Beta-endorphin-related peptides in the human pituitary. Isolation and characterization of major immunoreactive peptides, including the formerly unrecognized peptide beta-endorphin 1-18.

The qualitative and quantitative aspects of beta-endorphin-related peptides present in the human pituitary gland were investigated using autopsy pituitaries as starting material. Beta-endorphin-related peptides were isolated from two batches of autopsy pituitaries (40 and 51 glands) by acid acetone extraction, acetone precipitation, gel filtration (Sephadex G-75 or Bio Gel P 4/P 10), cation exchange chromatography (SP-Sephadex C-25) and different types of reversed phase high performance liquid chromatography (RP-HPLC). Purification was monitored using beta-endorphin radioimmunoassay, which is specific to the 6-17 sequence of beta-endorphin. Methods suitable for microscale structural analyses of the isolated peptides were developed. Amino acid analysis was performed using quantitative precolumn dansylation and RP-HPLC separation of the dansyl amino-acids. Amino-terminal sequences were determined using semi-microscale manual Edman degradation. An RP-HPLC system was set up for sensitive and unambiguous identification of the released phenylthiohydantoin amino acids. The immunoreactive peptide with the highest apparent molecular weight was identified as human beta-lipotropin on the basis of its amino acid composition. The results of amino-terminal sequence analysis, amino acid compositions of the HPLC-isolated tryptic peptides and carboxy-terminal residue analysis were fully compatible with the previously suggested structure of human beta-lipotropin. About 20% of isolated pituitary beta-lipotropin appeared to be in deamidated form. No proteolytically degraded forms of beta-lipotropin were detected. The yield of pure beta-lipotropin was 6.7-7 nmol/gland. A peptide with properties similar to human beta-endorphin was isolated with a yield of 1.5-5 nmol/gland. Amino acid composition, amino-terminal sequence, carboxy-terminal residue and immunochemical properties were identical to those of human beta-endorphin as suggested previously. About 25% of beta-endorphin appeared to be in methionine sulphoxide form as isolated. Several minor components of immunoreactivity with molecular weight similar to beta-endorphin were detected but none of these were obtained pure even after three RP-HPLC purification steps. Further purification was impossible due to the scarcity of the starting material. A previously unidentified beta-endorphin-related peptide with an apparent molecular weight of about 2000 daltons was isolated from the autopsy pituitaries. Amino acid composition, amino-terminal residue, carboxy-terminal sequence and immunochemical properties showed the peptide to be beta-endorphin 1-18. The yield of isolated peptide was 0.4 nmol/gland. This peptide has not been pr