Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.     

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.     

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

Purification and properties of bovine nasal hyaline cartilage collagenase

1. Collagenase from bovine nasal hyaline cartilage was extracted with 1 and 3 M NaCl in Tris-CaCl2 buffer. 2. Two peaks of collagenase activity were revealed on DE52 ion exchange column, collagenase 1 and collagenase 2. 3. The apparent mol. wt of collagenase 1 and 2 as determined by SDS-PAGE were 68 and 43 kDa, respectively. 4. Both enzymes degrade native collagen type II into two characteristic products, TCA(3/4) and TCB(1/4), at 25 degrees C and pH 7.6. 5. Trypsin and aminophenylmercuric acetate were capable of increasing the collagenase 1 activity. 6. The two enzymes can be characterized as metalloproteinases since they were inhibited by EGTA and 1,10-phenanthroline. The use of proteinase inhibitors (N-ethylmaleimide, iodoacetic acid, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, pepstatin, dithiothreitol) showed that the enzymes do not contain serine, cysteine or aspartic acid in their active sites.     

Purification and characterization of a collagenase extracted from rabbit tumours.

A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.     

The isolation and properties of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.     

Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology.

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.     

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.     

Collagenolytic serine proteinase from Euphausia superba Dana (Antarctic krill).

1. A serine proteinase isolated from E. superba shows collagenolytic properties: it acts on collagens from Achilles tendon (type I and V) and reconstituted fibrils of calf skin collagen under conditions that do not denature the substrates. 2. At 25 degrees C and pH 7.5 the enzyme both splits the calf skin collagen in solution to the fragments TCA and TCB and catalyses the conversion of dimeric molecules to monomeric chains. 3. The enzyme exhibits strong chymotrypsin-like and lower trypsin-like activities. 4. All the enzyme activities are inhibited to the same degree by diisopropylfluorophosphate (DFP), phenylmethylsulphonyl fluoride (PMSF), N alpha-tosyl-L-lysine chloromethyl ketone (TLCK), soybean trypsin inhibitor (SBTI), chicken ovomucoid (CHOM), chymostatin and leupeptin. None of the activities is inhibited by chelating agents and L-cysteine. 5. pH-Optima of the proteinase in protein substrates hydrolysis (6.0-6.2) are lower than those of synthetic substrates cleavage (7.8-8.0 in the case of BzTyrOEt and 8.7-8.9 for BzArgOEt). 6. Four from nine cysteine residues present in the enzyme molecule possess free thiol-groups. Since the enzyme is inhibited by p-chloromercuribenzoate (pCMB), N-ethylmaleimide (NEM) and iodoacetic acid (IAA), the role of its thiol-groups has been discussed.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.     

Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with collagenous substrates

The collagenolytic protease from Uca pugilator was studied with respect to its catalytic properties on collagen types I-V. The crab protease degraded all five collagen types, producing multiple cleavages in the triple helix of each native collagen at 25 degrees C. The major early cleavage in the alpha 1 polypeptide chain of collagen types I-III occurred at a 3/4:1/4 locus, resulting in fragments electrophoretically similar to the TCA and TCB products of mammalian collagenase action. Interestingly, a propensity toward this same cleavage was observed even following thermal denaturation of the substrates. The ability of the crab protease to degrade all native collagen types and to catalyze cleavages at multiple loci in the triple helix distinguishes its action from that of mammalian collagenases. The collagenolytic activity of the crab protease was also examined on fibrillar collagen and compared to that of human skin fibroblast collagenase. Enzyme concentrations of fibroblast collagenase which resulted in the saturation of available substrate sites failed to show such an effect in the case of the crab protease. Binding studies of the crab protease to fibrillar collagen likewise indicated substantially reduced levels of enzyme binding in comparison to fibroblast collagenase. These data suggest that the affinity of the crab protease for native collagen is considerably less than the affinity of mammalian collagenase for this substrate.     

The subunits of human hexosaminidase A.

Previous studies of the subunit structure of hexosaminidase gave ambiguous results, but suggested that the enzyme was composed of six equally sized subunits. Dissociation of hexosaminidase A with p-chloromercuribenzoate produces an alkylated fragment with mol.wt. approx. 50000, which is converted into hexosaminidase S by treatment with dithiothreitol. Treatment of native hexosaminidase A with sodium dodecylsulphate results in the formation of a large and a small fragment. However, although the native enzyme has a sedimentation coefficient of 5.8S, dissociation by S-carboxymethylation and maleic anhydride treatment results in subunits exhibiting a single schlieren boundary on analytical ultracentrifugation with a sedimentation coefficient of 2.18S. These results indicate that the enzyme is composed of four subunits, each with molwt. approx. 25000-27000. The mol.wt. of the native enzymes is calculated to be approx. 110000. Our data are consistent with the subunit structures of hexosaminidases A, B and S as being alpha2beta2, beta4 and alpha4 respectively.     

Purification and properties of a specific collagenase from rabbit synovial fibroblasts.

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.     

Purification, characterization and inhibition of human skin collagenase

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32mug of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5-8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25 degrees C, producing the two characteristic products TC(A)((3/4)) and TC(B)((1/4)). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25 degrees C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37 degrees C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the alpha-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37 degrees C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins alpha(2)-macroglobulin and beta(1)-anti-collagenase both inhibited the enzyme, but alpha(1)-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.     

Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase.

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5'-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.     

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.     

Collagenolytic enzymes from the starfish,Pycnopodia helianthoides

• 1.1. Two collagenolytic proteinases have been isolated from the starfish, Pycnopodia helianthoides and partially characterized.
• 2.2. The larger of these two enzymes, with a molecular weight of approx 60,000, resembles the vertebrate collagenases in that it is inhibited by ethylenediamine tetraacetate and is able to cleave the collagen triple-helix.
• 3.3. The smaller enzyme, with a mol. wt of approximately 16,500, resembles the vertebrate chymotrypsins in its ability to hydrolyse acetyl-tyrosine-ethylester and its cleavage of collagen in the telopeptide region.
• 4.4. Other properties of the enzymes, including alkaline pH optima, acidic isoelectric point and heat stability are similar for both proteinases.

     

Collagenase activity in cultures of rat prostate carcinoma.

A specific collagenase (EC 3.4.24.3) has been found and purified from serum-free culture medium of 11095 epidermoid carcinoma of rat prostate. The molecular weight of this collagenase was estimated at 71 000 and the pH optimum was approx. 7. At 26 degrees C, the collagenase cleaved collagen at a site 3/4 the length from the N-terminus. At 37 degrees C, this collagenase degraded collagen to smaller peptides. The enzyme activity was inhibited by serum, cysteine and EDTA, but not by protease inhibitors. The presence of collagenase in rat tumor tissue suggests that this enzyme might play a significant role in tissue invasion by cancer cells.     

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.     

The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan.

1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.