Breakdown of enkephalin and endorphin analogs by brain extracts

Abstract

Analogs of enkephalin [LPH (61–65)] containing D-amino acids in position 62, or amides in position 65, were exposed to extracts prepared from mouse brain and found to be highly resistant to breakdown. The resistance of these analogs when incubated for short periods corresponded well with their enhanced analgesic activities in vivo. After incubation for protracted periods, the analog D-Ala 62, Met-NH 2 65- LPH(61–65) was more resistant to breakdown than D-Met 62, Pro-NH 2 65-LPH(61–65) as determined by the release of N-terminal Tyr. Studies with singly substituted enkephalin analogs showed that D-Ala 62 alone did not prevent aminopeptidase action but retarded the action of aminopeptidase as compared to the unsubstituted pentapeptide. The presence of D-amino acids in positions 62–65 led to the production of a tetrapeptide intermediate that was resistant to the action of carboxypeptidases. When injected intracisternally into rats, the doubly substituted enkephalin analog, D-Ala 62, Met-NH 2 65, and also D-Ala 62-β-endorphin retained analgesic potency that was reversible by naloxone. Substitution with D-Ala 62 in the α, γ and β-endorphins [LPH(61–76), (61–77 and (61–91)] significantly retarded the action of brain aminopeptidases but did not prevent internal cleavages by endopeptidases. The patterns of amino acid release point to considerable differences in the susceptibility to breakdown of all three polypeptides, implying the existence of conformational restraints affecting enzymatic specificity.