Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes.
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.
Marie
J
J
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Seyer
R
R
Lombard
C
C
Desarnaud
F
F
Aumelas
A
A
Jard
S
S
Bonnafous
J C
JC
D000595:Amino Acid Sequence; D000804:Angiotensin II; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000138:chemical synthesis / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006882:Hydroxyapatites; D008099:Liver / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D019809:Streptavidin
D001426:Bacterial Proteins; D006882:Hydroxyapatites; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D001360:Avidin; D001710:Biotin; D019809:Streptavidin