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Antibodies Specific for α‐ N ‐Acetyl‐β‐Endorphins: Radioimmunoassays and Detection of Acetylated β‐Endorphins in Pituitary Extracts
Author(s) -
Weber Eckard,
Evans Christopher J.,
Chang JawKang,
Barchas Jack D.
Publication year - 1982
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1982.tb08648.x
Subject(s) - endorphins , radioimmunoassay , acetylation , chemistry , antiserum , antibody , posterior pituitary , biochemistry , peptide , carbodiimide , pituitary gland , chromatography , endocrinology , biology , hormone , immunology , gene
Abstract: Antibodies specific for α‐ N ‐acetyl‐β‐endorphins have been prepared by injecting into rabbits either α‐ N ‐acetyl‐β‐endorphin(1‐31) or [α‐ N ‐acetyl, ε‐acetyl‐Lys 9 ]‐β‐endorphin(1‐9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α‐ N ‐acetyl‐β‐endorphins. The radioimmunoassays were used to measure α‐ N ‐acetylated β‐endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β‐endorphin and α‐ N ‐acetyl‐β‐endorphin immunoreactivity, a relative ratio of β‐endorphin acetylation was obtained. The relative acetylation of β‐endorphin was highest in rat posterior‐intermediate lobe extracts (>90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β‐endorphin. Human pituitary extracts did not contain acetylated β‐endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β‐endorphin were resolved in extracts of rat posterior‐intermediate pituitaries. Two of these peptides corresponded to α‐ N ‐acetyl‐β‐endorphin(1‐31) and ‐(1‐27). One acetylated β‐endorphin fragment had the same size as α‐ N ‐acetyl‐β‐endorphin(1‐27) but was eluted earlier from the cation exchange column. This peptide had full cross‐reactivity with antibodies directed against the middle and amino‐terminal parts of β‐endorphin. Compared with α‐ N ‐acetyl‐β‐endorphin(1‐27), it had much less cross‐reactivity with antibodies directed against the COOH‐terminal part of β‐endorphin, suggesting that it was a COOH‐terminally modified derivative of β‐endorphin(1‐27). The remaining N ‐acetylated β‐endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y‐endorphin, i.e., β‐endorphin(1‐17), but smaller than β‐endorphin(1‐27). They had full cross‐reactivity in an amino‐terminally directed β‐endorphin radioimmunoassay and a greatly diminished cross‐reactivity with antibodies to the middle region of β‐endorphin.