The use of proteolysis to study the structure of nardilysin.
Treatment of a 128 kDa mouse nardilysin with trypsin initially produced an active 105 kDa N-terminally cleaved form. Continued trypsin digestion occurred at the C-terminus, producing inactive core species of approximately 92, 76.5, and 62 kDa. Protease V8 digestion generated a stable approximately 105 kDa form, nardilysin(V8), that was cleaved near the N-terminal trypsin site. The approximately 105 kDa nardilysin(V8) exhibited the same K(m) as did the uncleaved enzyme for substrates of the type Abz-GGFX(1)X(2)X(3)VGQ-EDDnp, where X residues were varied. However, k(cat) for nardilysin(V8) was 5-6 times greater. Both uncleaved nardilysin and nardilysin(V8) are inhibited by NaCl; however, nardilysin(V8) exhibits an IC(50) of approximately 2 mM compared to an IC(50) of approximately 50 mM for uncleaved nardilysin. Nardilysin(V8) is more sensitive to inhibition by phosphate buffer. Treatment of nardilysin(V8) with trypsin generated primarily the 92 kDa form which was inactive. Attempts to express nardilysin as a 105 kDa truncated N-terminal form or as a C-terminally truncated form led to inactive proteins.
Ma
Zhangliang
Z
Department of Molecular and Cellular Biochemistry, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536-0298, USA.
Chow
K Martin
KM
Csuhai
Eva
E
Hersh
Louis B
LB
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014357:Trypsin
D009842:Oligopeptides; D010446:Peptide Fragments; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D008666:Metalloendopeptidases; C087976:nardilysin