Characterization of pro-ACTH/endorphin-derived peptides in rat hypothalamus

Abstract

The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of β-endorphin, γ-lipotropin (γ-LPH), corticotropin-like intermediate lobe peptide (CLIP), α-melanotropin (γ-MSH), joining peptide, and glycosylated γ3-MSH all represent predominant end products. Equimolar amounts of β-endorphin-, α-MSH-, CLIP-, γ-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (~ 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of β-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to β-endorphin(1-31), β-endorphin(1-27), and β-endorphin(1-26). The α-N-acetylated forms of endorphin represent less than 10% of the total β-endorphin immunoreactivity. Analyses of hypothalamic α-MSH-sized molecules with acetyl- and amide-directed α-MSH antisera suggest that hypothalamic α-MSH is fully amidated, but largely not α-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that > 85% of the α-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to α-MSH [α-N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic γ-LPH-sized immunoreactive material is indistinguishable from γ-LPH synthesized by pituitary melanotropes. The minor extent of α-N-acetylation of α-MSH and β-endorphin, the limited carboxyl-terminal proteolysis of β-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia.