Purification of pig synovial collagenase to high specific activity.
Ontology highlight
ABSTRACT:
SUBMITTER: Cawston TE
PROVIDER: S-EPMC1161646 | biostudies-other | 1979 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.
| S-EPMC1162004 | biostudies-other
Project description:1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.
| S-EPMC1172413 | biostudies-other
Project description:Three isolectins from cultivated Eucheuma denticulatum were isolated. They were commonly monomeric proteins of about 28 kDa with a range of averaged molecular weights from 27,834 to 27,868 Da among the isolectins and shared almost the same 20 N-terminal amino acid sequences. Complementary DNA (cDNA) cloning based on the rapid amplification cDNA ends (RACE) methods elucidated the full-length sequence of EDA-2 which encodes 269 amino acids, including initiating methionine, with four tandemly repeated domains of about 67 amino acids. The primary structure of EDA-2 is highly similar to those of the high-mannose N-glycan specific lectins including Oscillatoria agardhii (OAA) and Burkholderia oklahomensis EO147 (BOA) from cyanobacteria, Myxococcus xanthus (MBHA) and Pseudomonas fluorescens Pf0-1 (PFL) from bacteria, and ESA-2 from a macro red alga. The hemagglutination activities were commonly inhibited by the glycoproteins bearing high-mannose N-glycans, but not by monosaccharides examined, including mannose. In a direct binding experiment with pyridylaminated oligosaccharides, an isolectin EDA-2 exclusively bound to high-mannose type N-glycans, but not to other glycans that include complex types and a core pentasaccharide of N-glycans, indicating that it recognized the branched oligomannoside moiety. Its binding activity was subtly different among the oligomannoside structures examined, showing that the lectin has preference affinity for high-mannose type N-glycans with an exposed (?1-3) mannose residue in the D2 arm. Interestingly, EDAs, the mixture of three isolectins inhibited the growth of shrimp pathogenic bacterium, Vibrio alginolyticus, although it did not affect the growth of V. parahaemolyticus and V. harveyi. Growth inhibition of V. alginolyticus with EDAs was not observed in the presence of yeast mannan bearing high-mannose N-glycans, suggesting that EDAs caused the activity through binding to the target receptor(s) on the surface of V. alginolyticus. These results indicate that cultivated carrageenophyte E. denticulatum is a good source of a lectin(s) that may be useful as a carbohydrate probe and an antibacterial reagent.
| S-EPMC7088313 | biostudies-literature
Project description:Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.
| S-EPMC5010206 | biostudies-literature
Project description:The transfer of glycosylphosphatidylinositol (GPI) anchors onto eukaryotic proteins is catalyzed by the transamidase complex, which is composed of at least five subunits (PIG-K, PIG-S, PIG-T, PIG-U and GPAA1). Here, the recombinant protein PIG-S(71-467) from Saccharomyces cerevisiae, including residues 71-467 of the entire 534-residue protein, was cloned, expressed and purified to homogeneity. The monodisperse protein was crystallized by the vapour-diffusion method. A diffraction data set was collected to 3.2?Å resolution with 91.6% completeness. The crystals belonged to space group C2, with unit-cell parameters a = 106.72, b = 59.33, c = 124.3?Å, ? = 114.19°, and contained two molecules in the asymmetric unit.
| S-EPMC3151122 | biostudies-literature
Project description:A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2--3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.
| S-EPMC1161203 | biostudies-other
Project description:Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.
| S-EPMC5624855 | biostudies-literature
Project description:1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.
| S-EPMC1162866 | biostudies-other
Project description:Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity.
| S-EPMC8395246 | biostudies-literature
Project description:An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.
| S-EPMC3608664 | biostudies-literature