Collagenase from the culture medium of rabbit colon wall with special reference to the latent type and substrate specificity

Oyamada, Ikuko; Kitaoka, Mitsuhiko; Araki, Chotaro; Yoshioka, Hidekatsu; Usuku, Gentaro

doi:10.1016/0016-5085(83)90327-x

Cited by 7 publications

(3 citation statements)

References 26 publications

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“…We have previously shown the substrate specificity for FIG. 17. Cytopathic effect of 56K protease on human embryonic lung fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

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Degradation of protease inhibitors, immunoglobulins, and other serum proteins by Serratia protease and its toxicity to fibroblast in culture

Molla¹,

Matsumoto²,

Oyamada³

et al. 1986

Infect Immun

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We investigated the effect of the extracellular protease of Serratia marcescens on human serum constituents such as immunoglobulins, fibronectin, alpha 1-protease inhibitor, alpha 2-macroglobulin, lysozyme, and transferrin. At a very low concentration of Serratia 56-kilodalton protease (56K protease), purified human plasma fibronectin was degraded rapidly into three structural domains or small fragments. Immunoglobulin G3 (IgG3) and IgA1 were also degraded within 30 min with 1 microgram of this protease per ml, more rapidly than their other subclass of IgG or IgA. alpha 1-Protease inhibitor, which did not inhibit the 56K protease, was degraded similarly by the protease. These events were demonstrated by fluorescence polarization and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protease was considerably inhibited by human alpha 2-macroglobulin and chicken ovomacroglobulin. However, when there was a 2 M excess of ovomacroglobulin or a 4 M excess of alpha 2-macroglobulin over the 56K protease, about 25 or 40% proteolytic activity remained, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protease degraded the alpha 2-macroglobulin extensively during prolonged incubation, which paralleled with regeneration of the protease activity. The protease also cleaved human lysozyme, although moderately. Human serum transferrin was degraded slightly, and human serum albumin was almost resistant to the 56K protease. The enzyme seemed to have no effect on reconstituted collagen, but it degraded rat tropocollagen and yielded fragments of beta and gamma chains by cleaving the intramolecular cross-links. Most of the above proteolysis by the 56K protease appears to result in a limited type of substrate specificity. Thus, the present study demonstrates that the protease is capable of degrading defense-oriented humoral proteins and tissue constituents. Furthermore, it is toxic to fibroblasts. These findings also clarified the possible role of Serratia protease as a virulence factor in the pathogenesis of serratial infections. We recently demonstrated this notion in vivo with rabbit cornea (R. Kamata et al., Ophthalmology 92:1452-1459, 1985).

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“…We have previously shown the substrate specificity for FIG. 17. Cytopathic effect of 56K protease on human embryonic lung fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

“…Then 2 ml of Serratia protease solution (1.0 mg/ml) was added, and the mixture was incubated at 21°C. Viscometry was carried out by using an Ostwald capillary viscometer as described previously (17). Furthermore, the same digest was subjected to analysis by SDS-PAGE as described above.…”

mentioning

confidence: 99%

Degradation of protease inhibitors, immunoglobulins, and other serum proteins by Serratia protease and its toxicity to fibroblast in culture

Molla¹,

Matsumoto²,

Oyamada³

et al. 1986

Infect Immun

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“…Such differences may relate to the presence of variable carbohydrate moieties in the enzyme molecule or to the existence of different genes for each tissue enzyme. The variations in physicochemical properties do not apparently affect the primary action of the enzyme on its substrate, but may explain the subtle differences observed for preferential enzymic activity against some collagen types (15,16) and the susceptibility to certain natural inhibitors (14,17). As yet it is not known whether the physicochemical properties of a collagenase from specific tumor cells might differ from that produced by the normal cells from which the tumor was derived.…”

Section: Extracellular Enzymesmentioning

confidence: 93%

Collagenolytic mechanisms in tumor cell invasion

Woolley¹

1984

Cancer Metast Rev

130

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Connective tissue stroma and basement membrane structures probably present natural barriers to the migration of tumor cells. It has therefore been proposed that collagenolytic enzymes are required to facilitate the spread and invasion of tumor cells into host tissues. The collagenases and cathepsin B-like enzymes are thought to be involved, but the cellular source of collagenolytic activity at the tumor: host interface or 'invasion zone' remains obscure in most cases. The 'invasion zone' of different tumors is very variable with regard to the type and numbers of host or tumor cells, as well as the type of collagenous matrix, and few generalities can be made. The existence within a tumor of specialised subpopulations of cells which have different metastatic potential has been postulated. As a consequence it seems plausible that the phenotypic expression of highly invasive or metastatic tumor cells should include the potential for generating collagenolytic activity. Immunolocalisation studies have demonstrated the production of type I and type IV collagenases at sites of tumor invasion, but it does not appear to be a continuous process and only a small proportion of tumor and/or host cells elaborate enzyme at any one moment. Collagenase production is invariably microenvironmental in nature and it seems likely that local host:tumor cell interactions are important in modulating collagenolysis. Macrophages and mast cells have been shown to stimulate collagenase expression by tumor and stromal cells in vitro, and it is proposed that these cells may assume a contributory role for the induction of collagenolytic activity in vivo. The collagenolytic mechanisms that operate at micro-foci of host:tumor junctions probably depend upon the type of collagen, the cellular composition and the extracellular ionic conditions of each invasion site. Either tumor or host cells may elaborate enzymes, this being dependent upon the type and/or tissue location of the invasive tumor.

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Direct measurement of collagenase in colonic anastomosis

Chowcat

Savage

Lewin

et al. 1990

British Journal of Surgery

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Collagenase has been implicated in colonic anastomotic dehiscence but the enzyme has not previously been specifically measured in colonic healing. A 72 h tissue culture method for colonic tissue and a radiochemical assay for collagenase were adapted to measure the enzyme in healing rabbit colon, with specificity of the assay confirmed by sodium dodecylsulphate polyacrylamide gel electrophoresis. Normal and postoperative colon secreted collagenase, predominantly in a latent form, in the first 24 h of culture. Total activity reached a plateau after 48 and 72 h in culture, when 50-70 per cent of the enzyme was in an active form. At these times in culture, activity was significantly higher than after 24 h (P less than 0.001). One day after anastomosis the total amount of collagenase secreted in culture was higher than normal but the increase did not achieve significance. Three days after anastomosis the colon secreted more collagenase than explants from 1 day postoperative tissue (P less than 0.002). The proportion of active enzyme in the first 24 h in culture was also increased. Since active collagenase can be measured in culture medium from both normal and postoperative colon, the tissue may be secreting plasminogen activator which allows plasmin to activate the enzyme. The increase in collagenase after operation coincided with a decrease in collagen concentration in the colon wall, measured by hydroxyproline. This supports previous suggestions that collagenase contributes to anastomotic dehiscence. However, the findings must be interpreted with caution as the variance of the results was shown to be predominantly due to time in culture, suggesting this could be a bigger influence than the operation itself. In addition, our previously reported immunohistochemical study of this system indicated that collagenase only occurred in a localized region, restricted to the everted portion of the anastomosis, with the activity being tightly controlled by its inhibitor, tissue inhibitor of metalloproteinases.

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Collagenase from the culture medium of rabbit colon wall with special reference to the latent type and substrate specificity

Cited by 7 publications

References 26 publications

Degradation of protease inhibitors, immunoglobulins, and other serum proteins by Serratia protease and its toxicity to fibroblast in culture

Degradation of protease inhibitors, immunoglobulins, and other serum proteins by Serratia protease and its toxicity to fibroblast in culture

Collagenolytic mechanisms in tumor cell invasion

Direct measurement of collagenase in colonic anastomosis

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