Studies of the in vitro human plasma degradation of methionine-enkephalin

Abstract

1. 1. Incubation of [ 3H]tyrosine methionine-enkephalin (6 × 10 −9 M final concentration) with human platelet-poor plasma (1:9 ratio to Trizma Base buffer, pH 7.4) results mostly (>95%) in hydrolysis of the tyrosyl-glycine peptide bond. This enzymatic reaction is essentially completed within 90 min, showing a half-life, K m and V max of 12.8 ± 2.5 min, 0.70 ± 0.01 mM and 17.90 ± 1.05 μmol/L/min, respectively. These values are comparable to those previously reported for the human plasma degradation of leucine-enkephalin. 2. 2. As expected hydrolysis of the methionine-enkephalin tyrosyl-glycine peptide bond was blocked by the known aminopeptidase inhibitors bestatin and puromycin (IC 50 1.2 ± 0.4 and 4.3 ± 2.4 μM, respectively) but not by either thiorphan or captopril. 3. 3. Neither the storing (up to 60 days) nor the freezing and thawing (up to ten times during a 60 days periods) significantly changed the above kinetic parameters, showing the stability of the plasma methionine-enkephalin degrading aminopeptidase.