Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts

• Published in: Regulatory peptides 2008-06, Vol.148 (1), p.54-61
• Main Authors: Szemenyei, Erzsébet, Barna, István, Mergl, Zsuzsa, Keresztes, Attila, Darula, Zsuzsanna, Kató, Erzsébet, Tóth, Géza, Rónai, András Z.
• Format: Article
• Language: English
• Subjects: [Gly 5]-endomorphins; Amino Acid Sequence; Animals; beta-Endorphin - immunology; Bioassays; Biological and medical sciences; Brain - immunology; Chromatography, High Pressure Liquid; Dynorphins - immunology; Endomorphin; Enkephalins - immunology; Fundamental and applied biological sciences. Psychology; Immune Sera - immunology; Male; Mice; Narcotic Antagonists - immunology; Novel endomorphin-2-immunoreactive species; Oligopeptides - immunology; Peptides - immunology; Peptides - isolation & purification; Rabbits; Radioimmunoassay; Radioimmunoassay - methods; Rat brain; Rats; Rats, Wistar; Vertebrates: endocrinology
• ISSN: 0167-0115; 1873-1686
• DOI: 10.1016/j.regpep.2008.03.001

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5 ± 7.5 pg/tube ( n = 7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, β-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6 ± 40.0 ( n = 3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1 ± 30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a μ-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA&GTPγS binding and mouse vas deferens), whereas the latter was a weak μ-opioid receptor agonist with a significant δ-opioid receptorial action as well and a definite indication of partial agonism.