Cyclic adenosine 3',5'-monophosphate suppresses interleukin 1-induced synthesis of matrix metalloproteinases but not of tissue inhibitor of metalloproteinases in human uterine cervical fibroblasts.

Interleukin 1 (IL-1) mediates many cellular functions, but the signal transduction mechanisms of its actions are not clearly understood. Here, we have examined the exact participation of cAMP in the IL-1-induced production of the precursors of matrix metalloproteinase (MMPs) and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP) in human uterine cervical fibroblasts. IL-1 significantly augmented the production of proMMP-1 (vertebrate procollagenase), proMMP-3 (prostromelysin), and TIMP without detectable changes in the intracellular level of cAMP. Dibutyryl cAMP (Bt2cAMP) and the cAMP elevating agent (forskolin) did not replace IL-1 as MMP inducers. On the contrary, the IL-1-mediated induction of proMMP-1 and proMMP-3 was significantly suppressed by treatment of the cells with Bt2cAMP, forskolin, or theophylline. The suppressive effect of Bt2cAMP on the IL-1-induced production of proMMP-1 and -3 was not due to the inhibition of zymogen secretion, but resulted from the decrease in the steady-state levels of proMMP-1 and proMMP-3 mRNAs. In contrast, Bt2cAMP slightly enhanced the IL-1-induced production of TIMP. The synthesis of proMMP-2 (72-kDa progelatinase/type IV procollagenase) was not altered by IL-1 and/or Bt2cAMP. These results suggest, first, that induction of proMMP-1 and -3 synthesis may share similar transduction pathways but they are distinct from those for proMMP-2 and TIMP synthesis and, second, that cAMP does not function as a second messenger in the MMPs' induction upon IL-1 stimulation in human uterine cervical fibroblasts. Thus, it is further suggested that the system that increases the intracellular cAMP level may be involved in negative regulation of proMMP-1 and -3 production.     

The enzymatic evaluation of procollagenase and collagenase inhibitors in crude biological media

The validity of the enzymatic assay of procollagenase within crude biological media containing also the collagenase inhibitor TIMP (tissue inhibitor of metalloproteinases) as well as other (pro)metalloproteinases and sometimes, metalloproteinase-TIMP complexes, has been reevaluated. To be enzymatically assayed, procollagenase has to be activated. The standard activation procedures by either trypsin or 4-aminophenylmercuric acetate (APMA) both allow an optimal recovery of collagenase from procollagenase when the media do not contain free TIMP. However, they do not destroy TIMP nor do they reactivate the collagenase present in enzyme-inhibitor complexes. Therefore, the collagenase formed by the activation of procollagenase in the presence of free TIMP is immediately inactivated by binding to the inhibitor. As a result, both the bound collagenase and TIMP can no longer be assayed by enzymatic methods. An optimal recovery of collagenase can, however, be obtained if free TIMP is neutralized by the binding of other tissue metalloproteinases (such as those present in culture media of rabbit bone marrow-derived macrophages) prior to the activation and assay of procollagenase. Similarly, it is possible to recover under an active free form a large part of the TIMP present in collagenase- (or other metalloproteinase-)TIMP complexes by heating the complexes at acid pH under conditions which inactivate the collagenase.     

Human hepatoma cells produce an 85 kDa gelatinase regulated by phorbol 12-myristate 13-acetate

Several human cell lines were studied for the production of gelatinases. Diploid fibroblasts, the melanoma cell line Bowes, the MG-63 osteosarcoma cell line and the human hepatoma cell line Malavu all constitutively produced a 67 kDa gelatinase. Gelatinolytic enzymes were quantified by a sensitive zymographic substrate conversion assay. Upon induction with phorbol 12-myristate 13-acetate (PMA), the human hepatoma cell line secreted considerable amounts of an 85 kDa gelatinase activity. The induction process was time- and dose-dependent. It represented a true increase in production per individual cell and was associated by a marked change of the cell morphology. The effect of various proteinase inhibitors and the maximal activity of the enzyme near neutral pH demonstrate that it is a neutral metalloproteinase. Characterization studies showed the 85 kDa gelatinase to be transformed to lower molecular weight, active forms by treatment with p-aminophenylmercuric acetate (APMA) or trypsin.     

Modulation by interleukin 1 and tumor necrosis factor alpha of production of collagenase, tissue inhibitor of metalloproteinases and collagen types in differentiated and dedifferentiated articular chondrocytes

The actions of interleukin 1 (IL1) and tumor necrosis factor alpha (TNF alpha) on several parameters of the collagen metabolism of rabbit articular chondrocytes were studied by comparing the responses of either differentiated chondrocytes in primoculture or dedifferentiated cells in late passage culture to human recombinant (hr) IL1 alpha, hr-TNF alpha and cytokine-enriched fractions of rabbit macrophage-conditioned media. In response to IL1 or TNF alpha, differentiated chondrocytes (i.e., producing the cartilage-specific collagens, types II and XI, but no type I), sharply reduced their synthesis of collagen, a reduction which involved both types II and XI collagens, without consistently changing their production of non-collagenous proteins; they also incorporated a smaller proportion of collagen into the matrix. Similar levels of response were obtained for hr-IL1 alpha at picomolar and for hr-TNF alpha at nanomolar concentrations. However, the action of TNF alpha, but not of IL1, was manifested only in the presence of serum. Simultaneously, IL1, but not TNF alpha, induced the chondrocyte production of procollagenase (a difference which contrasted with the similar levels of procollagenase induced by both cytokines in synovial and skin fibroblasts) but neither cytokine influenced the accumulation of the collagenase inhibitor TIMP. These effects were not affected by indomethacin and are thus unlikely to be prostaglandin-mediated. During their dedifferentiation in monolayer subcultures, chondrocytes became more sensitive to the procollagenase-inducing ability of IL1 and TNF alpha, but their response to TNF alpha was lower than to IL1. They also increased their production of TIMP, which remained unaffected by the cytokines. Simultaneously, they decreased their production of collagen and substituted progressively the synthesis of fibroblast-specific collagens, types I, III and V, for types II and XI. Acting on dedifferentiated cells, even in the presence of indomethacin, IL1 and TNF alpha further decreased the synthesis of collagen, reducing the production of both typical type I (i.e. [alpha 1(I)]2 x alpha 2(I) molecules) and type V collagens as well as their incorporation into the matrix, but increasing the synthesis of type III collagen. Therefore not only IL1, but also TNF alpha can exert profound influences on the collagen degradation and repair processes occurring in the pathology of articular cartilage.     

Procollagenase activator produced by rabbit uterine cervical fibroblasts.

Culture medium from rabbit uterine cervical fibroblasts contained a procollagenase and a neutral proproteinase which acts as a procollagenase activator. These two proenzymes have been purified by a combination of ion-exchange, affinity and gel chromatographies. The purified neutral proproteinase showed Mr 60,000 with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This neutral proproteinase was activated by trypsin, 4-aminophenylmercuric acetate (APMA) and plasmin, and the active species of the proteinase had Mr 53,000 when activated by APMA; kallikrein and urokinase did not activate this proproteinase. The purified neutral proteinase was inhibited by EDTA, 1,10-phenanthroline and rabbit plasma, but not by serine proteinase inhibitors, suggesting that this proteinase is a metal-dependent proteinase. The purified enzyme could also degrade gelatin, casein, proteoglycan and type IV procollagen. The purified procollagenase had Mr 55,000 and was activated by trypsin, APMA and the active neutral proteinase. These activations were accompanied by decrease in Mr, and the activated species had an Mr which was approx. 10,000 less than that of the procollagenase. In particular, procollagenase activation with neutral proteinase depended on incubation time and proteolytic activity of proteinase. These results indicate that activation of procollagenase by the rabbit uterine neutral proteinase is related to limited proteolysis in the procollagenase molecule.     

Activation of human progelatinase A by collagenase and matrilysin: Activation of procollagenase by matrilysin

Proteolytic and nonproteolytic methods were used to investigate the mechanism(s) by which human fibroblast progelatinase A and fibroblast-type procollagenase can be activated. Both collagenase and matrilysin were able to activate progelatinase A, resulting in an amino terminus in gelatinase A of Tyr.⁸¹ The cleavage occurred distal to Cys⁷³ within the sequence of PR_CGNPDVAN⁸⁰-Y⁸¹NFFPRKP. While several nonproteolytic reagents were tested, only the heavy metal Hg() andp-chloromercuribenzoate (PCMB) were able to induce activation of progelatinase A and resulted in the conversion of the latent 72-kDa gelatinase A to an active form of about 64.5 kDa. Matrilysin was also able to activate procollagenase and resulted in an amino terminus in collagenase of Phe.⁸¹ These results suggest that fibroblast-type collagenase and matrilysin may be physiologically relevant activators of progelatinase A; the maintenance of latency and the process of activation for progelatinase A may occur through the cysteine-switch mechanism, and the proteolytic activation of procollagenase by matrilysin resulted in the same amino terminus as produced by stromelysin-1.Abbreviations APMA p-aminophenylmercuric acetate - Bistris [bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane] - CAPS 3-(cyclohexylamino)-1-propane sulfonic acid - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - GSSG oxidized glutathione - HFC human fibroblast-type collagenase, MMP-1 - HFG human fibroblast gelatinase A/72-kDa type IV collagenase, MMP-2 - HFS human fibroblast stromelysin-1, MMP-3 - MMP matrix metalloproteinase - MT-MMP membrane-type matrix metalloproteinase, MMP-14 - NEM N-ethylmaleimide - PCMB p-chloromercuribenzoate - PMA phenylmercuric acetate - PMC phenylmercuric acid - PMSF phenylmethanesulfonyl fluoride - PTH phenylthiohydantoin - RTT rat tail tendon - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tes [tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid] - TIMP tissue inhibitor of metalloproteinase - Tris tris(hydroxymethyl)aminomethane - Tricine N-[tris(hydroxymethyl)methyl]glycine     

Pancreatic trypsin activates human promatrix metalloproteinase-2

In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.     

Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo.     

Natural human monocyte gelatinase and its inhibitor.

Gelatinases produced by stimulated peripheral blood monocytes were detected by substrate zymography and were compared with those derived from tumor cells. Stimulated monocytes were found to produce an 85 kDa gelatinase which co-migrated upon electrophoretic separation and cross-reacted in immunoprecipitation experiments with a phorbol ester inducible metalloprotease from human tumor cells. The intact natural gelatinase (85 kDa), a high molecular weight and complexed gelatinase as well as a proteolytic fragment (25 kDa) were purified by substrate- and antibody-affinity chromatography techniques. Aminoterminal sequence analysis showed that natural monocyte gelatinase occurs as a truncated form of tumor cell gelatinase/type IV collagenase. Furthermore, peripheral blood monocytes were found to also produce a tissue inhibitor of metalloproteases (TIMP). TIMP was co-purified with gelatinase on gelatin sepharose and identified by microsequencing. The balanced and regulated production of gelatinase and TIMP might be important in monocyte migration and tissue remodeling.     

Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans.

In the leukemic macrophage cell-line THP-1, a fraction of the secreted matrix metalloproteinase 9 (MMP-9) is linked to the core protein of chondroitin sulfate proteoglycans (CSPG). Unlike the monomeric and homodimeric forms of MMP-9, the addition of exogenous CaCl2 to the proMMP-9/CSPG complex resulted in an active gelatinase due to the induction of an autocatalytic removal of the N-terminal prodomain. In addition, the MMP-9 was released from the CSPG through a process that appeared to be a stepwise truncation of both the CSPG core protein and a part of the C-terminal domain of the gelatinase. The calcium-induced activation and truncation of the MMP-9/CSPG complex was independent of the concentration of the complex, inhibited by the MMP inhibitors EDTA, 1,10-phenanthroline and TIMP-1, but not by general inhibitors of serine, thiol and acid proteinases. This indicated that the activation and truncation process was not due to a bimolecular reaction, but more likely an intramolecular reaction. The negatively charged chondroitin sulfate chains in the proteoglycan were not involved in this process. Other metal-containing compounds like amino-phenylmercuric acetate (APMA), NaCl, ZnCl2 and MgCl2 were not able to induce activation and truncation of the proMMP-9 in this heterodimer. On the contrary, APMA inhibited the calcium-induced process, whereas high concentrations of either MgCl2 or NaCl had no effect. Our results indicate that the interaction between the MMP-9 and the core protein of the CSPG was the causal factor in the calcium-induced activation and truncation of the gelatinase, and that this process was not due to a general electrostatic effect.     

Evidence that human rheumatoid synovial matrix metalloproteinase 3 is an endogenous activator of procollagenase

The treatment of crude culture medium from human rheumatoid synovial cells with 4-aminophenylmercuric acetate (APMA) or trypsin results in the activation of procollagenase. This process was shown to be dependent on the presence of matrix metalloproteinase 3 (MMP-3). MMP-3 can directly activate procollagenase without changing the apparent molecular weight of procollagenase. This activity was accelerated in the presence of APMA. We propose that MMP-3 plays an important role in connective tissue destruction through the activation of procollagenase in addition to its direct action on components of the extracellular matrix.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties.

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).     

Human gelatinase/type IV procollagenase is a regular plasma component

Gelatin zymograms revealed in human plasma a constant 66 kDa proteolytically active polypeptide. In most plasma samples other major proteolytic activities were seen at Mr 92,000, 130,000 and 225,000. All four proteases were Ca2+-dependent metalloproteases and bound quantitatively to gelatin-Sepharose. Immunoblotting results indicated that the 66 kDa protease was the human fibroblast gelatinase/type IV procollagenase and that the other three proteases were macrophage/granulocyte-derived gelatinase components. The 66 kDa protease did not bind to conA- nor lentil lectin-Sepharose allowing its separation from the 92, 130 and 225 kDa proteases. During the isolation procedure the plasma gelatinase/type IV procollagenase tended to form a proteolytically active spontaneous disulfide-bonded dimer and a 62 kDa component that could also be obtained by digestion with trypsin. The same polypeptide changes occurred also in stored preparations of the corresponding protease isolated from fibroblast culture medium while the freshly purified protein contained only the 66 kDa proform.     

Protease expression in the supernatant of Chinese Hamster Ovary cells grown in serum-free culture

The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B.     

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1 beta and a protein kinase C activator.

The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1 beta (rIL-1 beta) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1 beta alone at concentrations of 1-100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1 beta (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1 beta or PKC agonists alone or in combination. Recombinant tumor necrosis factor alpha (rTNF alpha) did not substitute for IL-1 beta in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1 beta or TNF alpha alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.     

Characteristics of 92 kDa type IV collagenase/gelatinase produced by granulocytic leukemia cells: structure,expression of cDNA in E. coli and enzymatic properties

Human neutrophils can be triggered to release the collagenolytic metalloenzymes, interstitial collagenase and 92 kDa type IV collagenase/gelatinase. We have isolated and sequenced a 2.3 kb cDNA from a chronic granulocytic leukemia cDNA library that encodes for human neutrophil type IV collagenase. With the exception of one amino-acid substitution at position 280 (Arg → Gln), the deduced amino-acid sequences of neutrophil gelatinase are identical to the amino-acid sequences of the enzyme isolated from fibrosarcoma cells. Expression of the cDNA in E. coli yielded a 72 kDa protein having a gelatinolytic activity on zymogram gel. The recombinant enzyme was activated with APMA and trypsin. The activation was accompanied by a reduction in molecular weight of ≈ 10 kDa; such a reduction is characteristic of matrix metalloproteinases. The recombinant gelatinase cleaved native type V and XI collagens. Native type I collagen was not a substrate for the enzyme. These data suggest that native and recombinant 92 kDa type IV collagenase produced in E. coli have similar biochemical properties. The successful expression of the collagenase in a prokaryotic system will greatly facilitate the structure-function characterization of the enzyme and allow a more precise analysis of its physiological and pathological roles.     

The sensitivity of versican from rabbit lung to gelatinase A (MMP-2) and B (MMP-9) and its involvement in the development of hydraulic lung edema.

Large chondroitinsulphate-containing proteoglycan (versican) isolated from rabbit lung was cleaved by purified gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as by crude enzyme extract from rabbit lung with hydraulic edema. Gelatine zymography, performed after purification of gelatinases by affinity chromatography, demonstrated that the enzyme extract contained two main gelatinolytic bands at about 92 kDa and 72 kDa, identified by specific antisera as the latent proMMP-9 and proMMP-2, respectively. Moreover, enzyme extract from edematous lung showed an increased amount of the proteolytically activated forms of both gelatinases with respect to normal controls. These results suggest that MMP-2 and MMP-9 are involved in the breakdown of versican occurring in rabbit lung during the development of hydraulic edema.     

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.