A new and highly sensitive fluorescence assay for collagenase-like peptidase activity

Kojima, Kohichi; Kinoshita, Hideko; Kato, Takeshi; Nagatsu, Toshiharu; Takada, Katsumi; Sakakibara, Shumpei

doi:10.1016/0003-2697(79)90106-4

Cited by 69 publications

(20 citation statements)

References 24 publications

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“…A variety of commercially available peptides were also examined for their ability to serve as the substrate for ScpA (Table 3). However, none of them, including Suc-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide (MCA), which was a general substrate for bacterial collagenases (8), acted as a substrate for ScpA. To obtain specific peptide substrates suitable for the kinetic characterization of ScpA, collagen was digested with purified ScpA, and the resultant peptides were separated by reversed-phase HPLC.…”

Section: Resultsmentioning

confidence: 99%

Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Tsuruoka

Nakayama

Ashida

et al. 2003

Appl Environ Microbiol

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Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k cat , 5.41 s ؊1 ; K m , 32 M) and Met-Gly-ProArg*Gly-Phe-Pro-Gly-Ser (k cat , 351 s ؊1 ; K m , 214 M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.Collagen is an insoluble structural protein that accounts for approximately 30% of the total weight of animal proteins and is produced in large quantities as a by-product in livestock industries. It has recently been shown that the enzymatic degradation of collagen as well as that of gelatin, a denatured form of collagen, allows efficient utilization of these structural proteins. This degradation produces peptides, the collagen peptides, which have been shown to have several biological activities of industrial and medical interest (27), leading to the establishment of a wide variety of applications, e.g., an immunotherapeutic agent (7, 9), a moisturizer for cosmetics, a preservative (2), and seasoning and dietary materials (4).Generally, the enzymatic degradation of collagen is not affected by ordinary digestive proteinases but requires the collagen-specific, Zn 2ϩ -dependent metalloproteinases (collagenases). Although microbial collagenases have been found in a wide variety of mesophilic bacterial strains (3), the industrialscale application of known bacterial collagenases for collagen peptide production has been hampered because of their insufficient stability. In 2000...

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Section: Resultsmentioning

confidence: 99%

Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Tsuruoka

Nakayama

Ashida

et al. 2003

Appl Environ Microbiol

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“…17). Kojima et al (1979) employed a fluorescence assay for collagenaselike peptidase using succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA) as a synthetic substrate. Barrett et al (1989) assayed a clostridial collagenase using N-(2,4-dinitrophenyl)-ProLeu-Gly-Pro-Trp-Lys substrate.…”

Section: Synthetic Peptides and Fluorescent Assaymentioning

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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“…After unbroken tissue was separated, the homogenate was centrifuged at 13,000 × g for 30 min at 4°C and the supernatant was used for measurement of protease activities in bone. Cathepsin Kand collagenase-like activities were determined by measuring hydrolysis of their synthetic amidecoumaryl substrates, benzylmoxycarbony-Phe-Arg-4-methylcoumaryl-7-amide (Z-Phe-Arg-NH-Mec) and succinyl-GlyPro-Leu-Gly-Pro-NH-Mec, respectively, as described previously (Brömme et al, 1996;Kojima et al, 1979;Nikawa et al, 1989).…”

Section: Protease Activities In Bonementioning

confidence: 99%

A Cysteine Protease Inhibitor Prevents Suspension-Induced Declines in Bone Weight and Strength in Rats.

Nikawa

Ikemoto

Watanabe

et al. 2002

J. Physiol. Anthropol.

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In this study, we examined the effects of a potent cysteine protease inhibitor, N-(L-3-transcarboxyoxirane-2-cabonyl)-L-leucine-4-aminobutylamide (E-64a), on bone weight and strength in tail-suspended rats. We first administered a vehicle or 4 or 8 mg/rat of E64a to rats fed with a low calcium diet for 7 wks to determine effective doses of E-64a on bone resorption in vivo. Femoral cathepsin K-like activity and serum hydroxyproline level in rats fed with a low calcium diet were significantly higher than those in rats fed with a standard diet. The intraperitoneal injection of 8 mg/rat of E-64a to rats decreased their serum calcium and hydroxyproline concentrations after 3 to 6 hrs in parallel with changes in femoral cathepsin K-like activity, while 4 mg/rat of E-64a had weaker effects on these parameters. Based on these results, we injected 8 mg/rat of E-64a to tail-suspended rats twice a day for 2 wks and compared the results with those of treatment with 1 mg/rat of etidronate, a bisphosphonate, twice a week. In tailsuspended rats, femoral weight and strength, assessed by three-point bending test, significantly decreased from Day 5 to 21, while femoral cathepsin K-like activity and serum calcium and hydroxyproline concentrations did not change. E-64a inhibited femoral cathepsin K-like activity in tail-suspended rats, but etidronate did not. E64a as well as etidronate significantly prevented the suspension-induced declines in bone weight and strength. However, more frequent injection and higher doses were required for E-64a to exhibit significant efficacy of antiresorption, compared with those of etidronate. Our results suggest that a cysteine protease inhibitor could improve suspension-induced osteopenia by inhibiting cathepsin K-like activity in bone; however, it needs several improvements in the effect as a clinical drug.

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A new and highly sensitive fluorescence assay for collagenase-like peptidase activity

Cited by 69 publications

References 24 publications

Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Collagenolytic Serine-Carboxyl Proteinase fromAlicyclobacillus sendaiensisStrain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression

Extraction and Purification of Collagenase Enzymes: A Critical Review

A Cysteine Protease Inhibitor Prevents Suspension-Induced Declines in Bone Weight and Strength in Rats.

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