Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

Journal Article · Fri Dec 01 04:00:00 EST 1989 · Endocrinology; (USA)

DOI:https://doi.org/10.1210/endo-125-6-3068· OSTI ID:5037951

Grigoriadis, D E; Zaczek, R; Pearsall, D M; De Souza, E B ^[1]

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1. National Institute on Drug Abuse, Baltimore, MD (USA)

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

• OSTI ID: 5037951
• Journal Information: Endocrinology; (USA), Journal Name: Endocrinology; (USA) Vol. 125:6; ISSN ENDOA; ISSN 0013-7227
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACTH
ANIMALS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
BRAIN
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CEREBRAL CORTEX
CEREBRUM
CHEMICAL COMPOSITION
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
HORMONES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MOLECULAR WEIGHT
NERVOUS SYSTEM
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HORMONES
PITUITARY HORMONES
PROTEINS
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SOLUBILITY
TEMPERATURE DEPENDENCE
TIME DEPENDENCE
TRACER TECHNIQUES
VERTEBRATES