Radioimmunoassay and characterization of enkephalins in rat tissues
A highly sensitive and specific radioimmunoassay for three enkephalins (opiate-like pentapeptides) has been developed. The assay utilizes /sup 125/I-labeled enkephalins and antisera raised in rabbits, to synthetic enkephalins coupled with glutaraldehyde to bovine serum albumin. These antisera show <1% cross-reactivity between H/sub 2/N-Tyr-Gly-Gly-Phe-Met-OH ((Met/sup 5/)enkephalin) and H/sub 2/N-Tyr-Gly-Gly-Phe-Leu-OH ((Leu/sup 5/)enkephalin) and even lower cross-reactvity to ..beta..-endorphin. Morphine shows no cross reactivity at all. The assay can detect as little as 10 fmol of enkephalin. Dose response curves for synthetic enkephalin and enkephalin-like immunoreactivity in acid extracts of brain are superimposable. Enkephalin-like immunoreactivity in tissue extracts is destroyed by treating extracts with leucine aminopeptidase or carboxypeptidase, enzymes which also destroy synthetic enkephalin. An opiate receptor binding assay based on the binding of /sup 125/I-labeled (DAla/sup 2/Leu/sup 5/)-enkephalin to N4TG1 neuroblastoma cells has also been developed. Gel filtration of acid extracts of brain or pituitary gland shows that both contain opiate-like material. However, whereas in brain most of opiate-like material co-chromatographs with enkephalin, in the pituitary no opiate-like material was observed to co-chromatograph with enkephalin but was of higher molecular weight. Enkephalin is widely distributed in the brain. High concentrations of both (Met/sup 5/)- and (Leu/sup 5/)enkephalins are found in the striatum (approximately 5 pmol/mg of protein), lower concentrations in the thalamus and midbrain, and very low concentrations in the cerebellum. The ratio of (Met/sup 5/)enkephalin to (Leu/sup 5/)enkephalin also differs in different brain areas. The ratio is very high in the hippocampus (15.2) and hypothalamus (13.6), but lower in other areas such as the cortex (1.4).
- Research Organization:
- Wellcome Research Labs., Research Triangle Park, NC
- OSTI ID:
- 6934508
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 253:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ENKEPHALINS
RADIOIMMUNOASSAY
TISSUE DISTRIBUTION
BRAIN
CHROMATOGRAPHY
IMMUNE SERUMS
IODINE 125
LABELLED COMPOUNDS
PITUITARY GLAND
RATS
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CENTRAL NERVOUS SYSTEM
DAYS LIVING RADIOISOTOPES
DISTRIBUTION
ELECTRON CAPTURE RADIOISOTOPES
ENDOCRINE GLANDS
GLANDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
NERVOUS SYSTEM
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PEPTIDES
POLYPEPTIDES
PROTEINS
RADIOASSAY
RADIOISOTOPES
RODENTS
SEPARATION PROCESSES
TRACER TECHNIQUES
VERTEBRATES
550601* - Medicine- Unsealed Radionuclides in Diagnostics
551001 - Physiological Systems- Tracer Techniques