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Fragmentation of human polymorphonuclear-leucocyte collagenase
V Knäuper1, A Osthues, Y A DeClerck
1University of Bielefeld, Faculty of Chemistry, Department of Biochemistry, Federal Republic of Germany.
The Biochemical Journal
|May 1, 1993
Summary
Human polymorphonuclear-leucocyte collagenase degrades into catalytic and non-catalytic fragments. The catalytic fragment retains specificity but loses collagen-cleaving ability, while the C-terminal fragment is crucial for collagen binding.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Human polymorphonuclear-leucocyte collagenase (M(r) 64,000) is a key enzyme in extracellular matrix degradation.
- This enzyme undergoes autoproteolytic degradation into smaller fragments.
Purpose of the Study:
- To characterize the functional domains of human polymorphonuclear-leucocyte collagenase after degradation.
- To investigate the enzymatic activity and substrate specificity of the resulting fragments.
Main Methods:
- Autoproteolytic degradation of collagenase.
- N-terminal sequencing of degradation fragments.
- Substrate specificity analysis of fragments.
- Inhibition assays using tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2.
Main Results:
- Collagenase degraded into M(r) 40,000 (catalytic domain) and M(r) 27,000 (non-enzymatic) fragments.
- The M(r) 40,000 fragment exhibited specificity but lost collagenolytic activity.
- The M(r) 27,000 fragment was essential for collagen binding and collagenolysis.
- Both TIMP-1 and TIMP-2 inhibited the catalytic fragment in a 1:1 molar ratio.
Conclusions:
- The M(r) 40,000 fragment represents the catalytic domain of collagenase, retaining specificity but not collagen cleavage.
- The M(r) 27,000 fragment is critical for substrate binding and collagenolysis.
- Understanding these domains aids in developing targeted inhibitors for metalloproteinases.