Purification, primary structure, and neuronal localization of cerebral peptide 1 from Aplysia.
We are investigating peptidergic neurotransmission between ganglia in the Aplysia CNS. Neurons in the cerebral ganglia of Aplysia synthesize [35S]methionine-labeled peptides, which are transported to other central ganglia by fast axonal transport. We report the characterization of one of these peptides, termed cerebral peptide 1 (CP1). Transported [35S]methionine-labeled CP1 was used as a probe for the purification of CP1 from the pooled extracts of 1000 cerebral ganglia using three sequential modes of RP-HPLC. Amino acid sequence analysis by automated Edman degradation yielded the following sequence: Phe-Ser-Gly-Leu-Met-Ser-Glu-Gly-Ser-Ser-Leu-Glu-Ala. This sequence was consistent with data from amino acid composition analysis and FAB-MS of the purified peptide. In addition, a synthetic peptide with the proposed sequence coeluted with [35S]methionine-labeled native CP1 using the three RP-HPLC conditions used in the purification. Antisera raised against synthetic CP1 stained about 90 neuronal cell bodies in the cerebral ganglion including those of the asymmetric H cluster. Extracts of the H cluster dissected from ganglia incubated in [35S]methionine synthesized a radiolabeled peptide that coeluted with synthetic CP1, indicating that these neurons indeed synthesize authentic CP1. The other major ganglia each contained only a few CP1-immunoreactive neurons. The neuropil of each ganglion contains both smooth and varicose-immunoreactive fibers. Thus, CP1 is broadly distributed within the Aplysia CNS and may be a new peptide transmitter.
Phares
G A
GA
Department of Pharmacological and Physiological Sciences, University of Chicago, IL 60637, USA.
Lloyd
P E
PE
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001048:Aplysia / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D017952:Ganglia, Invertebrate / Q000737:chemistry* / Q000648:ultrastructure; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D007447:Invertebrate Hormones / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D008715:Methionine / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D009842:Oligopeptides / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D011817:Rabbits; D017421:Sequence Analysis; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D007106:Immune Sera; D007447:Invertebrate Hormones; D009479:Neuropeptides; D009842:Oligopeptides; D013462:Sulfur Radioisotopes; C103535:cerebral peptide 1; D008715:Methionine