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 ∼92, 76.5, and 62 kDa. Protease V8 digestion generated a stable ∼105 kDa form, nardilysin V8, that was cleaved near the N-terminal trypsin site. The ∼105 kDa nardilysin V8 exhibited the same K m as did the uncleaved enzyme for substrates of the type Abz-GGFX 1X 2X 3VGQ-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 ∼2 mM compared to an IC 50 of ∼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.