Partial purification of a photoaffinity-labeled mouse Ah-receptor

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5020842

Perdew, G; Glover, E; Poland, A

(/sup 125/I)-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, a high affinity ligand for the Ah receptor (K/sub D/ = 0.76 nM) with a photolabile arylazide functional group has been previously shown to photoaffinity label two peptides in the mouse liver cytosol. The two peptides: (1) have apparent M/sub r/ of 95,000 and 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), (2) are labeled in a 1:1 ratio and, (3) comigrate under nondenaturing conditions on ion exchange and size-exclusion chromatography. The 40-55% ammonium sulfate precipitated fraction of mouse hepatic cytosol was photoaffinity labeled with the (/sup 125/I)-ligand, and subjected to chromatography on a column of polyethyleneimine linked to Sepharose, using a NaCl gradient. This produced a 24 fold purification of the labeled peptides with a 50% yield. The enriched fraction was further purified by preparative SDS-PAGE, the 95 kDa and 70 kDa bands were cut from the gel and electroeluted, yielding a 700 fold purification (i.e., relative to their cytosolic concentrations). Each peptide fraction was then subjected to reverse-phase high performance liquid chromatography, resulting in a further 10-15 fold purification, (overall 10,000 fold enrichment, or about 10% of estimated homogeneity). The use of denaturing conditions to purify this soluble receptor was found necessary because following initial chromatography under nondenaturing conditions the labeled Ah receptor aggregated.

• Research Organization: Univ. of Wisconsin, Madison
• OSTI ID: 5020842
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DIOXIN
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
GLANDS
HETEROCYCLIC COMPOUNDS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
ISOTOPES
LABELLING
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
MEMBRANE PROTEINS
MICE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANS
PEPTIDES
PROTEINS
PURIFICATION
RADIOISOTOPES
RECEPTORS
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES