Processing and degradation of met-enkephalin by peptidases associated with rat brain cortical synaptosomes.
The generation of met-enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5) from met-enkephalin-Arg6-Phe7 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 Met5-Arg6 peptide bond, generating met-enkephalin and the dipeptide Arg-Phe. Captopril and EDTA inhibit this reaction. 2. Hydrolysis of the Tyr1-Gly2 peptide bond, generating Tyr and a tetrapeptide. Puromycin (ID50 = 5 X 10(-5) M) and parahydroxymercuribenzoate (ID50 = 5 X 10(-4) M) inhibit this reaction. 3. Hydrolysis of the Gly3-Phe4 peptide bond. Parahydroxymercuribenzoate (ID50 = 5 X 10(-4) M) inhibits this reaction completely. 1 mmol liter-1 Puromycin does not inhibit this reaction. 4. Hydrolysis of the Phe4-Met5 peptide bond. 5. Hydrolysis of the Gly2-Gly3 peptide bond. The pH optimum of all cleavage reactions was found to be around 7.8.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002540:Cerebral Cortex / Q000201:enzymology* / Q000648:ultrastructure; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D004744:Enkephalin, Methionine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology* / Q000648:ultrastructure; D013997:Time Factors
D000596:Amino Acids; D010446:Peptide Fragments; D011480:Protease Inhibitors; D004744:Enkephalin, Methionine; D010447:Peptide Hydrolases