Processing and degradation of met-enkephalin by peptidases associated with rat brain cortical synaptosomes

Abstract

The generation of met-enkephalin (Tyr 1-Gly 2-Gly 3-Phe 4-Met 5) from met-enkephalin-Arg 6-Phe 7 and subsequent degradation of the liberated peptides to the free amino acids by rat brain cortical synaptosomes in vitro was demonstrated by HPLC and amino acid analyses. Kinetic measurements of the individual steps of met-enkephalin processing and degradation upon incubation with synaptosomes revealed the following sequence of cleavage: 1. Hydrolysis of the Met 5-Arg 6 peptide bond, generating met-enkephalin and the dipeptide Arg-Phe. Captopril and EDTA inhibit this reaction. 2. Hydrolysis of the Tyr 1-Gly 2 peptide bond, generating Tyr and a tetrapeptide. Puromycin (ID 50 = 5 × 10 −5 m) and parahydroxymercuribenzoate (ID 50 = 5 × 10 −4 m) inhibit this reaction. 3. Hydrolysis of the Gly 3-Phe 4 peptide bond. Parahydroxymercuribenzoate (ID 50 = 5 × 10 −4 m) inhibits this reaction completely. 1 mmol liter −1 Puromycin does not inhibit this reaction. 4. Hydrolysis of the Phe 4-Met 5 peptide bond. 5. Hydrolysis of the Gly 2-Gly 3 peptide bond. The pH optimum of all cleavage reactions was found to be around 7.8.