Inactivation of enkephalin by brain enzymes

Two distinct enzymatic activities capable of hydrolyzing enkephalin are present in the brain. The major activity was shown to be a neutral aminopeptidase which hydrolyzes Leu-enkephalin (Leu-Enk) with a Km of 2 X 10(-5) M. This activity was inhibited by heavy metal ions (i.e. Zn++, Cd++), by sulfhydryl blocking reagents, and by the antibiotics bacitracin and puromycin. In contrast, these two antibiotics had no effect on the hydrolysis of Leu-Enk by either rat serum or commercial leucine aminopeptidase. The integrity of both the glycosidic and peptide bonds in the puromycin molecule was required for its inhibitory activity. On the other hand, modifications of the sugar moiety had relatively little effect, allowing the design of puromycin analogs which were inactive with regard to protein synthesis inhibition but still capable of inhibiting brain aminopeptidase. Puromycin was also shown to inhibit enkephalin degradation by homogenates of guinea pig ileum and to prolong the depressant effect of enkephalin on the electrically induced contractions of the ileum. The second enzymatic activity in brain homogenate was found to sediment with the synaptic plasma membrane fraction and cleave Leu-enkephalin into Tyr-Gly-Gly and Phe-Leu with a Km of 2.2 X 10(-5) M and pH optimum between 6.5 and 7.0. This endopeptidase was inhibited by metal chelating agents and by thiols but was insensitive to puromycin and to p-chloromercuribenzoate. In contrast to the aminopeptidase some cleavage of (D-Ala2)Met-Enk x amide by the endopeptidase was observed.