The use of proteolysis to study the structure of nardilysin
- 作者：Ma ; Zhangliang ; Martin Chow ; K. ; Csuhai ; Eva ; Hersh ; Louis B.
- 关键词：Peptidase ; Proteolysis ; Protein structure ; Regulatory domain ; Activation ; Protein folding
- 刊名：Archives of Biochemistry and Biophysics
- 出版年：2002
- 期刊代码：116_00039861
- 类别：ch
- 出版时间：May 15, 2002
- 卷：401
- 期：2
- 页码：198-204
- 文件大小：344 K
摘要
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.
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.