PURIFICATION, SEQUENCE-ANALYSIS, AND CELLULAR-LOCALIZATION OF A PRODYNORPHIN-DERIVED PEPTIDE RELATED TO THE ALPHA-NEO-ENDORPHIN IN THE RHYNCHOBDELLID LEECH THEROMYZON TESSULATUM

Cells immunoreactive to an antiserum specifically directed against ver tebrate alpha-Neo-endorphin (alpha-NE) were detected in the internal w all of anterior and posterior suckers of the rhynchobdellid leech Ther omyzon Tessulatum. These cells have morphological and ultrastructural characteristics close to the ''releasing gland cells'' of adhesive org ans. The epitope recognized by anti-alpha-NE was contained in granules having a diameter of 0.2-0.3 mu m. Previous works involving the brain of this leech demonstrate the existence of similar to 14 neurons immu noreactive to the anti-alpha-NE. Following an extensive purification i ncluding high pressure gel permeation and reversed-phase high performa nce Liquid chromatography, epitopes contained in both suckers and cent ral nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determin ed by a combination of automated Edman degradation, electrospray mass spectrometry measurement, and coelution experiments in reversed-phase high performance liquid chromatography with synthetic alpha-NE. The re sults demonstrate that epitopes recognized by the anti-alpha-NE in the suckers and the central nervous system are identical to vertebrate al pha-NE (YGGFL-RKYPK). This finding constitutes the first biochemical c haracterization of a prodynorphin-derived peptide in invertebrates. Mo reover the isolation of this peptide in the annelida establishes the v ery ancient phylogenetic origin of alpha-NE as well as its conservatio n in evolution.