Characterization of the individual collagenases from Clostridium histolyticum

Bond, Michael D.; Wart, Harold E. Van

doi:10.1021/bi00308a036

Cited by 212 publications

(153 citation statements)

References 29 publications

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“…Binding of ColH was not inhibited by low temperature unlike activity, suggesting that binding is independent of hydrolysis. In a commercial preparation of C. histolyticum collagenase, Bond and Van Wart (22)(23)(24) reported the presence of multiple forms of collagenases which showed similar peptide maps and amino acid analyses. They suggested that this organism harbors multiple collagenase genes (4).…”

Section: Discussionmentioning

confidence: 96%

A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

Matsushita¹,

Jung²,

Minami³

et al. 1998

Journal of Biological Chemistry

120

108

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The Clostridium histolyticum 116-kDa collagenase consists of four segments, S1, S2a, S2b, and S3. A 98-kDa gelatinase, which can degrade denatured but not native collagen, lacks the C-terminal fragment containing a part of S2b and S3. In this paper we have investigated the function of the C-terminal segments using recombinant proteins. Full-length collagenase degraded both native type I collagen and a synthetic substrate, Pzpeptide, while an 88-kDa protein containing only S1 and S2a (S1S2a) degraded only Pz-peptide. Unlike the fulllength enzyme, S1S2a did not bind to insoluble type I collagen. To determine the molecular determinant of collagen binding activity, various C-terminal regions were fused to the C terminus of glutathione S-transferase. S3 as well as S2bS3 conferred collagen binding. However, a glutathione S-transferase fusion protein with a region shorter than S3 exhibited reduced collagen binding activity. S3 liberated from the fusion protein also showed collagen binding activity, but not S2aS2b or S2b. S1 had 100% of the Pz-peptidase activity but only 5% of the collagenolytic activity of the fulllength collagenase. These results indicate that S1 and S3 are the catalytic and binding domains, respectively, and that S2a and S2b form an interdomain structure.Collagens are the major protein constituents of the extracellular matrix and the most abundant proteins in all higher organisms (1). The tightly coiled triple helical collagen molecule assembles into water-insoluble fibers or sheets which are cleaved only by collagenases, and are resistant to other proteinases. Various types of collagenases, which differ in substrate specificity and molecular structure, have been identified and characterized. Bacterial collagenases differ from vertebrate collagenases in that they exhibit broader substrate specificity (2, 3). Clostridium histolyticum collagenase is the best studied bacterial collagenase (4) and is widely used as a tissuedispersing enzyme (5, 6). This enzyme is unique in that it can degrade both water-insoluble native collagens and water-soluble denatured ones, can attack almost all collagen types, and can make multiple cleavages within triple helical regions (4). Kinetic studies of collagenases have provided insight into the high-ordered structure of collagens (7,8). However, the structure-function relationship of this unique enzyme is not known.

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Section: Discussionmentioning

confidence: 96%

A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

Matsushita¹,

Jung²,

Minami³

et al. 1998

Journal of Biological Chemistry

120

108

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“…Harper et al (1965) isolated two collagenases from Clostridium histolyticum with molecular weights of 105 and 57 kDa. Bond and Van Wart (1984) isolated six different collagenases from the same species with molecular weights ranging from 68-128 kDa. Matsushita et al (1994) reported that collagenases isolated from a related species of Clostridium perfringens had molecular weights ranging from 80-120 kDa.…”

Section: Collagenase and Collagenolytic Enzymesmentioning

confidence: 99%

“…Bond and Van Wart (1984) suggested that some of the variation in reported molecular weights of collagenases may be simply due to proteolysis of a larger collagenase precursor. For use in industry, such variation will be less important than overall collagenolytic activity but the potential for variation should be noted.…”

Section: Collagenase and Collagenolytic Enzymesmentioning

confidence: 99%

Extraction and Purification of Collagenase Enzymes: A Critical Review

Daboor¹,

Budge²,

Ghaly

et al. 2010

American Journal of Biochemistry and Biotechnology

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Problem statement: Enzymes have vital roles in several industrial processes (foods, cosmetics, nutraceuticals and pharmaceuticals) due to their highly selective nature and high activity at very low concentrations. Recent efforts to identify new sources of useful enzymes have been concentrated on the marine environment because of the potential to make use of processing wastes. About 35-50% of the mass of the fish caught is a waste that is disposed off at sea or in landfills. The extraction of enzymes from fish processing waste can reduce environment problems and improve the economics of the fish industry. Collagenases are a group of enzymes that can be extracted from fish waste. Approach: Comprehensive reviews of the literature on the extraction, purification, characterization and use of collagenases was carried out. Results: Collagenases have different molecular weights based on their types and sources. They have the ability to break down the peptide bonds in collagen at physiological pH. They are classified into two types: serine and metallocollagenase. Collagenolytic activities have been shown at a wide range of temperatures (20-40°C) and pH (6-8). Many activators can be used to achive collagenase activity including 4-Aminophenylmercuric Acetate (APMA), trypsin, potassium or sodium thiocyanate, iodoacetamide and potassium iodide. Dithiothreitol (DTT), mercaptoethanol, ethylendiaminetetracetic acid, ophenanthroline and cysteine inactivate the enzyme. Collagenases enzymes can be extracted with a variety of techniques using different buffering systems (tris-HCl, sodium bicarbonate, calcium chloride and cacodylate). All techniques involve the use of ammonium sulphate fractionation and centrifugation to precipitate the enzyme. Collagenases are normally purified using chromatographic techniques such as gel-filtration, ion-exchange and affinity column chromatography. Collagenase can be assayed with a number of methods, including: colorimetric absorbance, viscometry, radioactivity and fluorescence spectroscopy. Collagenases are partly responsible for toughness in red meats and are used as tenderizers in food industry, have application in the fur and hide tanning to ensure uniform dying of leather, used in medicine to treat burns and ulcers, eliminate scar tissues, transplantation of organs. Conclusion: Understanding of the nature of the enzymes and identifying the most suitable resources and the methods for their extraction and purification will have significant impact on the fish processing, food and medical industries.

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“…Thus, synergistic cleavage of collagen fibers suggested for different fractions (16,33) is as yet uncertain. Even the question of whether all the isozymes arise from cleavage of a single progenitor molecule or if some are coded by similar but different genes (5,7) remains to be answered.…”

mentioning

confidence: 99%

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase

et al. 1994

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The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving of the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.

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Characterization of the individual collagenases from Clostridium histolyticum

Cited by 212 publications

References 29 publications

A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

A Study of the Collagen-binding Domain of a 116-kDaClostridium histolyticum Collagenase

Extraction and Purification of Collagenase Enzymes: A Critical Review

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase

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