Immunoenzymology of clostridium histolyticum collagenase

Soru, E; Zaharia, O

doi:10.1007/bf01770294

Cited by 3 publications

(3 citation statements)

References 23 publications

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“…Collagenase inactivated with EDTA reacted with the antiserum as did enzyme in the presence of Ca2+, although the effect of Ca2+ on the interaction has not been examined systematically. This is different from bacterial collagenase, where in the absence of Ca2+ an antiserum raised against the Ca2+-activated enzyme does not precipitate the enzyme (Soru & Zaharia, 1974).…”

Section: Relationship Between Immunoinhibition and Immunoprecipitatiomentioning

confidence: 60%

Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase

Werb¹,

Reynolds²

1975

Biochemical Journal

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1. Antisera were raised against the collagenase from rabbit synovial fibroblasts and characterized by immunoprecipitation and immunoinhibition reactions. 2. Immunoglobulins from the antisera were potent inhibitors of the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. 3. The antibody-binding fragment, Fab', produced by digesting the IgG (immunoglobulin G) with pepsin, inhibited collagenase activity just as well as whole IgG. 4. A specific antiserum to the rabbit collagenase was raised by a multi-step procedure. An initial antiserum was made by injecting partially purified collagenase as a complex with sheep alpha2-macroglobulin into a sheep. The non-specific antiserum so obtained was used to produce precipitin lines with the purified enzyme, and these lines were used as antigen for the production of the specific antiserum. 5. An IgG preparation from the specific antiserum was a specific and potent inhibitor of the rabbit synovial fibroblast collagenase. Neutral metallo-proteinase activity secreted by the rabbit fibroblasts was not inhibited by the antibody to the rabbit collagenase. 6. Criteria for determination of the specificity of antisera are discussed.

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Section: Relationship Between Immunoinhibition and Immunoprecipitatiomentioning

confidence: 60%

Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase

Werb¹,

Reynolds²

1975

Biochemical Journal

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“…Clostripain Each class of collagenases consists of multiple forms of has been shown to be heat stable in the presence of calthe enzyme that vary in molecular weight from as low cium ions and it also is inhibited by EDTA (14). Thereas 66,000 Da to as high as 147,000 Da (8,17). The domifore, to prevent proteolysis of collagenase samples by nant collagenase bands are shown in the upper regions clostripain, it is essential to add EDTA to the sample at ϳ125 and 131 kDa, and they show no signs of degrabuffer.…”

Section: Resultsmentioning

confidence: 99%

Evaluating the Effect of Serine Proteases on Collagenase Activity during Human Islet Isolation

2002

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Inconsistencies in human islet yields after collagenase digestion have been attributed to the activation of endogenous enzymes of the donor pancreas. It has been suggested that pancreatic serine proteases contribute to the proteolysis of collagenase. This study defined the effects of endogenous enzymes within the pancreas on pancreas dissociation during collagenase digestion. Levels of collagenase activity from samples taken throughout several steps in islet isolation procedures, both with and without the addition of the serine protease inhibitor Pefabloc, were determined by a spectrophotometric assay using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala as the substrate. Results clearly demonstrated that the level of collagenase activity remains stable throughout the isolation procedure despite differences in the donor factors from several cadaveric donor pancreases. This was further demonstrated by observing no difference in activity levels after incubating commercial collagenase preparations with serine proteases and analyzing by means of collagenase activity and SDS-PAGE. These data show that the presence of serine proteases does not affect the level of collagenase activity; however, they likely damage the islet cells upon prolonged digestion of the pancreatic tissue. Further efforts at examining exogenous and endogenous enzyme levels may result in the development of an enzyme cocktail that is both stable and effective for digesting the human pancreas while preserving islet function and viability.

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“…Thermal inactivation of collagenase was determined at 49 ~ Half the activity of the Worthington preparation was lost in 4 minutes, whereas the Boehringer preparation was stabler, with a half-life of 15 minutes. These experiments were carried out in Ca-free buffer, since in the presence of calcium, purified collagenase is stable for at least 2 hours at 50 ~ [25]. Incubation of collagenase in 0.1 M Na2ED-TA for 1/2 hour resulted in loss of over % of the hydrolytic activity on the pentapeptide substrate.…”

Section: (4) Inhibition Of Enzyme Activitymentioning

confidence: 99%

Haemorrhagic and inflammatory properties of collagenase fromC. histolyticum

1976

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Collagenase from Clostridium histolyticum induced haemorrhages when applied to the surface of dog lung; it exerted a similar effect on mouse lung when injected intrathoracically. Injected into rat paws, bacterial collagenase induced haemorrhage and oedema. Effects of collagenase were prevented by several procedures that inhibit collagenolytic activity (heating at various temperatures and incubation with metal-complexing agents such as EDTA, penicillamine and dithiothreitol). Protein protease inhibitors, dexamethasone and standard acidic anti-inflammatory drugs had only a slight or no effect on collagenase-induced haemorrhages; dexamethasone and acidic anti-inflammatory drugs blocked collagenase-induced oedema. Inhibition of endogenous kinin-releasing mechanisms by administration of hexadimethrine, a recognized inhibitor of the activation of clotting Factor XII, and depletion of kininogen by administration of carrageenin blocked collagenase-induced oedema. Collagenase did not increase permeability of rat skin vessels, nor did it release potential inflammatory mediators, such as bradykinin or prostaglandins, from plasma or platelets. Bacterial collagenase-induced haemorrhage presumably resulted from enzymatic destruction of membranous structures; at least a portion of the inflammatory response may be due to activation of a kinin-like system.

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Immunoenzymology of clostridium histolyticum collagenase

Cited by 3 publications

References 23 publications

Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase

Immunochemical studies with a specific antiserum to rabbit fibroblast collagenase

Evaluating the Effect of Serine Proteases on Collagenase Activity during Human Islet Isolation

Haemorrhagic and inflammatory properties of collagenase fromC. histolyticum

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