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Results for “Microbial Collagenase”

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PubMed1984

Experimental synovitis induced by intraarticular administration of alpha 2-macroglobulin-proteinase complexes.

Rheumatoid synovial collagenase was prepared from tissue cultures from rheumatoid arthritis patients, obtained after synovectomy. Alpha 2-macroglobulin was isolated from human plasma and complexed with collagenase or trypsin. Formation of both types of complexes was proven by sodium dodecyl sulfate and rate electrophoresis. Normal rabbits were injected intraarticularly into the right knee, on days 0, 3, and 6, with either alpha 2-macroglobulin-collagenase or alpha 2-macroglobulin-trypsin complexes. Control injections of alpha 2-macroglobulin, trypsin, or rheumatoid synovial collagenase were applied to the left knee joint cavity. Groups of rabbits were killed 18 hours, 1 week, or 3 weeks after the last injection, and cellular exudation into synovial fluid and morphologic alterations of synovium were investigated. Joints injected with alpha 2-macroglobulin showed no synovitis, while joints injected with collagenase showed an experimental synovitis. Alpha 2-macroglobulin-proteinase complexes, however, induced a synovitis, which was more severe than that occurring after injection of proteinases only. In the early stages, synovium showed perivascular accumulation of inflammatory cells, infiltration with neutrophils, proliferation of synovial cells, and exudation of inflammatory cells into synovial fluid. Later stages were characterized by infiltration with mononuclear cells and fibroplasia.

Animals↗
PubMed1991

Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase.

The fibronectin-related region of the 72 kDa type IV procollagenase has been expressed in E. coli as a beta-galactosidase fusion product. The fragment containing the three type II units of the protein was found to have affinity for denatured collagen, suggesting that these domains may be responsible for the collagen-affinity of type IV collagenase. We have also shown that segment Ala-Ala-His-Glu of type IV collagenase (residues 372-375), which is similar to a fibronectin-segment previously implicated in collagen-binding, is not essential for binding activity.

Amino Acid Sequence↗
PubMed1989

Procollagenase associated with the noncalcified matrix of bone and its regulation by parathyroid hormone.

Collagenolytic enzyme activity associated with the noncalcified pool of collagen was studied using calvarial matrices from which the periosteal cell envelope had been removed. Aminophenylmercuric acetate (APMA) stimulated degradation of about 5% of the noncalcified collagen in matrices prepared from freshly dissected bone. Significantly more activity was detected if intact calvaria were cultured 24 h before removal of the cells, in which case 20-30% of the noncalcified collagen was degraded following treatment with APMA. Trypsin elicited a similar response. The collagen being degraded was representative of the entire pool of noncalcified collagen and was not underhydroxylated. Treatment of intact calvaria with parathyroid hormone (PTH) before removal of the cells increased the level of both active collagenase and procollagenase activity associated with the matrix. Enhanced 3H release was noted for PTH treated intact bone in the prior 24 h. Inactivation of endogenous procollagenase by phenanthroline had no effect on the ability of isolated calvarial cells to resorb the bone upon treatment with resorptive agents. The data show that PTH-stimulated collagenolysis of noncalcified collagen involves increased deposition of procollagenase onto the noncalcified matrix in addition to activation of the enzyme.

Animals↗
PubMed1984

Human gingival fibroblast collagenase: purification and properties of precursor and active forms.

Human gingival fibroblast procollagenase has been purified to apparent homogeneity from serum-free and serum-supplemented fibroblast culture medium by a combination of ammonium sulfate precipitation, CM-cellulose chromatography, and gel-filtration on Bio-Gel P-150. Sodium dodecylsulfate polyacrylamide gel electrophoretic studies suggests that the purified fibroblast proenzyme is comprised of two closely related zymogens with the estimated Mr of 57,000 and 52,000. Upon densitometric scanning of the gels, the ratio of the two proenzyme forms was about 1 : 4 (57 : 52 kdal). Limited proteolysis of the fibroblast procollagenase with trypsin resulted in the conversion of both proenzyme forms into active enzyme forms of Mr 48,000 and 44,000, respectively. Amino acid analysis of the active enzymes and proenzyme forms revealed that the active enzymes contained fewer basic amino acids than do the proenzyme forms. The purified trypsin-activated fibroblast collagenase hydrolyzed type I collagen fibrils, cleaved tropocollagen in solution at 24 degrees C into TCA and TCB fragments, and cleaved the synthetic peptide substrate, DNP-peptide III, at the Gly-Ile bond. The gingival fibroblast collagenase exhibited a pH optimum of 7.5, was completely inhibited by EDTA or dithioerythritol but was not inhibited by N-ethylmaleimide or phenylmethylsulfonyl fluoride, and appeared to cleave human type III collagen approximately 10-fold faster than homologous type I collagen. In addition, comparison of the biochemical properties of the precursor and active forms of human gingival fibroblast collagenase with the precursor and active forms of human skin fibroblast collagenase, previously characterized by Stricklin and co-workers (Biochemistry 17: 2331-2337, 1978), revealed that they were similar in Mr, amino acid composition, and substrate specificity. Furthermore, the human gingival and skin fibroblast procollagenases were immunologically identical.

Amino Acids↗
PubMed1985

Human skin fibroblast collagenase: interaction with substrate and inhibitor.

Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.

Chromatography, Gel↗
PubMed2002

Enzymatic debridement of burn wound with collagenase in children with partial-thickness burns.

Seventy-eight pediatric burn patients treated by enzymatic debridement with collagenase clostridiopeptidase A (CCA), were compared to 41 patients those burn wounds were excised surgically. Patients whose burn wounds were initially assessed as partial-thickness at admission were enrolled in the study. Total removal of eschar was achieved in 49 of 78 (62.8%) patients by CCA only (group D). In 29 patients (37.2%), therapy with CCA was ceased because of the development of burn wound infection or a manifest need for grafting of the wound, therefore, these patients underwent tangential wound excision (group DS). The records of 41 patients, treated by early tangential excision, having similar burn wounds by extent and depth with groups D and DS were used as controls (group S). There was no significant difference between the time to achieve a clean wound bed in groups D, DS and S (mean 7.8, 8, and 7 days, respectively, P>0.05). In group D, none of the patients required blood transfusion, except one. Patients in group DS were found to have fewer excisions (mean 1.1) when compared to those in group S (mean 1.5, P<0.05). The shortest hospital stay was found in group D (12.5 days, P<0.01). In conclusion, the use of CCA, provided a short hospital stay, reduced the overall need for surgery and blood transfusions in patients with partial-thickness burns. Thus, CCA should be considered as an initial treatment of choice for removal of eschar in children, having a partial-thickness burn wound without infection.

Analysis of Variance↗
PubMed1988

Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme.

This study describes 11 monoclonal antibodies (Mabs) against human fibroblast collagenase that (i) inhibit the specific catalytic activity of the enzyme and/or (ii) react with one or more forms of the enzyme on Western blots. Each of the Mabs specifically immunoprecipitated the Mr 57,000/52,000 procollagenase from [35S]methionine-labeled culture medium. Five Mabs, designated VI-3, VI-4, 2C5, 4A2, and 7C2, inhibited the activity of fibroblast-type collagenase against soluble monomeric collagen and against reconstituted collagen fibrils but did not inhibit the genetically distinct human PMN leukocyte collagenase. The interstitial collagenase produced by human mucosal keratinocytes (SCC-25) was also inhibited, whereas the corresponding enzyme from rat was not. Assignment of epitopes to structural domains within the molecule based on immunoperoxidase staining of Western blots of collagenase and its autocatalytic fragments revealed that 9 of 11 epitopes, including those recognized by 4 inhibitory Mabs, were clustered in a 169-residue domain, which constitutes the NH2-terminal part of the Mr 46,000/42,000 active enzyme. One Mab (X-2a) specifically recognized the Mr 57,000/52,000 zymogen species and failed to react with the active Mr 46,000/42,000 form. The inhibitory Mab VI-3 was used for immunoaffinity purification of procollagenase from culture media with a recovery better than 80% and a yield of approximately 1.4 mg of enzyme/L of medium.

Antibodies, Monoclonal↗
PubMed1983

Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.

Animals↗
PubMed1991

Hormonal regulation of collagenolysis in uterine cervical fibroblasts. Modulation of synthesis of procollagenase, prostromelysin and tissue inhibitor of metalloproteinases (TIMP) by progesterone and oestradiol-17 beta.

Rabbit uterine cervical fibroblasts produced a large amount of matrix metalloproteinases (MMPs) such as collagenase (MMP-1) and stromelysin (MMP-3) and a small relatively amount of tissue inhibitor of metalloproteinases (TIMP). When cells were treated with progesterone or oestradiol-17 beta, both steroids concurrently decreased the level of procollagenase and prostromelysin in the culture media and the steady-state levels of the respective mRNAs. On the other hand, the level of TIMP in the culture media and the steady-state level of its mRNA were simultaneously increased by these steroids. Similarly, the suppression of production of MMPs and the augmentation of TIMP production by both steroids were observed with interleukin 1 (IL-1)-treated cells, but the action of progesterone was more effective than that of oestradiol-17 beta in the IL-1-untreated and -treated cells. These results suggest that collagenolysis in uterine cervical fibroblasts is negatively regulated by steroid hormones via the acceleration of TIMP production and the suppression of synthesis of MMPs at the pretranslational level.

Animals↗