Fig. 8.

DARTS analysis of His-Tyr and Nln interaction. (A and B) present concentration-dependent effect of His-Tyr (at 30, 100, and 300 µM) on degradation of recombinant Nln (A; 2.6 µM) and NEP (B; 2.6 µM) by subtilisin A (Sbt). In the presence of His-Tyr statistically significant protection of Nln digestion is observed (A), which, however, is lacking in NEP under the same experimental conditions (B). (C) presents concentration-dependent effect of Nln inhibitor dynorphin A (1-13) (DynA, at 30, 100, and 300 µM) on degradation of recombinant Nln by subtilisin A. Here too, digestion of Nln is diminished in the presence of DynA, which is a known competitive inhibitor of the peptidase (n = 4 independent experiments with duplicate samples for controls and individual samples for each concentration of a ligand; **, P < 0.01, ***, P < 0.001 compared with digested peptidase incubated with vehicle and subtilisin A). The black line within the scattered dots for each experimental group indicates the mean. (D-F) representative results of immunoblotting experiments based on which the degree of degradation (i.e., density) for Nln and NEP were measured in the experimental groups.