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 vertebrate alpha-Neo-endorphin (alpha-NE) were detected in the internal wall of anterior and posterior suckers of the rhynchobdellid leech Theromyzon tessulatum. These cells have morphological and ultrastructural characteristics close to the "releasing gland cells" of adhesive organs. The epitope recognized by anti-alpha-NE was contained in granules having a diameter of 0.2-0.3 microm. Previous works involving the brain of this leech demonstrate the existence of approximately 14 neurons immunoreactive to the anti-alpha-NE. Following an extensive purification including high pressure gel permeation and reversed-phase high performance liquid chromatography, epitopes contained in both suckers and central nervous system were isolated. Purity of the isolated peptides was controlled by capillary electrophoresis. Their sequences were determined 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 results demonstrate that epitopes recognized by the anti-alpha-NE in the suckers and the central nervous system are identical to vertebrate alpha-NE (YGGFLRKYPK). This finding constitutes the first biochemical characterization of a prodynorphin-derived peptide in invertebrates. Moreover the isolation of this peptide in the annelida establishes the very ancient phylogenetic origin of alpha-NE as well as its conservation in evolution.
Salzet
M
M
Centre de Biologie Cellulaire, Groupe de Neuroendocrinologie des Hirudinées, SN3, Université des Sciences et Technologies de Lille, Lille, France.
Verger-Bocquet
M
M
Bulet
P
P
Beauvillain
J C
JC
Malecha
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002490:Central Nervous System / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D004723:Endorphins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004745:Enkephalins / Q000737:chemistry*; D007150:Immunohistochemistry; D007865:Leeches / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004723:Endorphins; D004745:Enkephalins; D011498:Protein Precursors; C025681:alpha-neoendorphin; C033660:preproenkephalin