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 ¹²⁵I-labeled enkephalins and antisera raised in rabbits, to synthetic enkephalins coupled with glutaraldehyde to bovine serum albumin. These antisera show <1% cross-reactivity between H₂N-Tyr-Gly-Gly-Phe-Met-OH ([Met⁵]enkephalin) and H₂N-Tyr-Gly-Gly-Phe-Leu-OH ([Leu⁵]enkephalin) and even lower cross-reactivity to β-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 ¹²⁵I-labeled [D-Ala² D-Leu⁵]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 [Met⁵]- and [Leu⁵]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 [Met⁵]enkephalin to [Leu⁵]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).