Effect of dexamethasone, indomethacin, and lipopolysaccharide on the secretion of interstitial collagenase and type V collagenase by cultured macrophages

The rabbit alveolar macrophage secretes at least two collagenolytic metalloproteinases in vitro including an interstitial collagenase and a type V collagenase. Using assays previously shown to discriminate between these two activities, the secretion of these two enzyme activities was investigated. Both enzyme activities accumulated in culture over 11 days and the release of both were similarly inhibited by cycloheximide. Collagenolytic activity was negligible in cell lysates. The interstitial collagenase was found in a latent form but the type V collagenase activity was active in the culture medium. When cultured in the presence of dexamethasone, the secretion of both the enzymes were identically inhibited in a dose-dependent manner. Indomethacin was an effective inhibitor of secretion of both collagenases at a concentration of 10(-5) M but not at lower concentrations. Finally, bacterial lipopolysaccharide stimulated the secretion of both type V and interstitial collagenase by these cells. These studies indicate that, like the interstitial collagenase, the type V collagenase is released from the cell as synthesized and is not stored intracellularly. Protein synthesis is necessary for the release of both these collagenases. Furthermore, the release of type V collagenase responded to dexamethasone, indomethacin, and lipopolysaccharide in a manner identical to the secretion of the interstitial collagenase suggesting that synthesis and secretion of these two enzymes are regulated in a similar manner.     

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.     

Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase

Feeder-cell-independent serially propagating keratinocytes from rat oral mucosa (tongue) dissolved reconstituted type I [3H]collagen fibrils, although rather slowly. Analysis of the conditioned medium from such cultures revealed secretion of a Mr = 65,000 collagenase which remained almost entirely latent in the absence of exogenous protease activity. Addition of trypsin (0.1-1.0 microgram/ml) or plasmin (1.0-4.0 micrograms/ml) resulted in substantial acceleration of the collagenolytic process in stimulated secretion of latent collagenase and, at higher concentrations, in conversion of the latent enzyme to the catalytic form. The keratinocyte collagenase was indistinguishable from interstitial, fibroblast-type collagenases by several criteria including: cleavage of native type I collagen in solution at the characteristic collagenase-sensitive locus at 22 degrees C and dissolution of reconstituted type I collagen fibrils at 35 degrees C; activation by trypsin and by organomercurials and inhibition by Zn2+ and Ca2+ chelators; and cross-reaction with antibody to fibroblast-type procollagenase. Expression of collagenolytic activity in keratinocyte cultures was effectively regulated by cell density. The activity (on a per cell basis) was maximal at 10-20% confluence and was more than 95% "contact-inhibited" at subconfluent and early confluent densities (2-4 X 10(5)/cm2). Our findings show that mucosal keratinocytes possess a potent enzymatic apparatus for degradation of interstitial collagen fibrils which includes a classical vertebrate collagenase.     

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.     

Secreted forms of human neutrophil collagenase

Collagenase in human neutrophils is found within intracellular granules which can be stimulated to be secreted with phorbol myristic acetate. This extracellular secreted form of neutrophil collagenase was isolated by immunoaffinity chromatography using a monoclonal antibody previously shown to specifically recognize neutrophil collagenase. The enzyme efficiently bound to this column and was eluted with NaSCN as three major species of 75, 57, and 22 kDa, respectively. These proteins were closely related immunologically since, after radiolabeling and separation by gel filtration, each of the three proteins was precipitated by the monoclonal antibody. Also, the 75- and 57-kDa proteins exhibited collagenase activity after elution from polyacrylamide gels run under nondenaturing conditions. Further, the 57-kDa protein autodegraded into a 22-kDa protein with time. Polyclonal antibody, prepared to the 57-kDa enzyme, also recognized the 75- and 22-kDa proteins using an immunoblot technique. When crude neutrophil supernatants containing latent collagenase were immunoblotted, both the 75- and the 57-kDa enzymes were present. Our immunoaffinity purified active enzymes, although activated during the course of purification, resemble the latent enzymes in crude neutrophil supernatants. The multiple forms of secreted collagenase from degranulated leukocytes may resemble more closely that seen in inflammation.     

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.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils

Collagenase released from rheumatoid synivial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24°C and 37°C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibril can be degraded. Binding was not inhibited by the presence of α₂ macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding the substrate, and that activation by proteases such as plasmin intiates collagen breakdown.     

A precursor form of latent collagenase produced in a cell-free system with mRNA from rabbit synovial cells

mRNA extracted from rabbit synovial fibroblasts which had been induced to produce large amounts of collagenase (EC 3.4.23.7) by urate crystals was translated in a cell-free wheat germ system. Collagenase was identified by immunoprecipitation using mono-specific antibody to rabbit synovial collagenase. In the absence of microsomal membranes, a single precursor with Mr = 59,000 was synthesized. This polypeptide was susceptible to proteolytic degradation. In the presence of canine pancreatic microsomes, the nascent protein was processed to a polypeptide with Mr = 57,000 (identical in mobility on sodium dodecyl sulfate-gel electrophoresis to the major latent collagenase secreted from cells) and was protected from tryptic digestion unless a detergent was used to disrupt the membranes. In addition to Mr = 57,000 material, cells secreted immunologically reactive latent collagenase with Mr = 61,000. High molecular weight collagenase was separated from Mr = 57,000 species by binding to concanavalin a-Sepharose, suggesting that this enzyme was a product of post-translational glycosylation. Both latent enzymes were activated by trypsin and human plasma kallikrein to Mr = 45,000 and 49,000. The evidence indicates that rabbit synovial fibroblast collagenase is synthesized and secreted as a single polypeptide zymogen, not as an enzyme-inhibitor complex.     

The activation of latent pig synovial collagenase

Latent pig synovial collagenase (EC 3.4.24.7) can be activated by a variety of different treatments to give an active enzyme form of lower molecular weight which rapidly degrades collagen. Trypsin and plasmin effectively activated the latent collagenase whilst elastase and cathepsin G degraded most of the latent enzyme before it was activated. A number of mercurials were compared and maximum activation was achieved using 4-aminophenylmercuric acetate and phenylmercuric chloride. The latent collagenase bound to a mercurial-Sepharose column and was eluted in the active form with NaCl. The latent collagenase also activated spontaneously and the conditions which encouraged and prevented this activation were studied. High NaCl concentration, diisopropylphosphofluoridate, soybean trypsin inhibitor, low Zn2+ concentration and high and low pH all prevented the spontaneous activation of latent pig synovial collagenase.     

Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.     

Purification to homogeneity of latent and active 58-kilodalton forms of human neutrophil collagenase

Latent and active 58-kDa forms of human neutrophil collagenase (HNC) have been purified to homogeneity. Buffy coats were extracted in the presence and absence of phenylmethanesulfonyl fluoride to generate crude starting preparations that contained latent and active HNC, respectively. The buffers used in preparing these extracts and for all subsequent chromatographic steps contained NaCl at a concentration of 0.5 M or greater, 0.05% Brij-35, concentrations of CaCl2 of 5 mM or greater, and (when feasible) 50 microM ZnSO4 to stabilize the HNC. The collagenase activity in the buffy coat extracts was adsorbed to a Reactive Red 120-agarose column at pH 7.5 in 0.5 M NaCl and was eluted when the NaCl concentration was increased to 1 M. The active and p-(chloromercuri)benzoate-activated latent enzymes were next adsorbed to a Sepharose-CH-Pro-Leu-Gly-NHOH affinity resin in 1 M NaCl at pH 7.5 and desorbed at pH 9 to give a fraction containing only HNC and a small amount of neutrophil gelatinase. The latter enzyme was removed by passage over a gelatin-Sepharose column in 1 M NaCl at pH 7.5. The purified samples of active and latent HNC were obtained with typical cumulative yields of 32 and 82% and specific activities toward soluble rat type I collagen at 30 degrees C of 7200 and 12,000 micrograms min-1 mg-1, respectively. These specific activities are markedly higher than previously reported for HNC. Both active and latent HNC exhibit a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the presence and in the absence of 2-mercaptoethanol. The mobility of latent HNC is consistent with a molecular weight of approximately 58K, with the active form exhibiting a slightly lower (less than 1-2K) molecular weight.     

Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens.

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.     

Purification and characterization of three forms of collagenase from Clostridium histolyticum

Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified. Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar. Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3. This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes. Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity. C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site. The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related. Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3. C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining.     

Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen

A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices.     

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.     

Identification of a new metalloproteinase inhibitor that forms tight-binding complexes with collagenase.

Connective-tissue cells produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that the levels of this inhibitor controlled the extracellular activity of these enzymes. We recently detected a new metalloproteinase inhibitor present in culture media of WI-38 fibroblasts. The inhibitor, named 'large inhibitor of metalloproteinases' (LIMP), can be separated from TIMP by gel filtration on Ultrogel AcA 44, where it is eluted with an apparent Mr of 76,000. A portion of this inhibitor-containing peak binds to concanavalin A-Sepharose, indicating that at least some of the inhibitor contains carbohydrate. LIMP inhibits collagenase (MMP-1), stromelysin (MMP-3) and gelatinase (MMP-2) in a dose-dependent fashion. Collagenase forms tight-binding complexes with LIMP, which can be separated from free collagenase on gel-filtration columns. The complex is eluted with Mr 81,600 (AcA 44) or Mr 60,000 (Superose 12). This complex is larger than that formed between collagenase and TIMP, which has Mr 52,800 (Aca 44) or 41,000 (Superose 12). Polyclonal antibody to TIMP does not recognize LIMP by immunoblotting, and will not block the inhibition of collagenase by LIMP, showing that LIMP is not a multimeric form of TIMP. The role of this new inhibitor in connective-tissue breakdown studies and its relationship to previously described inhibitors of metalloproteinases is discussed.     

Latent and active forms of collagenase in rat uterine explant cultures: regulation of conversion by progestational steroids

A latent collagenase, activatable by trypsin, has been identified in the culture media of postpartum rat uterus explants. Progesterone at a concentration of 25 × 10^?6m reduced the level of active collagenase by approximately 50%, whereas, total enzyme levels (active + latent) remained essentially constant during the first 3 days of culture. In addition, medroxyprogesterone acetate at a concentration of 1 × 10^?6m reduced active enzyme by approximately 75% while only small decreases in total enzyme were observed. After the third day of culture, total enzyme levels were also significantly decreased. These data suggest that during the first 3 days in culture the progestins prevent the conversion of latent collagenase to its active form. A fraction capable of promoting the activation of explant collagenase was detected in the culture medium and was partially separated from the collagenase. Progesterone (25 × 10^?6m) or medroxyprogesterone acetate (1 × 10^?6m) caused a 50 or 71% decrease, respectively, in the levels of the activator.     

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.     

Matrix degrading proteinases from human granulocytes: type I, II, III collagenase, gelatinase and type IV, V-collagenase. A survey of recent findings and inhibition by gamma-anticollagenase

Three human matrix degrading leukocyte proteinases, type I collagenase, gelatinase and a new type IV collagenase were isolated in latent and active form. Activation of all three latent enzymes could be achieved by treatment with either organomercurials or with trypsin. In addition the 90 kDa latent type I-collagenase could be activated by disulfides, while a newly discovered 70 kDa latent form could be activated with organomercurials or with trypsin. The active type I collagenase was inhibited by gamma-anticollagenase from human serum (and the leukocyte type I collagenase inhibitor, while the newly found type IV collagenase was inhibited only partially. The complexes formed from gamma-anticollagenase with type I collagenase, i. e. latent enzyme, are not reactive site associated complexes. The binding is not of a substrate-like and competitive manner. After inhibition of the enzyme though inactive against its natural substrates it is still hydrolyzing the synthetic low molecular weight octapeptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH.