The identity of the ZP3 receptor molecules on sperm therefore remains unknown. However, studies using other receptor-ligand systems (Marie et al, 1990;Eppler et al, 1992) suggest that saturation of isolated membranes with ~-Z P~~E B G D followed by membrane solubilization may provide a viable experimental approach for identification and affinity purification of ZP3a-specific receptors.…”

N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

“…Photoaffinity labelling of WT and mutant receptors were carried out using the iodinated photoreactive probe ["#&I]Sar",(4h-N $ )Phe)-AII. This agonist probe of the AT " receptor has been shown be suitable for specific covalent labelling of the AT " receptor [19] and has been used for molecular-mass determination of AT " receptors from different tissues and species [19,21,22]. As shown in Figure 3, photoaffinity labelling of c-Myc-epitopetagged receptor expressed in COS-7 cells followed by SDS\PAGE and autoradiography analysis led to the detection of a protein with a molecular mass of 153p12 kDa, similar to the WT receptor ( Figure 3A, lanes a and b).…”