title: Expression of the acidic stretch of nardilysin as a functional binding domain.
abstract: Kinetic evidence suggests an acidic region in nardilysin binds polyamines and acts as a regulatory domain. The binding of approximately 5 mol of spermine/mol of nardilysin was demonstrated. The binding curve was sigmoidal exhibiting an IC(50) of approximately 118 microM and a Hill coefficient of 1.8. Spermine diminished the tryptophan fluorescence of the enzyme and increased its sensitivity to protease V8. The acidic stretch from mouse and human nardilysin were expressed as glutathione transferase fusion proteins. All fusion proteins bound spermine with an IC(50) of 40 to 110 microM. The mouse fusion protein bound approximately 7 mol of spermine exhibiting a sigmoidal binding curve and a Hill coefficient of 1.4. The human acidic stretch, containing fewer acidic residues, bound approximately 5 mol of spermine/mol with a hyperbolic binding curve. Chimeric fusion proteins containing the N-terminus of the mouse acidic region fused to the C-terminus of the human acidic region bound approximately 10 mol of spermine, while the opposite chimera bound approximately 4 mol of spermine/mol. The N-terminal region of the mouse acidic domain binds 3--4 mol 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 sensitivity of the mouse acidic domain, but not the human acidic domain, to protease V8. Together the data support a model where the acidic stretch of nardilysin functions as an autonomous domain.
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Ma
authors.forename: E
authors.initials: E
authors.lastname: Csuhai
authors.forename: K M
authors.initials: KM
authors.lastname: Chow
authors.forename: L B
authors.initials: LB
authors.lastname: Hersh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013096:Spermine / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013096:Spermine; D005982:Glutathione Transferase; D008666:Metalloendopeptidases; C087976:nardilysin