In:
European Journal of Biochemistry, Wiley, Vol. 244, No. 1 ( 1997-02), p. 81-88
Abstract:
Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases‐1 (rTIMP‐1) and wild‐type and mutant human collagenase type I (rMMP‐1) proteins in SF9 cells by the baculovirus expression system. Wild‐type MMP‐1, as well as the MMP‐1 mutant lacking the C‐terminal hemopexin‐like domain [des‐(248–450)‐MMP‐1], exhibit enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4‐aminophenylmercuric acetate. Enzyme activity of both proteins can be inhibited by addition of rTIMP. Deletion of the complete active‐site [des‐(161–228)‐MMP‐1] within the catalytic domain, or mutation of a single His residue of the Zn 2+ binding domain (His199), generates stable forms of MMP‐1 proteins which are unable to digest collagen type I or β‐casein. In addition to communo precipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized rTIMP to determine the structural requirements of MMP‐1 to form complexes with its inhibitor. Only the activated and not the latent forms of wild‐type and C‐terminal mutant des‐(248–450)‐MMP‐1 proteins are able to form complexes with TIMP. Neither mutation of Hisl99, nor deletion mutants des‐(161–228)‐MMP‐1 and des‐(161–228/248–450)‐MMP‐1, interact with TIMP. This demonstrates that the C‐terminal hemopexin domain of MMP‐1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP‐1. In summary, we have shown that the integrity of the catalytic domain of MMP‐1 and its ability to bind Zn 2+ is absolutely required for complex formation with TTMP‐1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP‐1.
Type of Medium:
Online Resource
ISSN:
0014-2956
,
1432-1033
DOI:
10.1111/ejb.1997.244.issue-1
DOI:
10.1111/j.1432-1033.1997.00081.x
Language:
English
Publisher:
Wiley
Publication Date:
1997
detail.hit.zdb_id:
1398347-7
detail.hit.zdb_id:
2172518-4
SSG:
12
