Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.

Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.     

Clinical and Pathological Characteristics of Intraocular Cysticercosis

This study aimed to explore the clinical, radiological, and pathological characteristics of intraocular cysticercosis due to Taenia solium metacestode infection. Total 8 patients diagnosed with intraocular cysticercosis at the Red Cross Hospital of Yunnan Province, China were examined retrospectively. Patients with clear dioptic media had undergone fundus chromophotography. All patients underwent B ultrasonography of the ocular region (CT) successive scanning of the orbit and cerebral tissues. Parasites were extracted surgically and then examined pathologically. The fundus chromophotography showed a white and condensing scolex package in the vesicle. The B ultrasonic examination showed a vesicle-like echogenic mass in the vitreous chamber, in which the high-level echo spot was the cysticercus scolex. The pathological examinations showed that the vesicle wall exhibited hyaline degeneration, inflammatory cell infiltration, neuroglial fiber, and glial cell proliferation layers from the inside to the outside. The scolex is round and is composed of the outer tissue (the body wall) and the inner furrow tissue; these tissues migrated together. Primordially differentiated sucking discs were found in one case, but no hooklets were found. The inner scolex tissue was folded like a paper flower. The severity of intraocular disease is closely correlated with the pathophysiological processes of the cysticercus worm. Pathological examination of the intraocular lesions can help to evaluate the course of the disease as well as to provide a scientific basis for effective antiparasitic medication.     

Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.

We report the isolation of a cDNA clone providing the first and complete sequence of mouse 72-kDa type IV collagenase. The clone contains 2800 nucleotides with a 1986-nucleotide open reading frame coding for 662 amino acids. The amino acid sequence includes a 29-residue signal peptide, an 80-residue propeptide, and a 553-residue enzyme proper. The sequence identity between the mouse and human enzymes is 96% with all cysteine residues conserved. The carboxyl-terminal domain of the mouse enzyme contains two more residues than the human enzyme. Northern hybridization analysis revealed considerable expression of the enzyme gene in newborn mouse lung, heart, kidney, and psoas muscle tissues, whereas only weak or no signals were observed in liver, spleen, and brain. Expression of the gene was substantially reduced in the same tissues of 3-month-old mice. In situ hybridization analysis of 72-kDa type IV collagenase expression in 10-15-day-old mouse embryos showed that the gene was intensely expressed in mesenchymal cells. Brain and surface ectoderm were completely negative. The epithelial tissue component of developing organs was negative with the exception of salivary gland. Although the expression varied somewhat between different mesenchymal tissues, no temporal or spatial changes could be associated with the advancement of epithelial branching morphogenesis. These findings together with our previous data on the expression of 72-kDa type IV collagenase in human tumors indicate that this enzyme has some very specific roles both in the physiological and pathological degradation of extracellular matrix. Furthermore, it has become clear that the closely related 92-kDa type IV collagenase differs completely with respect to expression pattern as well as gene regulation. The mouse cDNA clones reported in this study may provide important tools unraveling the actual roles of these enzymes in vivo.     

Cloning and nucleotide sequence analysis of the colH gene from Clostridium histolyticum encoding a collagenase and a gelatinase.

The colH gene encoding a collagenase was cloned from Clostridium histolyticum JCM 1403. Nucleotide sequencing showed a major open reading frame encoding a 116-kDa protein of 1,021 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, HEXXH. A 116-kDa collagenase and a 98-kDa gelatinase were copurified from culture supernatants of C. histolyticum. While the former degraded both native and denatured collagen, the latter degraded only denatured collagen. Peptide mapping with V8 protease showed that all peptide fragments, except a few minor ones, liberated from the two enzymes coincided with each other. Analysis of the N-terminal amino acid sequence of the two enzymes revealed that their first 24 amino acid residues were identical and coincided with those deduced from the nucleotide sequence. These results indicate that the 98-kDa gelatinase is generated from the 116-kDa collagenase by cleaving off the C-terminal region, which could be responsible for binding or increasing the accessibility of the collagenase to native collagen fibers. The role of the C-terminal region in the functional and evolutional aspects of the collagenase was further studied by comparing the amino acid sequence of the C. histolyticum collagenase with those of three homologous enzymes: the collagenases from Clostridium perfringens and Vibrio alginolyticus and Achromobacter lyticus protease I.     

Significance of the distribution of 57Co-vitamin B12 in Spirometra mansonoides (Cestoidea) during growth and differentiation in mammalian intermediate and definitive hosts

The distribution of labeled cyanocobalamin (CN-[57Co]Cbl = [57Co]-vitamin B12) in pleurocercoids and adult tapeworms of Spirometra mansonoides was studied during development in mice 22 days days PI, respectively. Plerocercoid scolices, obtained by cutting away their bodies or by in vitro enzymatic dissolution of the bodies, were pulsed with CN- magnitude of 57Co Cbl for 1h at 37 degrees C and reimplanted subcutaneously into mice or given per os to cats. In regenerated plerocercoids, the highest concentration of magnitude of 57Co Cbl occurred in the scolex and then decreased posteriorly in the newly-formed tissues of the body. Approximately 60% of the total magnitude of 57Co Cbl present remained concentrated in the scolex following body regeneration plerocercoids and adult tapeworms of Spirometra mansonoides was studied during development in mice 22 days post-infection (PI) and in cats 16 days PI, respectively. Plerocercoid scolices, obtained by cutting away their bodies or by in vitro enzymatic dissolution of the bodies, were pulsed with CN-[57Co]Cbl for 1 h at 37 degrees C and reimplanted subcutaneously into mice or given per os to cats. In regenerated plerocercoids, the highest concentration of [57Co]Cbl occurred in the scolex and then decreased posteriorly in the newly-formed tissues of the body. Approximately 60% of the total [57Co]Cbl present remained concentrated in the scolex following body regeneration for up to 109 days PI. This high [57Co]Cbl concentration in the plerocercoid scolex was bound to protein and appears to be maintained by a complex homeostatic mechanism in association with directional transport of [57Co]Cbl to the scolex with ultimate depletion along the length of the body.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase.

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.     

Purification and Characterization of a Nonvesicular Vesamicol-Binding Protein from Electric Organ and Demonstration of a Related Protein in Mammalian Brain

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.     

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.     

The terminology of larval cestodes or metacestodes

The terminology associated with the nomenclature of larval or metacestodes is reviewed as well as the various morphological and developmental characters used to define different types of larval cestodes. Based on a review of the literature, the key characters differentiating the types of larval cestodes are the presence of a primary lacuna and the invagination/retraction of the scolex. The presence of a cercomer and of a bladder-like enlargement of the larval cestode were considered to be useful secondary characteristics. Using these characters, six basic types of larval cestodes were identified: the procercoid, an alacunate form which cannot develop further until ingested by a second intermediate host; the plerocercus, an alacunate form with a retracted scolex; the plerocercoid, an alacunate form with an everted scolex; the merocercoid, an alacunate form with an invaginated scolex; the cysticercoid, a lacunate form with a retracted scolex; and the cysticercus, a lacunate form with an invaginated scolex. The diversity of larval types within the broad classifications of cysticercoid and cysticercus can be differentiated by the use of appropriate prefixes. Deficiencies in knowledge of specific types of larval cestodes are identified and further avenues of research are indicated.Pseudonym for the contributors of a workshop on metacestode terminology, chaired by I. Beveridge, which took place during the Third International Workshop for Tapeworm Systematics,Sofia, Bulgaria, 20-24 July, 1999 (Organiser: B.B. Georgiev).     

Interleukin 4 inhibition of prostaglandin E2 synthesis blocks interstitial collagenase and 92-kDa type IV collagenase/gelatinase production by human monocytes.

Activation of human monocytes results in the production of interstitial collagenase through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Inasmuch as interleukin 4 (IL-4) has been shown to inhibit PGE2 synthesis by monocytes, we examined the effect of IL-4 on the production of human monocyte interstitial collagenase. Additionally, we also assessed the effect of IL-4 on the production of 92-kDa type IV collagenase/gelatinase and tissue inhibitor of metalloproteinase-1 (TIMP-1) by monocytes. The inhibition of PGE2 synthesis by IL-4 resulted in decreased interstitial collagenase protein and activity that could be restored by exogenous PGE2 or dibutyryl cyclic AMP (Bt2cAMP). IL-4 also suppressed ConA-stimulated 92-kDa type IV collagenase/gelatinase protein and zymogram enzyme activity that could be reversed by exogenous PGE2 or Bt2cAMP. Moreover, indomethacin suppressed the ConA-induced production of 92-kDa type IV collagenase/gelatinase. These data demonstrate that, like monocyte interstitial collagenase, the conA-inducible monocyte 92-kDa type IV collagenase/gelatinase is regulated through a PGE2-mediated cAMP-dependent pathway. In contrast to ConA stimulation, unstimulated monocytes released low levels of 92-kDa type IV collagenase/gelatinase that were not affected by IL-4, PGE2, or Bt2cAMP, indicating that basal production of this enzyme is PGE2-cAMP independent. IL-4 inhibition of both collagenases was not a result of increased TIMP expression since Western analysis of 28.5-kDa TIMP-1 revealed that IL-4 did not alter the increased TIMP-1 protein in response to ConA. These data indicate that IL-4 may function in natural host regulation of connective tissue damage by monocytes.     

Immunological quantitation of phospholipid/Ca2+-dependent protein kinase and its fragments. Tissue levels, subcellular distribution, and ontogenetic changes in brain and heart

Levels of phospholipid/Ca2+-dependent protein kinase (protein kinase C, 80 kDa) and its presumed proteolytic fragments were quantified in a variety of animal tissues and cultured human leukemic cell lines (HL60 and K562) using an immunoblot analysis technique. Of many tissues examined, the rat brain and HL60 cells were by far the richest sources of the 80-kDa native enzyme, with its concentration estimated to be about 2-3 microM in both tissues. The major enzyme species detected in most tissues, however, was the 67-kDa fragment; the 80-kDa native enzyme was present in a smaller amount except in spleen which contained nearly equal amounts of both enzyme species. It was also found that HL60 and K562 leukemic cells contained the 50-kDa species instead of the 67-kDa species. A study of the subcellular distribution of the 80- and 67-kDa species showed the enzyme to be localized predominantly in the soluble fraction for some tissues (e.g. heart) and nearly equally distributed between soluble and particulate fractions in others (e.g. spleen). In the brain, however, the majority of the enzyme was present in the particulate fraction, in agreement with the findings made with immunocytochemical localization of the enzyme. The total enzyme content in developing rat brain and heart increased during the first 2 to 4 weeks following birth and decreased to 60% of peak levels in the adult. The present immunological method, showing for the first time that the tissue levels of phospholipid/Ca2+-dependent protein kinase and its fragments can be quantitated, would be useful for studies on the regulation of cellular events and pathophysiology of tissues thought to be involved in this multi-functional protein phosphorylation system.     

Comparison of microthrix ultrastructure and morphology on the plerocercoid and adult scolex of Calliobothrium cf. verticillatum (Tetraphyllidea: Onchobothriidae)

The distal bothridial surfaces of adult triloculate onchobothriids are covered with short structures that have been tentatively classified as very short filitriches, but this hypothesis has never been tested. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to investigate microthrix morphology in the plerocercoid and adult forms of Calliobothrium cf. verticillatum, a triloculate onchobothriid tapeworm from Long Island Sound (Connecticut). Plerocercoids of C. cf. verticillatum were collected from the anterior midgut ceaca of Pagurus pollicaris Say, 1817 (flat-clawed hermit crab), and adults were collected from the spiral intestine of the dusky smooth hound Mustelis canis (Mitchell, 1815). Two plerocercoids and 2 adults were examined using SEM; 2 plerocercoids and 2 adults were examined using TEM. Microthrix distribution and morphology (including measurements of total length, base length, shaft length, and base width) were investigated on all surfaces of the plerocercoid and adult scolex. Slender filitriches and large bladelike spinitriches were observed extending from the tegument of plerocercoid and adult forms. The filitriches were found to have significantly narrower bases than the spinitriches (65-167 nm vs. 466-1,936 nm, respectively). The scolex proper of the plerocercoid and adult forms were found to have filitriches of medium-length and bladelike spinitriches. The distal bothridial surfaces differed dramatically in microthrix morphology between plerocercoid and adult forms; on the distal surfaces of the plerocercoids were long filitriches and bladelike spinitriches. However, the distal surfaces of the adults had short structures (previously hypothesized to be short filitriches) and a few bladelike spinitriches. Serial transverse sections revealed that the short structures on the distal bothridial surfaces of the adults were homologous with filitriches. They included all of the structural components of a filithrix as well as a base width that conformed to the filitriches found on other surfaces. The bothridial margins of the plerocercoid and adult forms had a microthrix pattern similar to that seen on the proximal bothridial surfaces except that the filitriches on the margins were significantly longer than those found anywhere else on the bothridia. The most dramatic difference between the plerocercoid and adult forms occurred on the distal bothridial surfaces, where the filitriches of the adult cestodes were significantly shorter and narrower, and the spinitriches were almost entirely lacking.     

SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages

We have reported that SV40-transformed human lung fibroblasts secrete a 92-kDa metalloprotease which is not detectable in the parental cell line IMR-90. We now present the complete structure of this enzyme along with the evidence that it is identical to the 92-kDa metalloprotease secreted by normal human alveolar macrophages, phorbol ester-differentiated monocytic leukemia U937 cells, fibrosarcoma HT1080 cells, and cultured human keratinocytes. A similar, perhaps identical, enzyme can be released by polymorphonuclear cells. The preproenzyme is synthesized as a polypeptide of predicted Mr 78,426 containing a 19 amino-acid-long signal peptide and secreted as a single 92,000 glycosylated proenzyme. The purified proenzyme complexes noncovalently with the tissue inhibitor of metalloproteases (TIMP) and can be activated by organomercurials. Activation with phenylmercuric chloride results in removal of 73 amino acids from the NH2 terminus of the proenzyme, yielding an active form capable of digesting native types IV and V collagen. The in vitro substrate specificity of the enzyme using these substrates was indistinguishable from that of the 72-kDa type IV collagenase. The 92-kDa type IV collagenase consists of five domains; the amino-terminal and zinc-binding domains shared by all members of the secreted metalloprotease gene family, the collagen-binding fibronectin-like domain also present in the 72-kDa type IV collagenase, a carboxyl-terminal hemopexin-like domain shared by all known enzymes of this family with the exception of PUMP-1, and a unique 54-amino-acid-long proline-rich domain homologous to the alpha 2 chain of type V collagen.     

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.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Studies on the rat liver following iron overload: An analysis of iron and lysosomal enzymes in isolated parenchymal and non-parenchymal cells

Iron overload is known to affect the liver. In order to study the effect of iron on various liver cellular and subcellular compartments and the alterations due to mobilization of iron, an experimental model has been developed previously. In this study iron stores in parenchymal and non-parenchymal cells have been investigated during iron loading and unloading. Following completion of the experimental procedures, liver cells were isolated by means of collagenase perfusion (parenchymal cells) and pronase treatment (nonparenchymal cells). It was found that iron overload did not result in significantly increased levels of three lysosomal enzymes, and that the enzyme activities were not altered as iron was mobilized. Iron stores were localized largely in parenchymal cells, and these stores decreased after cessation of iron loading. The iron content was further lowered if the animals were bled. The non-parenchymal cells of the liver initially stored a relatively small part of the administered iron but this increased in the two months following iron loading. On the other hand if the animals were bled there was a pronounced decrease in iron content of these cells as well as in parenchymal cells. It is concluded that iron overload does not affect lysosomal enzymes and that iron stores in both parenchymal and non-parenchymal cells can be mobilized in response to increased demand.     

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.