Collagenolytic Enzyme Activity Research Articles

Resin-Dentin Bonding Interface: Mechanisms of Degradation and Strategies for Stabilization of the Hybrid Layer.

Several studies have shown that the dentin-resin interface is unstable due to poor infiltration of resin monomers into the demineralized dentin matrix. This phenomenon is related to the incomplete infiltration of the adhesive system into the network of exposed collagen fibrils, mainly due to the difficulty of displacement and subsequent replacement of trapped water between interfibrillar spaces, avoiding adequate hybridization within the network of collagen fibrils. Thus, unprotected fibrils are exposed to undergo denaturation and are susceptible to cyclic fatigue rupture after being subjected to repetitive loads during function. The aqueous inclusions within the hybrid layer serve as a functional medium for the hydrolysis of the resin matrix, giving rise to the activity of esterases and collagenolytic enzymes, such as matrix metalloproteinases, which play a fundamental role in the degradation process of the hybrid layer. Achieving better interdiffusion of the adhesive system in the network of collagen fibrils and the substrate stability in the hybrid layer through different strategies are key events for the interfacial microstructure to adequately function. Hence, it is important to review the factors related to the mechanisms of degradation and stabilization of the hybrid layer to support the implementation of new materials and techniques in the future. The enzymatic degradation of collagen matrix, together with resin leaching, has led to seeking strategies that inhibit the endogenous proteases, cross-linking the denudated collagen fibrils and improving the adhesive penetration removing water from the interface. Some of dentin treatments have yielded promising results and require more research to be validated. A longer durability of adhesive restorations could resolve a variety of clinical problems, such as microleakage, recurrent caries, postoperative sensitivity, and restoration integrity.

Substantivity of Carbodiimide Inhibition on Dentinal Enzyme Activity over Time

The use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) has recently been investigated for its effectiveness in the prevention of collagen degradation over time and the improvement of resin-dentin bond durability. The objective of the present study was to evaluate the effects of a 0.3 M EDC-containing conditioner on endogenous enzymatic activities within the hybrid layer (HL) created by a self-etch or an etch-and-rinse adhesive after 1 y. The activity within the HL was examined using in situ zymography and confocal laser scanning microscopy after 24 h or 1-y storage in artificial saliva. Dentin specimens were bonded with Clearfil SE Bond (CSE) or XP Bond (XPB). For CSE, the self-etching primer was applied and treated with 0.3 M EDC for 1 min, and then the bonding agent was applied. For XPB, dentin was etched and treated with 0.3 M EDC for 1 min and then bonded with the primer-bonding agent. Control specimens were prepared without EDC treatment. Slices containing the adhesive-dentin interface were covered with fluorescein-conjugated gelatin and observed with a multiphoton confocal microscope. Fluorescence intensity emitted by hydrolyzed fluorescein-conjugated gelatin was quantified, and the amount of gelatinolytic activity was represented by the percentage of green fluorescence emitted within the HL. After 24 h of storage, enzymatic activity was detected by in situ zymography within the HLs of both tested adhesives, with XPB higher than CSE (P < 0.05). Almost no fluorescence signal was detected when specimens were pretreated with EDC compared to controls (P < 0.05). After 1 y of storage, enzymatic activities significantly increased for all groups (excluding XPB control) compared to 24-h storage (P < 0.05), with EDC pretreated specimens exhibiting significantly lower activity than controls (P < 0.05). The present study showed, for the first time, that the use of EDC for both the self-etch and the etch-and-rinse approaches results in the reduction but not complete inhibition of matrix-bound collagenolytic enzyme activities over time in the HL.

Produção de protease colagenolítica por Bacillus stearothermophillus de solo amazônico

As proteases colagenolíticas são enzimas capazes de hidrolisar as ligações peptídicas de vários tipos de colágeno e têm grande importância na medicina e em aplicações terapêuticas. O objetivo desta pesquisa foi avaliar a produção de proteases colagenolíticas por Bacillus stearothermophilus. Os tratamentos foram realizados por meio de um planejamento fatorial completo 2³, a fim de avaliar a significância dos efeitos e interações das variáveis - pH inicial, concentração de substrato e temperatura - sobre a produção de protease colagenolítica. O ponto central foi executado em quadruplicata para fornecer uma estimativa dos erros experimentais. Ensaios enzimáticos com colágeno e azocaseína como substratos foram realizados para determinação das atividades colagenolítica e proteolítica respectivamente. A maior atividade enzimática colagenolítica foi 79,38 U mL-1, correspondendo a atividade específica de 136,86 U mg-1, em condições iniciais de fermentação, na concentração de substrato a 1% (p/v), pH 7,2 e 25 °C. A atividade proteolítica da enzima foi mais ativa em pH 9,0 e 50 °C e foi estável nas faixas de pH (6,0 - 9,0) e temperatura (45 °C - 50 °C). Bacillus stearothermophilus apresenta viabilidade para a produção de proteases colagenolíticas e a obtenção dessas enzimas tem grande importância para aplicações biotecnológicas.

Bioactive effects of a calcium/sodium phosphosilicate on the resin–dentine interface: a microtensile bond strength, scanning electron microscopy, and confocal microscopy study

This study evaluated, through microtensile bond strength (μTBS) testing, the bioactive effects of a calcium/sodium phosphosilicate (BAG) at the resin-dentine interface after 6 months of storage in phosphate buffer solution (PBS). Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were also performed. Three bonding protocols were evaluated: (i) RES-Ctr (no use of BAG), (ii) BAG containing adhesive (BAG-AD), and (iii) BAG/H (3) PO (4) before adhesive (BAG-PR). The dentin-bonded specimens were prepared for μTBS testing, which was carried out after 24 h or 6 months of storage in PBS. Scanning electron microscopy ultramorphology analysis was performed after debonding. Confocal laser scanning microscopy was used to evaluate the morphological and nanoleakage changes induced by PBS storage. High μTBS values were achieved in all groups after 24 h of storage in PBS. Subsequent to 6 months of storage in PBS the specimens created using the BAG-AD bonding approach still showed no significant reduction in μTBS. Moreover, specimens created using the BAG-AD or the BAG-PR approach showed an evident reduction of nanoleakage after prolonged storage in PBS. The use of BAG-containing adhesive may enhance the durability of the resin-dentine bonds through therapeutic/protective effects associated with mineral deposition within the bonding interface and a possible interference with collagenolytic enzyme activity (matrix metalloproteinases) responsible for the degradation of the hybrid layer.

Sub-clinical necrotic enteritis in broiler chickens: Novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue

The present study revealed several previously not recognized etiological details in the development of necrotic enteritis (NE) in broilers. We provide evidence that the pathological process leading to mucosal epithelium necrosis follows morphologically distinct phases commencing at the basal domain of the mucosal epithelium and then progressively invading the entire lamina propria. Initially mucosal epithelium appears normal, but as the pathological changes progress throughout the lamina propria, the adjacent enterocytes begin to show features of necrotic cell death and the necrotic process of the epithelium progresses from being focal to locally extensive. Ultra-structural examination showed that primary changes occur at the level of basal and lateral domains of the enterocytes, whereas the apical domain of enterocytes remains intact even in the face of advanced necrotic changes. This indicates that the mucosal necrosis does not result from direct damage to the mucosal epithelium. Rather, the necrotic death of enterocytes is a consequential effect of the destruction of lamina propria, the extra-cellular matrix, and intercellular junctions. The nature of these morphological changes indicates that initiation of the pathological process leading to NE involves proteolytic factors affecting the extra-cellular matrix and cellular junctions. Further studies revealed that, indeed, the elevated activity of collagenolytic enzymes in the mucosal milieu and in intestinal tissue represents an integral component of the pathological process leading to NE. In the first instance we discovered that Clostridium perfringens strains isolated from field cases of NE secrete several potent collagenolytic enzymes. In the second instance we observed that, in comparison to controls, broilers challenged with C. perfringens isolated from field cases of NE show high levels of several collagenolytic enzymes in the intestinal tissue. A major component of the overall collagenolytic activity detected in the intestinal tissue was identified by zymography as matrix metalloproteinases (MMPs). Dominant activity was associated with MMP-2. We confirmed using immuno-histochemistry that this enzyme is expressed at high levels in mucosal tissue showing signs of NE. The high levels of collagenolytic activities, in particular associated with MMP-2, demonstrated in our studies are consistent with the nature of morphological changes observed primarily in extra-cellular matrix (ECM) at the basal domain of enterocytes, as well lateral domains of enterocytes. The lack of changes at the level of apical domain of mucosal epithelium indicates that the lipolytic aspect of alpha toxin in NE is not an essential factor in primary lesions development. Taken together, our findings indicate that the early lesions leading to NE are associated with virulence factors that induce proteolytic activity, rather than lipolytic activity.

Genetic diversity and potential function of microbial symbionts associated with newly discovered species of Osedax polychaete worms.

We investigated the genetic diversity of symbiotic bacteria associated with two newly discovered species of Osedax from Monterey Canyon, CA, at 1,017-m (Osedax Monterey Bay sp. 3 "rosy" [Osedax sp. MB3]) and 381-m (Osedax Monterey Bay sp. 4 "yellow collar") depths. Quantitative PCR and clone libraries of 16S rRNA gene sequences identified differences in the compositions and abundances of bacterial phylotypes associated with the newly discovered host species and permitted comparisons between adult Osedax frankpressi and juveniles that had recently colonized whalebones implanted at 2,891 m. The newly discovered Osedax species hosted Oceanospirillales symbionts that are related to Gammaproteobacteria associated with the previously described O. frankpressi and Osedax rubiplumus (S. K. Goffredi, V. J. Orphan, G. W. Rouse, L. Jahnke, T. Embaye, K. Turk, R. Lee, and R. C. Vrijenhoek, Environ. Microbiol. 7:1369-1378, 2005). In addition, Osedax sp. MB3 hosts a diverse and abundant population of additional bacteria dominated by Epsilonproteobacteria. Ultrastructural analysis of symbiont-bearing root tissues verified the enhanced microbial diversity of Osedax sp. MB3. Root tissues from the newly described host species and O. frankpressi all exhibited collagenolytic enzyme activity, which covaried positively with the abundance of symbiont DNA and negatively with mean adult size of the host species. Members of this unusual genus of bone-eating worms may form variable associations with symbiotic bacteria that allow for the observed differences in colonization and success in whale fall environments throughout the world's oceans.

Reversibility of experimental rabbit liver cirrhosis by portal collagenase administration

The regression of cirrhosis is associated with increased intrahepatic collagenolytic enzyme activity. We investigated whether collagenase supplementation via portal vein infusion can retard cirrhosis development and/or reverse cirrhosis. In all, 35 rabbits were initially assigned to study. However, because of high surgical mortality and infection, only 15 animals completed study. Four normal controls (group I) received olive oil subcutaneously (SC) for 12 weeks followed by normal saline portal perfusion for 12 weeks. Four (group II) received CCl4 SC for 6 weeks followed by portal vein collagenase, 6?mg twice weekly, plus SC CCl4 for 6 additional weeks and then killed. Four rabbits (group III) received CCl4 SC for 12 weeks and then 6?mg of collagenase portally for 12 weeks, while three control rabbits (group IV) received CCl4 for 12 weeks followed by saline for 12 weeks. After 12 weeks of CCl4, liver hydroxyproline content of collagenase-treated group II (361.1±106.6?μg/g) was significantly reduced compared with group III+IV that had not yet received collagenase (589.0±162.9?μg/g; P<0.05). In the main comparison, hydroxyproline content of collagenase-treated group III (177.5±35.6?μg/g) was significantly decreased compared with saline-treated controls (446.3±150.1?μg/g; P<0.01). Further, liver histology showed complete regression of cirrhosis in the collagenase-treated animals. No toxicity of liver, kidney, lung, brain or heart was observed histologically. Anaphylaxis occurred in 2/35 original animals (one fatal). In conclusion, this study provides ‘proof of principle' that collagenase portal administration can retard cirrhosis development and speed regression of established cirrhosis in the rabbit CCl4 model. Potential application to humans is premature, but feasible, if these findings are confirmed in additional animal studies.

Pre-eclampsia (EPH-gestosis)-induced decrease of MMP-s content in the umbilical cord artery

Background: It was found in our previous paper that pre-eclampsia-associated accumulation of collagen in the umbilical cord artery (UCA) is a result of increased biosynthesis and decreased degradation of this protein. It is known that the activity of collagenolytic enzymes is a main factor regulating collagen degradation rate in various tissues. Methods: For this reason it was decided to evaluate the effect of pre-eclampsia on the content and activity of metalloproteinases by immunoenzymatic method (ELISA), zymographic technique and with the use of specific substrates. Results: A low amount of MMP-1 (collagenase 1), MMP-9 (gelatinase B) and MMP-3 (stromelysin 1) was detected in the extracts from the wall of the umbilical cord artery. MMP-2 (gelatinase A) is the main collagenolytic enzyme in UCA wall (both latent and active form). Pre-eclampsia is associated with a distinct reduction in those metalloproteinases content in comparison to control UCAs. It can be concluded from zymography that MMP-2 (gelatinase A) of the umbilical cord artery forms an inactive complex with a tissue inhibitor of metalloproteinases (TIMP). Such a complex dissociates under the action of p-aminophenylmercuric acetate (APMA) or sodium dodecyl sulphate. Conclusions: The decrease of metalloproteinases content and activity in the umbilical cord artery may be a factor that reduces the breakdown of collagen in the arterial wall and promotes the accumulation of this protein. The accumulation of collagen with simultaneous reduction in elastin content in the UCA may be the factor that reduces the elasticity of arterial wall and decreases the blood flow in the fetus of women with pre-eclampsia.

Textural Changes During Iced Storage of Salmon (Salmo salar) and Cod (Gadus morhua)

Textural properties, protein solubility, water holding capacity and activity of collagenolytic enzymes of salmon (Salmo salar) and cod (Gadus morhua) fillets were measured during iced storage. Breaking strength and hardness of fillets were reduced during iced storage. Elasticity of cod fillets was reduced, while cohesiveness of salmon increased during storage. Salmon was softer and less elastic than cod. During storage, total amount of extracted proteins was reduced for cod, and increased followed by a reduction for salmon. The fraction of salt soluble proteins increased during storage, and values for cod were higher than those for salmon. The activity of collagenolytic enzymes was higher in cod than salmon during the whole storage period.

EPH-gestosis (pre-eclampsia)-induced decrease of gelatinase activity may promote an accumulation of collagen in the umbilical cord artery

It was found in our previous paper that edema, proteinuria, hypertension (EPH)-gestosis-associated accumulation of collagen in the umbilical cord artery (UCA) is a result of increased biosynthesis and decreased degradation of this protein. It is known that the activity of collagenolytic enzymes is a main factor regulating collagen degradation rate in various tissues. For this reason it was decided to evaluate the effect of EPH-gestosis on the activity of proteolytic enzymes which may be involved in collagen degradation in the UCA wall. Proteolytic activity against bovine serum albumin, reconstituted collagen fibres and gelatin were evaluated. Latent forms of proteolytic enzymes were activated by the action of trypsin, p-chloromercuric benzoate (PCMB) and p-aminophenylmercuric acetate (APMA). A low activity of gelatinase (type IV collagenase) was detected in the extracts from the wall of the umbilical cord artery. This enzyme increased its activity several times after the action of trypsin, PCMB and APMA. EPH-gestosis results in a distinct reduction in gelatinase activity. Despite the action of activating agents the gelatinase from EPH-gestosis UCAs was considerably lower in comparison to control UCAs. It can be concluded that gelatinase of the umbilical cord artery forms an inactive complex with a tissue inhibitor of metalloproteinases. Such a complex dissociates under the action of trypsin, PCMB or APMA or sodium dodecyl sulphate. The decrease of gelatinolytic activity in the umbilical cord artery may be a factor that reduces the breakdown of collagen in the arterial wall and promotes an accumulation of this protein. The accumulation of collagen with simultaneous reduction in elastin content in the UCA may be the factors which reduce the elasticity of arterial wall and decrease the blood flow in the fetus of woman with EPH-gestosis.

A matrix metalloproteinase inhibitor reduces bone-type collagen degradation fragments and specific collagenases in gingival crevicular fluid during adult periodontitis.

To determine whether an inhibitor of matrix metalloproteinases (MMPs), administered to human subjects in a dental school research clinic, can reduce bone-type collagen degradation fragments in oral inflammatory exudates containing excessive levels of collagenase. Gingival crevicular fluid (GCF) was collected from 18 subjects with adult periodontitis whose clinical findings (gingival inflammation, pocket depth, and bone loss on radiographs) predicted excessive MMP activity in their periodontal pockets. One month before the baseline appointment, plaque and calculus were removed from the teeth by supra- and subgingival scaling. After collection of GCF from 8-12 pocket sites per subject and recording of clinical indices, 12 of the 18 subjects were treated with doxycycline at a low dosage (20 mg b.i.d.) known via an extensive literature to suppress mammalian MMP activity by a non-antimicrobial mechanism. The remaining 6 subjects were followed without drug treatment. At the baseline, 1 and 2-month appointments, GCF samples were analyzed for ICTP. (carboxyterminal peptide, a pyridinoline-containing fragment of Type I collagen) and osteocalcin by radioimmunoassay, as well as collagenolytic enzyme activity and MMP species (Western blot). Statistical analyses were determined by ANOVA. GCF ICTP and functional collagenase activity (but not osteocalcin levels) were significantly reduced (p < 0.05) in the doxycycline-treated subjects at both 1 and 2 month evaluations: there was no such change in the non-treated subjects. Western blots revealed that neutrophil-type collagenase (MMP-8) was the predominant MMP; MMP-13, which has been associated with pathologic collagenolysis including bone resorption, was detected in human GCF for the first time and was more substantially reduced than MMP-8. This is the first demonstration in human subjects of the simultaneous reduction of excessive MMP activity with concomitant reduction in levels of collagen degradation fragments. The findings are potentially applicable to a wide variety of human diseases characterized by excessive collagenase activity.

Effects of mast cell-macrophage interactions on the production of collagenolytic enzymes by metastatic tumor cells and tumor-derived and stromal fibroblasts.

Histological examination of the metastatic rat mammary adenocarcinoma line MTLn3 showed that macrophages and mast cells were frequently localized at the tumor periphery in the stromal tissues adjacent to the zones of tumor invasion. The interactions of these host cells with tumor cells and tumor-associated fibroblasts could be important in stimulating the production of extracellular matrix-degrading enzymes that facilitate tumor invasion and metastatic spread. Therefore, we examined the effects of isolated, activated macrophages and mast cells on the secretion of collagenolytic activities by normal fibroblasts, metastatic mammary adenocarcinoma cells and tumor-associated fibroblasts. Medium from activated macrophages or degranulated mast cells stimulated significant increases in production of collagenolytic activities by normal and tumor-associated fibroblasts and MTLn3 tumor cells. Medium from activated macrophages that had been pretreated with medium from degranulated mast cells, however, were less stimulatory to fibroblasts and tumor cell production of collagenolytic activities than medium from degranulated mast cells alone. We also examined the effects of two cytokines, interleukin-1 alpha and tumor necrosis factor-alpha on activated macrophage- and degranulated mast cell-stimulation of fibroblast and tumor cell collagenolytic activities. The two cytokines alone or in combination stimulated increased production of collagenolytic activities by fibroblasts and tumor cells. Addition of the cytokines to degranulated mast cell products resulted in secretion of higher collagenolytic enzyme activities by normal fibroblasts (but not by tumor-derived fibroblasts or tumor cells) than with degranulated mast cell product-treatment of either target cell alone. Cytokines used in combination with macrophage-conditioned medium were less effective in stimulating fibroblast and tumor cell collagenase activities than cytokines alone. Thus normal infiltrating host cells such as macrophages and mast cells can have profound effects on the production of degradative enzymes by tumor cells and tumor-associated stromal fibroblasts.

Gingival epithelial cells secrete a substance which increases the collagenolytic enzyme activity of periodontal ligament cells.

To examine whether the gingival epithelium provokes a loss of connective tissue attachment during periodontitis, periodontal ligament (PDL) cells and gingival fibroblasts (GF) were cultured with conditioned medium of gingival epithelial cells, and the collagenolytic activity of PDL cells and GF were examined, respectively. The epithelial cells were cultured in the presence or absence of lipopolysaccharide (LPS), and the respective conditioned media were added to the cultures of PDL cells and GF. The collagenolytic response of PDL cells to both of the conditioned media was 3- to 10-fold higher than that of each control, whereas the response of GF was only 1.4-fold higher. The LPS-stimulated epithelial conditioned medium showed a stimulation rate for collagenolytic activity similar to that of the LPS-free conditioned medium. These results suggest that human gingival epithelial cells, without LPS stimulation, secrete a substance which accelerates the collagenolytic enzyme activity of PDL cells for degradation of PDL fibers, in the absence of GF enzyme activity.

Assay for type III collagenolytic activity in lung cancer tissue

We developed a method for measuring the activity of type III collagenolytic enzyme in lung cancer tissue, using as substrate, type III collagen purified from human placenta. In this method [ 3H]propionate is used for labeling type III collagen, with bacterial collagenase used for making the standard curve. It, therefore, becomes possible to compare type III collagenolytic activity with those of other collagen subtypes (types I and IV). As this method is a fibril assay it is not susceptible to trypsin or other proteases. The average type III collagenolytic enzyme activity was higher in squamous cell carcinoma than in adenocarcinoma, while that of lung cancer tissue exceeded that of normal lung tissue. The activity of type III collagenase increased with the progression from one disease stage to the next.

Stimulation of collagenolytic enzyme release from cultured bone cells of normal and osteopetrotic (mi/mi) mice by parathyroid hormone and lipopolysaccharide

Collagenolytic enzyme release from bone cells was studied using cultured calvarial cells which are capable of degrading calcified and noncalcified collagen (cells from normal mice) or only noncalcified collagen (cells from osteopetrotic (mi/mi) mice). Treatment of cells from either normal or mi/mi mice with parathyroid hormone (PTH) or lipopolysaccharide (LPS) resulted in the appearance of latent collagenolytic enzyme activity in the medium. Chromatography of media from cells from normal mice treated with PTH on lysine-Sepharose resulted in the separation of latent collagenase and latent gelatinase. Further characterization of the enzymes showed that they were similar to those previously isolated from media of calvaria cultured with heparin. Collagenase activity of media of cells from normal or mi/mi mice treated with PTH or LPS yielded identical elution patterns upon chromatography on lysine-Sepharose. These results show that the mi allele has no effect on regulation of latent collagenolytic enzyme release. The data also suggest that previously described differences between PTH- and LPS-stimulated collagen degradation in cultured calvaria are due to factors other than differences in the ability of these agents to stimulate the release of collagenolytic enzymes.

Procollagenase associated with the noncalcified matrix of bone and its regulation by parathyroid hormone

Collagenolytic enzyme activity associated with the noncalcified pool of collagen was studied using calvarial matrices from which the periosteal cell envelope had been removed. Aminophenylmercuric acetate (APMA) stimulated degradation of about 5% of the noncalcified collagen in matrices prepared from freshly dissected bone. Significantly more activity was detected if intact calvaria were cultured 24 h before removal of the cells, in which case 20–30% of the noncalcified collagen was degraded following treatment with APMA. Trypsin elicited a similar response. The collagen being degraded was representative of the entire pool of noncalcified collagen and was not underhydroxylated. Treatment of intact calvaria with parathyroid hormone (PTH) before removal of the cells increased the level of both active collagenase and procollagenase activity associated with the matrix. Enhanced 3H release was noted for PTH treated intact bone in the prior 24 h. Inactivation of endogenous procollagenase by phenanthroline had no effect on the ability of isolated calvarial cells to resorb the bone upon treatment with resorptive agents. The data show that PTH-stimulated collagenolysis of noncalcified collagen involves increased deposition of procollagenase onto the noncalcified matrix in addition to activation of the enzyme.

Inhibition of ovulation, steroidogenesis and collagenolytic activity in rabbits by sulpiride-induced hyperprolactinaemia.

Ovulation induced by hCG in rabbits was reduced significantly (P less than 0.005) by sulpiride-induced hyperprolactinaemia. The pre- and post-ovulatory increases in peripheral and ovarian venous progesterone (but not oestradiol or testosterone) were suppressed in the treated animals. The condition of hyperprolactinaemia also prevented the usual changes in 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH peptidase (DNP-peptidase) and alpha-N-benzoyl-DL-Arg-beta-naphthylamide hydrolase (BANA-hydrolase) activities in follicular tissue that had been stimulated by an ovulatory dose of hCG. These results suggest that inhibition of progesterone production and collagenolytic enzyme activity by sulpiride-induced hyperprolactinaemia may be responsible for the ovulatory dysfunction that occurs when a mammal has a high level of circulating prolactin.

Nature of collagenolytic enzyme and inhibitor activities in crevicular fluid from healthy and inflamed periodontal tissues of beagle dogs.

We have previously described the occurrence of collagenolytic enzyme activity in crevicular fluids from beagle dogs in which experimental conditions of gingivitis and periodontitis had been induced (Kryshtalskyj, Sodek &amp; Ferrier 1986). In general, active collagenase activity was demonstrable in fluids from periodontitis sites, whereas in control and gingivitis sites various levels of latent collagenase and collagenase inhibitor activity were present. Analysis of the collagen digestion patterns by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) has shown that crevicular fluid samples with low collagenolytic activity generated ¾‐α and ¼‐α collagen fragments, typical of vertebrate collagenase. However, higher collagenolytic activity associated with periodontitis sites produced further degradation of the native collagen substrate. This activity was characterized by specific degradation products migrating between the ¾‐α and ¼‐α fragments. Similar digestion patterns are generated by enzymes in crevicular fluid from periodontitis sites in humans and by partially purified collagenase from osteoblastic cells (Otsuka, Sodek &amp; Limeback 1984), but not by bacterial collagenase. Analysis of plaque, crevicular cell debris, saliva and serum indicated that the collagenolytic activity is derived from connective tissue and/or inflammatory cells, whereas collagenase inhibitor could also be derived from saliva and serum.

Proactivator-dependent activation of procollagenase induced by treatment with EGTA.

A new mechanism for activation of the proactivator of procollagenase [Vater, Nagase & Harris (1983) J. Biol. Chem. 258, 9374-9382] has been found. Collagenolytic and other proteolytic enzyme activities in the medium of cultured rabbit synovial fibroblasts were found to be activated by a new mechanism: short-term incubation at 37 degrees C performed in the presence of EGTA followed by replacement of Ca2+ during enzyme assay. The crucial event in procollagenase activation is the production of a functional activator enzyme. Activation of procollagenase in the culture medium did not occur when proactivator was removed by immunoprecipitation. Proteolytic activity of proactivator was fully activated, whereas procollagenase alone could not be activated by the same sequence. EGTA treatment of the culture medium at 0 degrees C did not result in enzyme activation if Ca2+ was replaced before incubation at 37 degrees C. Certain other bivalent metal ions (e.g. Sn2+, Cd2+, Zn2+ and Mn2+) could substitute for Ca2+ to stabilize the proactivator as a zymogen and therefore prevent the appearance of proteolytic activity in culture medium. Isolation of proactivator and procollagenase from EGTA-treated radiolabelled culture medium by immunoprecipitation and subsequent analyses by fluorography revealed that a time-dependent proteolysis of both molecules occurred after replacement of Ca2+ and incubation at 37 degrees C. However, comparison of enzyme activity with fluorographic analyses showed that the maximal activation of both enzymes was achieved before any detectable decrease in Mr. The results suggest that the activation of proactivator and the subsequent activation of procollagenase may be initiated by conformational changes in structure of the proactivator molecule produced by removal of stabilizing bivalent metal ions.

Isolated osteoclasts resorb the organic and inorganic components of bone.

Osteoclasts are the principal resorptive cells of bone, yet their capacity to degrade collagen, the major organic component of bone matrix, remains unexplored. Accordingly, we have studied the bone resorptive activity of highly enriched populations of isolated chicken osteoclasts, using as substrate devitalized rat bone which had been labeled in vivo with L-[5-3H]proline or 45Ca, and bone-like matrix produced and mineralized in vitro by osteoblast-like rat osteosarcoma cells. When co-cultured with a radiolabeled substrate, osteoclast-mediated mineral mobilization reached a maximal rate within 2 h, whereas organic matrix degradation appeared more slowly, reaching maximal rate by 12-24 h. Thereafter, the rates of organic and inorganic matrix resorption were essentially linear and parallel for at least 6 d when excess substrate was available. Osteoclast-mediated degradation of bone collagen was confirmed by amino acid analysis. 39% of the solubilized tritium was recovered as trans-4-hydroxyproline, 47% as proline. 10,000 osteoclasts solubilized 70% of the total radioactivity and 65% of the [3H]-trans-4-hydroxyproline from 100 micrograms of 25-50 micron bone fragments within 5 d. Virtually all released tritium-labeled protein was of low molecular weight, 99% with Mr less than or equal to 10,000, and 65% with Mr less than or equal to 1,000. Moreover, when the 14% of resorbed [3H]proline-labeled peptides with Mr greater than or equal to 2,000 were examined for the presence of TCA and TCB, the characteristic initial products of mammalian collagenase activity, none was detected by SDS PAGE. In addition, osteoclast-conditioned medium had no collagenolytic activity, and exogenous TCA and TCB fragments were not degraded by osteoclasts. On the other hand, osteoclast lysates have collagenolytic enzyme activity in acidic but not in neutral buffer, with maximum activity at pH 4.0. These data indicate that osteoclasts have the capacity to resorb the organic phase of bone by a process localized to the osteoclast and its attachment site. This process appears to be independent of secretion of neutral collagenase and probably reflects acid protease activity.