Identification of -endorphins in the pituitary gland and blood plasma of the common carp (Cyprinus carpio) E H van den Burg, J R Metz, R J Arends, B Devreese 1 , I Vandenberghe 1 , J Van Beeumen 1 , S E Wendelaar Bonga and G Flik Department of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands 1 Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, KL Ledeganckstraat 35, B9000 Gent, Belgium (Requests for offprints should be addressed to G Flik; Email: gertflik@sci.kun.nl) Abstract Carp -endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the -endorphins. Forms of -endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chroma- tography (HPLC) coupled with radioimmunoassay, the -endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of -endorphins by the pituitary gland was studied after stimulation with corticotrophin- releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl -endorphin(1–33) coeluted with N-acetyl -endorphin(1–29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl - endorphin(1–15) (30·8% of total immunoreactivity) and N-acetyl -endorphin(1–10) (3·1%) via two different cleavage pathways. The acetylated carp homologues of mammalian - and -endorphin were also found. N-acetyl -endorphin(1–15) and (1–29) and/or (1–33) were the major products to be released in vitro, and were the only acetylated -endorphins found in blood plasma, although never together. CRF stimulated the release of opioid -endorphin from the pars distalis. This non- acetylated -endorphin represents the full length peptide and is the most abundant form in plasma. Journal of Endocrinology (2001) 169, 271–280 Introduction Beta-endorphin is a pro-opiomelanocortin (POMC)- derived peptide and is predominantly produced in the pituitary gland and the brain. Its bioactivity depends on C-terminal truncation and N-terminal acetylation. For instance, -endorphin(1–31) is a very potent opiate, whereas its acetylated congener is essentially devoid of opioid activity (Akil et al. 1981). On the other hand, C-terminal cleavage produces the very effective opioid antagonist -endorphin(1–27) (Nicolas & Li 1985). In addition to its opioid function, -endorphin was found to be involved in a variety of physiological processes (reviewed by Dalayeun et al. 1993), including regu- lation of the immune (Heijnen et al. 1987, Shahabi et al. 1991, 1996) and reproductive (e.g. Faletti et al. 1999) systems. Furthermore, -endorphin plays a role in the vertebrate stress response (e.g. Akil et al. 1985, Kjær et al. 1995, Wendelaar Bonga 1997, Mosconi et al. 1998). Since forms of -endorphin exert different biological effects, research so far has focused on the occurrence of -endorphins that may each have different actions. Post- translationally modified -endorphins have been described in a variety of vertebrates, including teleost fish (Takahashi et al. 1984) and mammals (Weber et al. 1981, Smyth 1984, Millington et al. 1992). However, data on the release and functions of -endorphins are very scarce. In human blood plasma, the major form is -endorphin(1–31) (Silberring et al. 1998). In fish, N-acetyl -endorphin immunoreactivity has been reported in plasma (Rodrigues & Sumpter 1984, Sumpter et al. 1985, Mosconi et al. 1998), although the exact nature of the forms responsible for this immuno- reactivity is unknown. Recent cloning of carp POMC (Arends et al. 1998) showed that full length -endorphin in this species consists of 33 amino acid residues, rather than 31 amino acids as in mammals and other vertebrates, and contains one potential dibasic and five potential monobasic cleavage sites. There- fore, identification of -endorphins in the pituitary gland 271 Journal of Endocrinology (2001) 169, 271–280 0022–0795/01/0169–271  2001 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org Downloaded from Bioscientifica.com at 06/06/2020 05:58:11PM via free access