HYPOTHALAMIC DEGRADATION OF GALANIN(1-29) AND GALANIN(1-16) - IDENTIFICATION AND CHARACTERIZATION OF THE PEPTIDOLYTIC PRODUCTS

The degradation of the neuropeptide galanin(1-29) and its fully active synthetic N-terminal fragment galanin(1-16) in hypothalamic tissue where these peptides potently affect feeding behaviour, is studied. Galanin(1-29) had a half-life of 100 min while galanin(1-16) had a half-life of 28 min when incubated with a hypothalamic membrane preparation. The putative sites of peptidolytic cleavage of the active N-terminal fragment galanin(1-16) were determined as being between amino acids Leu4 and Asn5, between Asn5 and Ser6, and between His14 and Ala15, respectively. The synthetic analogs of galanin(1-16) where Leu4, Asn5 or Ser6 was substituted by Ala were all more stable to peptidolysis; [Ala4]galanin(1-16) had a half-life of 55 min. Cleavage of the galanin(1-16) between His14-Ala15 yields a ligand - galanin(1-14) which binds to the receptor with high affinity (K(D) almost-equal-to 10(-7) M), while cleavage at amino acid residues Leu4, Asn5 and Ser6 results in inactive peptide fragments with affinities for the galanin receptor below 10(-4) M. The enzyme(s) responsible for degradation of galanin were identified as endopeptidase(s), which were partially inhibited by bacitracin (1 mg/ml) by up to 50%, but not significantly by EDTA (1 mM), phosphoramidon (1-mu-M), phenylmethylsulfonyl fluoride, (100-mu-M) or aprotinin (10-mu-g/ml).