Abstract
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 striatummore » (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).« less
Highlights
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 destroy synthetic enkephalin
This paper describes the development of a radioimmunoassay for the enkephalins and its use to measure the distribution of these peptides in various regions of the brain
Summary
Peptides - Peptides were synthesized by conventional solution methods or by the repetitive mixed anhydride procedure described by Tilak [15] or by VanZon and Beyerman [16]. Of peptide (enkephalin), 0.5 mg was mixed with 1 mg of bovine serum albumin (Pentex) in 200 ~1 of 0.1 M sodium phosphate buffer, pH 7.4. For use in opiate binding assays (see below) the 1251-labeled [nAla*, nLeu5]enkephalin was further purified on DEAE-Sephadex as detailed elsewhere.* This method separates the mono- and di-iodo derivatives of the peptide. The sample was lyophilized, suspended in the assay buffer, centrifuged, and assayed Recovery by this method was greater than. Procedure- To glass test tubes (12 x 75 mm) were added 20 ~1 of Y-labeled enkephalin To each assay tube were added 200 ~1 of cell suspension, 20 ~1 of 1251-labeled [nAlaZ,nLeuS]enkephalin Assay tubes were incubated at room temperature for 45 min (equilibrium conditions).
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