scholarly journals Fragments of human fibroblast collagenase. Purification and characterization

1989 ◽  
Vol 263 (1) ◽  
pp. 201-206 ◽  
Author(s):  
I M Clark ◽  
T E Cawston
Keyword(s):  
Active Site ◽  
Human Fibroblast ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Terminal Part ◽  
Purification And Characterization ◽  
Activity Profile ◽  
Fibroblast Collagenase ◽  
Sequencing Studies ◽  
Minor Form ◽  
Differential Binding

On purification, human fibroblast collagenase breaks down into two major forms (Mr22,000 and Mr 27,000) and one minor form (Mr 25,000). The most likely mechanism is autolysis, although the presence of contaminating enzymes cannot be excluded. From N-terminal sequencing studies, the 22,000-Mr fragment contains the active site; differential binding to concanavalin A shows the 25,000-Mr fragment is a glycosylated form of the 22,000-Mr fragment. These low-Mr forms can be separated by Zn2+-chelate chromatography. An activity profile of this column, combined with data from substrate gels, indicates no activity against collagen in the 22,000-Mr and 25,000-Mr forms, but rather, activity casein and gelatin. The 27,000-Mr form has no activity. The 22,000/25,000-Mr form can act as an activator for collagenase in a similar way to that reported for stromelysin. The activity of the 22,000/25,000-Mr form is not inhibited by the tissue inhibitor of metalloproteinases (TIMP). The 27,000-Mr C-terminal part of the collagenase molecule therefore appears to be important in maintaining the substrate-specificity of the enzyme, and also plays a role in the binding of TIMP.

Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site

Biochemistry ◽  
1990 ◽  
Vol 29 (28) ◽  
pp. 6670-6677 ◽  
Author(s):  
Gregg B. Fields ◽  
Sarah J. Netzel-Arnett ◽  
Lester J. Windsor ◽  
Jeffrey A. Engler ◽  
Henning Birkedal-Hansen ◽  
...  
Keyword(s):  
Active Site ◽  
Human Fibroblast ◽  
Fibroblast Collagenase ◽  
Proteolytic Activities ◽  
Hydrolysis Of

scholarly journals Strategy For Cellulase Immobilization And Its Partial Purification And Characterization

2021 ◽  
Author(s):  
Karina Komarova
Keyword(s):  
Active Site ◽  
Partial Purification ◽  
Purification Step ◽  
Purification And Characterization ◽  
Force Microscopy ◽  
Glycosidic Bonds ◽  
Atomic Force ◽  
Lower Glucose ◽  
Force Profile

Conversion of cellulose to glucose units by cellulases, called hydrolysis, is a very complex step in ethanol production. It requires the mixing of aqueous suspensions of cellulose/cellulases so that cellulases (majority composed of the active site domain and the binding site domain) can attach to cellulose chains, cut or hydrolyze ß(1-4) glycosidic bonds between glucose units, de-attach and move to another location. Mixing extent (insufficient or excessive agitation) might influence the attachment of cellulases and possibly lead to lower glucose yields. A long-term goal of this research is to determine the strength of mixing required to be applied during the cellulose-cellulase mixing cycle. For that purpose, one of the objectives was to purify CBH I exocellulase from the commercial cellulase mixture. A partial purification of the CBH I that was performed on a much smaller scale with uncontrolled flow rate was successful. Another objective was to propose a scheme that would covalently immobilize CBH I exoceullase via its active site domain (ASD) on an atomic force microscopy-compatible support, a silicon support. A theoretically-developed hypothetical scheme was constructed (with the provided detailed procedure). The approach of immobilizing the inhibitor specific to the ASD of CBH I enzyme led to the possibility that no purification of CBH I could be required. Skipping CBH I purification step would save time and hassle associated with purification step. Once the ASD of CBH I is immobilized on a silicon support, the AFM force profile between the free-floating CDB and substrate cellulose could be established.

scholarly journals Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain

1993 ◽  
Vol 295 (1) ◽  
pp. 273-276 ◽  
Author(s):  
A J Fosang ◽  
K Last ◽  
V Knäuper ◽  
P J Neame ◽  
G Murphy ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Polymorphonuclear Leucocyte ◽  
Gelatinase A ◽  
Cleavage Sites ◽  
Similar Extent ◽  
Human Sequence ◽  
Fibroblast Collagenase ◽  
Gelatinases A And B

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

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