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PubMed1977

The Seventh Frederick H. Verhoeff Lecture. Collagenase and collagenase inhibitors.

Besides EDTA and cysteine, cystine and penicillamine are the best collagenase inhibitors. The collagenase is produced by the leucocytes. The action mechanism of the collagenase inhibitors is due to the chelation of Zn ions. The best clinical indications for collagenase inhibitors are punctate epithelial keratitis, chemical burns, recurrent corneal erosions in keratoconus, trophic postinfectious ulcerations of the cornea (metaherpetic ulcers), and descemetoceles.

Animals↗
PubMed1975

Action of rheumatoid synovial collagenase on cartilage collagen. Different susceptibilities of cartilage and tendon collagen to collagenase attack.

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, alpha-1(II)-3, in solution at 25 degrees C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three-quarter and one-quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The alpha-chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000. Electron microscopy of segment-long-spacing crystallites of the reaction products revealed three-quarter (TC-a) and one-quarter (TC-b) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I-41). This cleavage site and the formation of TC-a and TC-b reaction products are very similar to those found for type-I collagen substrates. Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid-point melting temperatures (T-m) for lathyritic cartilage and tendon collagen were 40.5 and 41.5 degrees C, and for the collagenase-produced reaction products 38.5 and 37.5 degrees C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.

Animals↗
PubMed1977

Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors.

A radial diffusion assay for tissue collagenase (EC 3.4.24.3) has been devised which is simple, sensitive and capable of application to large numbers of samples. The assay employs an agarose matrix containing solubilized lathyritic rat skin collagen as substrate. Fibril formation is induced for 2 h at 37 degrees C subsequent to 41 h digestion at 28 degrees C. The procedure results in sharply defined zones of lysis which may be measured directly or after photography. The characteristics of the procedure are otherwise similar to those reported for other radial diffusion assays. The new method was used to examine the action of 10 compounds which were known or potential inhibitors of tadpole collagenase. The concentration of inhibitor required to produce 50% inhibition is reported for the following compounds: alpha2-macroglobulin, 142 microng/ml; N-acetylcysteine, greater than or equal to 100 mM; cysteine, 8.7 mM; EDTA, 0.46 mM; histidine, greater than or equal to 100 mM; 2,3-dimercaptopropanol, 0.5 mM and mercaptoacetic acid, 70 mM. The procedure also has potential for clinical determinations (e.g. tears, synovial fluid) since assay dishes may be prepared in advance and only 15 micronl of sample is required.

Animals↗
PubMed1977

Collagenase and collagenase inhibitors in osteoarthritic and normal cartilage.

In advanced osteoarthritis, all of the cartilaginous components are lost from the joint surface. Although mechanisms exist for proteoglycan degradation, there is not known to be any system for removal of the collagen. This study suggests that the loss of the collagen components may be a function of articular cartilage collagenase. The enzyme in normal human cartilage is bound to an inhibitor and appears to be present in very small amounts. Attempts to demonstrate collagenase activity in ground human articular cartilage or in its lysosomal fraction were unsuccessful. 7-Day cartilage tissue cultures also failed to demonstrate the presence of the enzyme; but the same culture fluid, incubated with trypsin, showed significant degradation of collagen, suggesting that trypsin destroyed the inhibitor. 7-Day culture fluids were then chromatographed on a heparin-charged Sepharose 4B affinity column that had been activated with cyanogen bromide. This removed the inhibitor, and the chromatographed fluid from osteoarthritic cartilage released 42% of the incorporated counts of the collagen substrate, whereas normal cartilage released 10.1% and a trypsin control, 6.4%. Electrophoresis of the degradation products of the enzyme-collagen complex incubated at 37 degrees C revealed breakdown was complete to small dialyzable fragments, while at 25 degrees C larger fragments were split off.

Cartilage, Articular↗
PubMed1979

Some newly characterized collagenases from procaryotes and lower eucaryotes.

Chemical and enzymatic properties of four collagenases newly isolated from anaerobic Clostridium histolyticum, aerobic Achromobacter iophagus, and from two lower eucaryotes, the fungus Entomophthora coronata and the insect Hypoderma lineatum are reviewed. The problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in Achromobacter by their macromolecular substrates are discussed. The two bacterial collagenases are Zn-metallo-enzymes; the highly purified Clostridium collagenase contains cyst(e)ine, serine phosphate and tryptophan additionally to amino acids reported previously. Achromobacter collagenase has the highest specific activity of all collagenases; it yields by autolysis enzymatically active degraded forms. The active dimer is composed of two identical subunits of molecular weight 35,000. Similarities between Achromobacter collagenase, thermolysin and Bacillus subtilis neutral proteinase in molecular weight, amino acid composition, and amino acids important for the active sites are discussed. The two collagenases from low eucaryotes are serine proteinases; Hypoderma collagenase is homologous to the trypsin family in the amino terminal sequence. The initial cleavage of native collagen by highly purified bacterial collagenases occurs in the central helical part of the alpha chains and not progressively from the amino terminal end. One of the two initial cleavages produced by Achromobacter collagenase is situated in the region cleaved specifically by vertebrate collagenases, but with different bond specificity. The same is true for the insect collagenase. Entomophthora collagenase is a proteinase of broad specificity which also cleaves collagen in its helical parts. All four collagenases also degrade other proteins according to their bond specificity.

Alcaligenes↗
PubMed1975

Rabbit collagenase. Immunological identity of the enzymes released from cells and tissues in normal and pathological conditions.

1. The immunological cross-reactivity between rabbit collagenases from a variety of normal and pathological sources was examined. The specific antibody raised against collagenase secreted from normal rabbit synovial fibroblasts gave reactions of complete identity with collagenases secreted from fibroblasts derived from rabbit skin, and from synovium from experimentally arthritic rabbits. 2. The rabbit fibroblast collagenase was immunologically identical with collagenases obtained from the organ culture medium of normal rabbit skin, synovium, ear fibrocartilage and subchondral bone. 3. Collagenases from the culture media of normal rabbit synovium and from hyperplastic synovium of rabbits made experimentally arthritic were identical. 4. The collagenase secreted from rabbit fibroblasts gave a reaction completely identical with that of a collagenase extracted directly from a rabbit carcinoma. 5. IgG (immunoglobulin G) from a specific antiserum to rabbit fibroblast collagenase was a potent inhibitor of the collagenases obtained from the culture media of the various rabbit cells and tissues. 6. Collagenases from human synovium and from mouse macrophages and bone were neither precipitated nor inhibited by antibodies to rabbit collagenase. 7. No immunoreactive material was found in lysates of rabbit polymorphonuclear leucocyte granules with the specific antisera to rabbit fibroblast collagenase. No evidence for inactive forms of rabbit collagenase in lysates of the rabbit synovial fibroblasts could be found, either by double immunodiffusion against the specific collagenase, or by displacement of active enzyme from inhibition by the IgG.

Arthritis↗
PubMed1977

Identification and partial characterization of an inhibitor of collagenase from rabbit bone.

Bone explants from foetal and newborn rabbits synthesize and release a collagenase inhibitor into culture media. Inhibitor production in the early days of culture is followed first by latent collagenase and subsequently active collagenase in the culture media. A reciprocal relationship exists between the amounts of free inhibitor and latent collagenase in culture media, suggesting strongly that the inhibitor is a component of the latent form of the enzyme. Over 90% of the inhibitory activity of culture media is associated with a fraction of apparent mol.wt. 30000 when determined by gel filtration on Ultrogel AcA 44. The inhibitor blocks the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. It inhibits the action of either active collagenase or latent collagenase activated by 4-aminophenylmercuric acetate. Latent collagenase activated by trypsin is usually much less susceptible to inhibition. The activity of the inhibitor is destroyed by heat, by incubation with either trypsin or chymotrypsin and by 4-aminophenylmercuric acetate. Collagenase activity can be recovered from complexes of enzyme (activated with 4-aminophenylmercuric acetate) with free inhibitor by incubation with either trypsin or 4-aminophenylmercuric acetate, at concentrations similar to those that activate latent collagenase from culture media. The rabbit bone inhibitor does not affect the activity of bacterial collagenase, but blocks the action of collagenases not only from a variety of rabbit tissues but also from other mammalian species.

Animals↗
PubMed1978

Purification and characterization of a marine bacterial collagenase.

A true collagenase was isolated from the culture fluid of a marine bacterium which has been designated Vibrio B-30 (ATCC 21250). Collagenase production was obtained only in media containing collagen or certain degradation products of collagen. Partial purification on DEAE-cellulose and Sephadex G-200 columns produced active enzyme which was free of nonspecific proteases but which contained two collagenases. The two collagenases have the same apparent molecular size, and evidence is presented to support the theory that one collagenase is derived from the other. Vibrio B-30 collagenase appears to be a tetramer with a molecular weight of about 105 000 composed of two different subunits (mol wt 24 000 and 28 000). Some of the properties of the Vibrio collagenase are compared with those of Clostridium histolyticum collagenase. Molecular weights, subunit structures, specificity and mode of collagen hydrolysis, insensitivity to diisopropyl fluorophosphate and calf serum, and sensitivity to certain metal ion complexing agents and isopropyl alcohol are similar for the collagenases from both organisms. However, Vibrio B-30 collagenase and Clostridium collagenase differ immunologically and electrophoretically.

Clostridium↗
PubMed1979

Collagenase from corneal cell cultures and its modulation by phagocytosis.

The uptake of latex by fibroblasts in confluent primary culture results in the secretion of collagenase at a linear rate for a prolonged period. Phagocytosis might therefore constitute an important level of collagenase regulation in corneal ulceration. The collagenase in cell cultures is present in a latent form (40,000 MW) like that obtained from organ cultures of ulcerating corneas and can be activated proteolytically. Production of the latent collagenase in cell culture depends upon the presence of serum and diminishes greatly when serum is removed from the medium. Collagenase activity can be demonstrated after the latent collagenase has been separated from serum antiproteases in the media. Alternatively, careful titration of the crude media with trypsin to saturate serum antiproteases, to release collagenase from the complex with alpha 2-macroglobulin, and to activate latent collagenase also results in measurable collagenase activity. The collagenase that is secreted cleaves fibrillar type I collagen and cleaves soluble type I collagen into the typical 3/4 and 1/4 length fragments, as demonstrated by SDS-gel electrophoresis and electron microscopy.

Animals↗
PubMed1976

Collagenase enzymes from Clostridium: characterization of individual enzymes.

Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized. The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00. Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose. The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity. At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000. The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain. All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive. Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell.

Clostridium↗
PubMed1978

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts.

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

Blood Proteins↗
PubMed1977

Proteolytic activation of rat liver adenylate cyclase by a contaminant of crude collagenase from Clostridium histolyticum.

Treatment of rat liver plasma membranes with various commercial preparations of crude collagenase from Clostridium histolyticum at concentrations as low as 1 mug/ml, resulted in activation of the adenylate cyclase system. Maximal activation occurred at 50 to 100 mug/ml of collagenase, and promoted a 2- to 3-fold increase in the basal activity as well as in the activities stimulated by catecholamines, glucagon, fluoride, or GTP. This was due to an increase in the maximal velocity of the cyclizing reaction without any increase in the affinity of the enzyme for its substrate. Treatment of plasma membranes with crude collagenase did not induce gross structural modifications as judged by electron microscopic examination. 5'-Nucleotidase activity was slightly inhibited and ATPase activity remained unaffected. The stimulatory substance was nondialyzable, thermolabile, and inhibited by both EDTA and -SH reagents, thus appearing to be a protein. The following observations suggest the effects observed were due to other protease(s) present in crude collagenase: (a) only crude collagenase was active on liver adenylate cyclase: treatment with purified collagenase from C. histolyticum or from Achromobacter iophagus gave no stimulation; (b) the stimulatory activity was irreversible since washing of the membranes after treatment was without effect; (c) crude collagenase contained no lecithinase or sphingomyelinase activity under our conditions of adenylate cyclase assay; (d) after chromatography on Sephadex G-100, the activator appeared as a peak in the 30,000-dalton region and was clearly separated from the collagenase and clostripain peaks, but coincident with elastolytic and caseinolytic activities; (e) the effect of crude collagenase could be prevented by addition of elastin in vitro and was mimicked by purified elastase from hog pancreas. It remains to be seen whether the effects observed result from an increase in the catalytic constant of adenylate cyclase, or an unmasking of new catalytic sites.

Adenosine Triphosphate↗
PubMed1976

Haemorrhagic and inflammatory properties of collagenase from C. histolyticum.

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.

Animals↗
PubMed1979

Total, latent and active collagenase during the course of post-partum involution of the rat uterus. Effect of oestradiol.

1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.

Animals↗
PubMed1978

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.

Chloramphenicol↗
PubMed1978

Different appearance of hepatic collagenase and lysosomal enzymes in recovery of experimental hepatic fibrosis.

1. Both activities of hepatic collagenase and lysosomal enzymes (acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase) have been observed in the recovery from experimental hepatic fibrosis in rats treated with carbon tetrachloride for 6 to 20 weeks, and compared with the disappearance of newly formed collagen fibers in the recovery process. 2. In the process of experimental hepatic fibrosis, collagenase activity reached maximum on sethe accumulation of collagen fibers in reversible hepatic fibrosis, but decreased to the same level as that of non-treated rat liver in cirrhotic stage. In the reocvery from reversible hepatic fibrosis, collagenase activity reached maximum on second day after the discontinuation of carbon tetrachloride, and decreased to the same extent of that of non-treated rat liver on seventh day. 3. Lysosomal enzyme activity was parallel to the activity of hepatic collagenase and to the accumulation of collagen fibers in the process of hepatic fibrosis. In the recovery stage, lysosomal enzyme activity in mesenchymal cells within the septa increased markedly on second day after the discontinuation of toxic agent but turned to the same level of that of non-treated rat liver seven days later, which was consistent with the appearance and disappearance of collagenase activity. On the other hand the appearance of lysosomal enzymes activities in Kupffer cells and hepatocytes was different from that of collagenase activity. That is lysosomal enzyme activity in Kupffer cells decreased in early days but increased five days later, and the enzyme activity in hepatocytes markedly decreased but gradually recovered to normal level seven days later. 4. The appearance of collagenase was observed at the beginning of the recovery stage. It indicates that mammalian collagenase initiates the collagen degradation and lysosomal enzymes might have a role in the subsequent degradation of collagen.

Animals↗