Expression of the acidic stretch of nardilysin as a functional binding domain

Kinetic evidence suggests an acidic region in nardilysin binds polyamines a nd acts as a regulatory domain. The binding of similar to5 mol of spermine/ mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibit ing an IC50 of similar to 118 muM and a Hill coefficient of 1.8. Spermine d iminished the tryptophan fluorescence of the enzyme and increased its sensi tivity to protease V8. The acidic stretch from mouse and human nardilysin w ere expressed as glutathione transferase fusion proteins. All fusion protei ns bound spermine with an IC50 of 40 to 110 muM. The mouse fusion protein b ound similar to7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound similar to5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acid ic region fused to the C-terminus of the human acidic region bound similar to 10 mol of spermine, while the opposite chimera bound similar to4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3-4 mo l spermine/mol exhibiting a Hill coefficient of 1.4, while the same region from human nardilysin binds 1 mol of spermine/mol. Spermine enhanced the se nsitivity of the mouse acidic domain, but not the human acidic domain, to p rotease V8. Together the data support a model where the acidic stretch of n ardilysin functions as an autonomous domain.