α-N-Acetyl β-Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1-13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1-13) treated with a water-soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β-endorphin and dynorphin(1-13) as well as by CDI-treated dynorphin(1-13), but only weakly by authentic dynorphin(1-13). Neither nonacetylated β-endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI-treated dynorphin(1-13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1-13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI-treated dynorphin(1-13) used as a trace and as a standard. Competition studies showed 0.5-1% cross-reactivity with α-N-acetyl β-endorphin(1-31), α-N-acetyl β-endorphin(1-27), and totally acetylated β-endorphin(1-31). Nonacetylated β-endorphins did not cross-react. Posterior-intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior-intermediate lobe extracts were also fractionated by cation-exchange chromatography. Fractionated extracts were analyzed by RIAs for β-endorphin, CDI-treated dynorphin(1-13), and authentic dynorphin(1-13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β-endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β-endorphins in the pituitary.