The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.     

Separation of collagenase and a metal-dependent endopeptidase of rat uterus that hydrolyzes a heptapeptide related to collagen.

1. The synthetic peptide, 2,4-dinitrophenyl-L-Pro-L-Leu-Gly-L-Ile-L-Ala-Gly-L-Arg-amide (DNP-peptide) was tested as a potential substrate for uterine collagenase. Rat uteri were homogenized and the insoluble fraction was extracted at 60 degrees C to obtain collagenase. The extracts were chromatographed on Sephadex G-150 to yield two peaks of DNP-peptide hydrolyzing activity. Peak I was completely inhibited by EDTA and had a molecular weight greater than 100 000. Peak II was inhibited about 90% by EDTA and had an apparent molecular weight of about 70 000. 2. Peak II coincided closely, but not exactly, with the peak of collagenase activity. It differed from collagenase in heat stability, binding properties on CM-Sephadex and failure to display latency. 3. Peak II represents a new endopeptidase activity. It has a pH optimum of 7 and it cleaves the DNP-peptide at the Gly-Ile and, possibly, the Leu-Gly bond. 4. The DNP-peptide is not a satisfactory substrate for the assay of impure collagenase preparations nor does it inhibit the action of collagenase on collagen substrate when added in 30-fold molar excess.     

A spectroscopic collagenase assay using peroxidase-labeled collagen

A quantitative collagenase assay detecting soluble collagen fragments is described in this paper. Using the reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) type I collagen was conjugated with horseradish peroxidase (POD) which was employed as a reporter enzyme. POD was preferentially linked to the TC B fragment in a ratio of 1.4 mol POD/mol collagen. The conjugation product was immobilized on AH-Sepharose via carbodiimide coupling to form the final collagenase substrate used in the assay. POD activity in the supernatants caused by liberated TC B fragments exhibited a linear relationship for collagenase concentrations up to 100 micrograms/ml bacterial collagenase. Over an incubation period of 4 h the lowest detection limits found were 20 ng/100 microliters for bacterial collagenase and 60 ng/100 microliters for human leukocyte collagenase. Incubation of the assay mixture with 5 micrograms trypsin resulted in 3.8% of the activity released by the equivalent amount of leukocyte collagenase. The assay developed here has been shown to be sensitive and specific for collagenase, with the additional advantage that this method is suited for simple and economic handling.     

The collagen substrate specificity of rat uterus collagenase

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.     

Guinea pig pancreatic acini prepared with purified collagenase

Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca²⁺. Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with <0.14 units of protease per unit of collagenase produced highly responsive acini; collagenase with >0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.     

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22-28 degrees C (RT) and for 24 h at 5 degrees C; centrifuged at 13000 g for 30 min; filtered through 5-microm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl(2); supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl(2). The lectins adsorbed on FRBC are desorbed using either 100-500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.     

Extraction of collagenase from the involuting rat uterus.

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.     

Synthesis of collagenase and collagenase inhibitors by osteoblast-like cells in culture

A rat osteosarcoma cell clone (ROS 17/2), and osteoblast-enriched populations from rat calvaria cultured in the presence of concanavalin A, have been shown to produce latent collagenase and collagenase inhibitors. The enzymes and inhibitor activities from the ROS 17/2 cells were concentrated by ammonium sulphate precipitation and separated by gel filtration on AcA 54 resin. The size of the latent collagenase (Mr approximately equal to 58000) was reduced on conversion to active enzyme (Mr approximately equal to 48000) by p-aminophenylmercuric acetate. Latent and active forms of gelatinase activity, similar in size to the corresponding forms of collagenase, were also resolved. The collagenase inhibitor activity, which was sensitive to organomercurials, was recovered in two peaks (Mr approximately equal to 68000 and 30000). The active collagenase cleaved interstitial collagens (type I = III greater than II) producing typical 3/4 and 1/4 fragments. This activity was inhibited by the metal ion chelators ethylenediaminetetraacetic acid and o-phenanthroline. Additional specific cleavages of native collagen were also observed which, from the susceptibility of this activity to phenylmethylsulphonyl fluoride, leupeptin and antipain, suggested the presence of a second collagenolytic enzyme. This synthesis of collagenolytic enzymes by these osteoblast-like cells suggests that individual osteoblasts, like fibroblasts, are capable of both synthesizing and degrading their respective organic matrices in vivo.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

A model for biased diffusion of collagenase along collagen fibrils

We present a model to describe the biased diffusion of a collagenase along collagen fibrils. Based on the structures of collagen fibril and collagenase, the interaction is described by a one-dimensional potential that is symmetric in the region of no cleavage and asymmetric in the cleavage region. We show that the mean velocity of the unidirectional diffusion of the collagenase depends on the three parameters: the asymmetric ratio of the local potential in the cleavage region, the chemical reaction rate of proteolysis and the jumping rate of collagenase between two neighboring tracks. We calculate the correlation function and the mean transport velocity for both wild-type and mutant collagenases along collagen fibrils, the results of which are consistent with the previous experiments.     

Activation of the 92 kDa type IV collagenase by tissue kallikrein

Type IV collagenases are secreted as latent 92 and 72 kDa proenzymes which are then activated extracellularly. The mechanisms by which they are activated in vivo are not clear. We have studied the activation of porcine endothelial cell type IV collagenases by tissue and plasma kallikrein, and found that tissue kallikrein was a very efficient activator of the 92 kDa type IV collagenase. Enzyme cleavage was observed at concentrations of tissue kallikrein as low as 0.1 μg/ml. Plasma kallikrein had no effect. By comparison, plasmin, which has been proposed to be the physiological activator of interstitial collagenase and stromelysin, and elastase were much less effective, and high concentrations (plasmin at 100–200 μg/ml and elastase at 20 μg/ml) were required to cause only a limited cleavage which was not associated with an increase in activity, as observed by the gelatin-gel lysis assay. In addition tissue kallikrein was found by immunohistochemistry to be present in the extracellular matrix of the intima of porcine aortic vessel wall. These findings suggest that tissue kallikrein can be a potential activator of the 92 kDa type IV collagenase in vivo. © 1993 Wiley-Liss, Inc.     

A specific collagenase from rabbit fibroblasts in monolayer culture

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.     

An improved viscosimetric assay for vertebrate collagenase activity

An improved viscosimetric assay for vertebrate collagenase acitivity is described. The assay is carried out at 35 degrees C in the presence of 1 M glucose to prevent fibril formation. The decrease in viscosity is linear with ime and proportional to enzyme concentration.     

A collagenase assay using [3H-methyl]collagen

A new assay procedure for collagenase is presented. Highly radioactive substrate is prepared by methylation of native collagen. The ³H-labelled protein is readily attacked by bacterial as well as by mammalian collagenase and resistant to other proteinases. The sensitivity of this assay is higher than that of the enzymic methods hitherto available.     

Application of the chloramphenicol acetyltransferase (CAT) diffusion assay to transgenic plant tissues.

Chloramphenicol acetyltransferase (CAT) activity was quantified in crude extracts from tobacco callus tissues using a modification of a previously reported diffusion assay. We describe here the alterations necessary in applying this rapid and simple assay procedure to plant materials. Due to the high concentration of nonspecific oxidases present in most plant tissues, some type of protective agent is required to maintain enzyme activity. We have tested beta-mercaptoethanol, cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone as protective agents within the initial extraction buffer. We also investigated the effect of heat (60 degrees C, 10 min) and 5 mM EDTA on CAT activity. The highest CAT activity was obtained using 5 mM cysteine plus 5 mM EDTA in 40 mM Tris-HCl (pH 7.8) as the initial extraction buffer followed by a heat treatment. Using this buffer, CAT activity was stable on ice for more than two hours. In our hands, total acetyl-coenzyme A concentration within the assay mixture was found to be saturating at 250 microM and the Km determined to be 100 microM. Assays performed using the same crude plant extract indicate that 1) duplicate assays show less than 1.5% variation in activities and 2) CAT activity increases linearly with respect to volume of extract used.     

Purification of rabbit bone inhibitor of collagenase.

1. Rabbit bones in tissue culture synthesize an inhibitor of collagenase during the first 4 days of culture. 2. The inhibitor was purified by a combination of gel filtration, concanavalin A--Sepharose chromatography, ion-exchange chromatography and zinc-chelate affinity chromatography. 3. The purified inhibitor migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had a mol.wt. of 28000. 4. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase, neutral proteinase III (proteoglycanase), human leucocyte collagenase and gelatinase, but not thermolysin or bacterial collagenase. The serine proteinases plasmin and trypsin were not inhibited. 5. The inhibitor interacted with purified rabbit bone collagenase with 1:1 stoichiometry. 6. The inhibitory activity was lost after incubation for 1 h at 90 degrees C, after treatment with trypsin (250 micrograms/ml) at 37 degrees C for 30 min and after reduction and alkylation.     

An assay of collagenase activity using enzyme-linked immunosorbent assay for mammalian collagenase

A new method was developed for the measurement of collagenase activity using the enzyme-linked immunosorbent assay (ELISA). Rabbit colon wall collagenase, pepsin-soluble rat skin type I collagen, and its antisera were used in the present experiment. After the collagenase-degraded portion of the collagen coated on the microwell was released, the immunoreaction of the residual collagen on the microwell to anticollagen sera was determined by ELISA. This method was approximately 10 times more sensitive than the conventional assay procedure using [14C]-glycine-labeled reconstituted collagen fibrils as substrate. It was suitable for screening a large number of samples without radioisotopes.     

Tissue collagenase: a simplified, semiquantitative enzyme assay

Tissue collagenase activity from the ulcerating rabbit cornea has been quantitated in a sensitive capillary tube assay system with an unlabeled, native collagen substrate. In this assay system, initial rates of gel lysis are proportional to enzyme concentration over a defined range of enzyme concentrations. Increased sensitivity to enzyme with an unlabeled substrate has been achieved by restricting diffusion of enzyme to one dimension, in a capillary gel. Corneal collagenase activity has been measured at concentrations down to 0.1 μg/μl. In addition to its high sensitivity to enzyme, the precision and simplicity of the assay and minimal equipment requirements all recommend its use for routine screening of biological fluids for collagenase activity and in the investigation of the effects of inhibitors and stimulators of collagenase activity.