Molecular characterization of opioid receptors
The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mg of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.
- Research Organization:
- New York Univ., NY (USA)
- OSTI ID:
- 6720388
- Country of Publication:
- United States
- Language:
- English
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550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
BETA DECAY RADIOISOTOPES
CENTRAL NERVOUS SYSTEM DEPRESSANTS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ION EXCHANGE CHROMATOGRAPHY
ISOTOPE APPLICATIONS
ISOTOPES
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NARCOTICS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PROTEINS
PURIFICATION
RADIOISOTOPES
RADIORECEPTOR ASSAY
RECEPTORS
SEPARATION PROCESSES
TRACER TECHNIQUES