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 ∼92, 76.5, and 62 kDa. Protease V8 digestion generated a stable ∼105 kDa form, nardilysinV8, that was cleaved near the N-terminal trypsin site. The ∼105 kDa nardilysinV8 exhibited the same Km as did the uncleaved enzyme for substrates of the type Abz-GGFX1X2X3VGQ-EDDnp, where X residues were varied. However, kcat for nardilysinV8 was 5-6 times greater. Both uncleaved nardilysin and nardilysinV8 are inhibited by NaCl; however, nardilysinV8 exhibits an IC50 of ∼2 mM compared to an IC50 of ∼50 mM for uncleaved nardilysin. NardilysinV8 is more sensitive to inhibition by phosphate buffer. Treatment of nardilysinV8 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.
|Number of pages||7|
|Journal||Archives of Biochemistry and Biophysics|
|State||Published - May 15 2002|
Bibliographical noteFunding Information:
This research was supported in part by grants from the NIH/NIDA DA02243 and DA07062 to LBH and DA05671 to EC.
- Activation Protein folding
- Protein structure
- Regulatory domain
ASJC Scopus subject areas
- Molecular Biology