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synthesis and collagenase inhibition of new glycosides of aranciamycinone: the aglycon of the naturally occurring antibiotic aranciamycin.glycosides of aranciamycinone were prepared by glycosylation with sugar acetates and trimethylsilyl triflate in dichloromethane. glycosides of the following sugars were prepared: alpha-l-rhamnopyranose, beta-d-glucopyranose, beta-d-ribopyranose, beta-d-xylopyranose, alpha-l-fucopyranose, 2-azido-2,6-dideoxy-alpha-l-mannopyranose, 2,6-dideoxy-alpha-l-arabino-hexopyranose, 3,6-dideoxy-alpha-l-arabino-hexopyranose, and 4,6-dideoxy-alpha-l-lyxo-hexopyranose. the new glycosides were tested for inhibi ...19921335359
potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide n-(phenylethylphosphonyl)-gly-l-pro-l-aminohexanoic acid.a phosphonamide peptide, n-(phenylethylphosphonyl)-gly-l-pro-l-aminohexanoic acid, previously shown to block clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (mcc)-pro-leu-gly-pro-d-lys-dinitrophenyl (dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with ki values of 0.3 and 0.9 nm respecti ...19921332678
kinetics of hydrolysis of type i, ii, and iii collagens by the class i and ii clostridium histolyticum collagenases.the kinetics of hydrolysis of rat tendon type i, bovine nasal septum type ii, and human placental type iii collagens by class i and class ii clostridium histolyticum collagenases (chc) have been investigated. to facilitate this study, radioassays developed previously for the hydrolysis of these [3h]acetylated collagens by tissue collagenases have been adapted for use with the chc. while the chc are known to make multiple scissions in these collagens, the assays are shown to monitor the initial p ...19921325155
identification of clostridium histolyticum collagenase hyperreactive sites in type i, ii, and iii collagens: lack of correlation with local triple helical stability.the class i and ii clostridium histolyticum collagenases (chc) have been used to identify hyperreactive sites in rat type i, bovine type ii, and human type iii collagens. the class i chc attack both collagens at loci concentrated in the n-terminal half of these collagens starting with the site closest to the n-terminus. the class ii chc initiate collagenolysis by attacking both collagens in the interior to produce a mixture of c-terminal 62,000 and a n-terminal 36,000 fragments. both fragments a ...19921325154
an analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation.crude clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of langerhans. the variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. in this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and pu ...19921324862
inhibition of collagenase by aranciamycin and aranciamycin derivatives.aranciamycin (1), an anthracycline antibiotic, was found to be an inhibitor of clostridium histolyticum collagenase, with an ic50 = 3.7 x 10(-7) m. elastase and trypsin were not inhibited at concentrations less than or equal to 10(-5) m. a number of aranciamycin derivatives 2-13 were prepared and tested for collagenase inhibition. while loss of activity was found for derivatives modified in the sugar ring or rings b and d of the aglycone, increased potency was found when the tertiary alcohol at ...19921322986
the collagen shield as a collagenase inhibitor and clinical indicator of collagenase activity on the ocular surface.collagen shields applied to the corneas of patients with bacterial keratitis degrade rapidly, often within a few hours. once treatment brings the infection under control, subsequently applied collagen shields degrade more slowly. in vitro models were established to evaluate the significance of these observations. twenty-four and 72-hour collagen shields were incubated with collagenase from clostridium histolyticum. the in vitro rate of digestion of the shields was directly proportional to the co ...19921313747
comparative action of cathepsins b and l on intramuscular collagen as assessed by differential scanning calorimetry.intramuscular beef collagen of different degrees of reticulation was treated with cathepsin l obtained from chicken liver and a commercial cathepsin b prepared from beef spleen. it was shown that, in the absence of calcium, both proteinases caused a decrease in the initial temperature of denaturation whereas the total enthalpy of denaturation was unaffected. treatment with cathepsin b resulted in the appearance of a new peak of denaturation at a lower temperature (˜ 44°c), a change wholly compar ...199222059816
effect of divalent metal ions on collagenase from clostridium histolyticum.the collagenase from clostridium histolyticum (ec 3.4.24.3) degrades type iv collagen with km 32 nm, indicating a high affinity for this substrate. ferrous and ferric ions can inhibit clostridium collagenase. inhibition by fe++ was of the mixed, non-competitive type, with ki 90 microm. the inhibitory effect of fe++ may be due to zn++ displacement from the intrinsic functional center of this metalloprotease, since in the presence of excess amounts of zn++ enzyme activity is retained. this inhibit ...19911663343
[studies on collagenase inhibitors. iv. inhibitors of bacterial collagenase in coptidis rhizoma].a hot aqueous extract of coptidis rhizoma had an inhibitory effect on the bacterial collagenase from clostridium histolyticum. active principles were isolated by silica gel column chromatography from the chcl3 extract. consequently, two inhibitors obtained were identified with the chloride of berberine and coptisine. the concentrations of the berberine and coptisine in the assay mixture to give 50% inhibition (ic50) were 0.73 mm and 0.16 mm, respectively. the type of inhibition by coptisine chlo ...19911662269
[anacollagenase by the action of amitriptyline on clostridium histolyticum collagenase].intraperitoneal injection of a mixture of collagenase (300 u) and amitriptyline (laroxyl*, 3 mg) induce no lesions in contrast with the severe effects of collagenase alone. also, a complete resistance to intraperitoneal collagenase injection is observed when preceded by 3 intramuscular injections of the same mixture (associated with freund's incomplete adjuvant). this is due to the development of collagenase antibodies, as demonstrated by nephelometry and immunodiffusion. these facts show that a ...19911666017
inhibition of clostridium histolyticum collagenases by phosphonamide peptide inhibitors.several phosphonamide peptides having the general structure r-po(oh)-xaa-yaa-zaa were synthesized and tested for inhibition of clostridium histolyticum collagenase. inhibition was found to depend on the nature of r, xaa, yaa and zaa such that the maximal affinity (ki = 5 nm) was observed when r = p-nitrophenylethyl, xaa = gly, yaa = pro and zaa = 2-aminohexanoic acid; this represents the tightest binding of inhibitor reported to date for any bacterial collagenase. substitution of the p-nitrophen ...19902167850
changes in the core of the mammalian-pyruvate dehydrogenase complex upon selective removal of the lipoyl domain from the transacetylase component but not from the protein x component.the dihydrolipoyl transacetylase (e2) component contains a cooh-terminal inner domain (e2i) and an extended nh2-terminal structure, which is composed of two lipoyl domains (the fragment containing both is designated as e2l) and a subunit-binding domain (e2b). the four domains are connected by hinge regions. a subcomplex, composed of an oligomer of e2 subunits, protein x (which also has an nh2-terminal lipoyl domain), and the [pyruvate dehydrogenase]-kinase catalytic and basic subunits (kc and kb ...19902167319
proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin.the interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. mouse alpha-macroglobulin formed complexes with thrombin, clotting factor xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pa ...19892481676
the ketone cinnamoyl-(1-13c-phe)-cgly-pro-pro is a tetrahedral transition state analog inhibitor of c. histolyticum collagenase.the ketone cinnamoyl-(1-13c-phe)-cgly-pro-pro [(4-13c-5-cinnamido-4-oxo-6-phenylhexanoyl)-pro-pro 2] competitively inhibits a mixture of collagenases from clostridium histolyticum with a ki of 40 +/- 6 nm. 13c-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13c resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm. ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm. the bound resonance is displaceable by anot ...19892539108
[study on the effect of some group-specific agents on clostridiopeptidase].the interaction of clostridiopeptidase of clostridium histolyticum with edc, tnm and ma, the specific reagents for cooh-groups, tyrosine and lysine residues was studied. it was shown that at ph 6.0 edc inactivates the enzyme. the inactivation process follows the pseudo-first order kinetics and is described by a second order rate constant equal to 1 m-1 min-1. the synthetic substrate does not prevent, in practical terms, the enzyme inactivation by edc. at ph 8.0 tnm modifies about 19 tyrosine res ...19892547458
novel method for the preparation of sterile collagen-agarose-plates for isolating collagenolytic bacteria.an assay method for bacterial collagenase using sterile collagen-agarose plates was developed to isolate collagenolytic bacteria. in order to avoid heat-denaturation of collagen, the plates were made at 37 degrees c mixing microwave-sterilized collagen with low melting point-agarose. sensitivities to various proteases were tested on the plates; it was shown that whereas the collagen in these plates was digested only by the collagenases, the collagen could also be hydrolyzed by other proteolytic ...19892561866
protease activity in pancreatic islet isolation by enzymatic digestion.commercial collagenase* prepared from clostridium histolyticum is widely used in isolation of pancreatic islets. it is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches. our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized. comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that th ...19892642834
human fibroblast collagenase-alpha-macroglobulin interactions. localization of cleavage sites in the bait regions of five mammalian alpha-macroglobulins.the interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin, and rat alpha 1-inhibitor 3) differing in primary and quaternary structure has been investigated. complex formation with each of these alpha-macroglobulins follows the course identified for many other proteinases, i.e. specific limited proteolysis in their bait regions inducing a set of conformational changes ...19892462561
production of human adrenocorticotropin by cleavage of alkaline-phosphatase-derived fusion proteins containing repetitive recognition sequences for collagenases.recombinant plasmids coding for fusion proteins which consist of human adrenocorticotropin joined to n-terminal sequences of escherichia coli alkaline phosphatase via collagenase-sensitive linkers were constructed and used for the production of these proteins by transformed e. coli cells. it was shown that repetitive linkers of the form -gly-(pro-xaa-gly)n-pro- with n greater than or equal to 2 were cleaved by clostridiopeptidase a (clostridium histolyticum) by orders of magnitude faster than co ...19892573529
[activation in vivo of the collagenase of clostridium histolyticum by erythromycin lactobionate].the authors demonstrate a previously not described increase, in vivo, in a mean proportion of 70%, of the enzymatic activity of the clostridium collagenase in rats by erythromycin lactobionate. the pathogenesis of this phenomenon cannot yet be explained.19892555034
new thiol inhibitors of clostridium histolyticum collagenase. importance of the p3' position.an extensive series of synthetic mercaptotripeptides (hs-ch2-ch2-co-pro-yaa) was prepared, and the inhibitory constants were determined on the clostridium histolyticum collagenase. among the factors which control the optimal binding of these inhibitors, we found that the presence of a free c-terminal carboxylate group in the position p3' of the compounds is of primary importance. in general, the esterification of this carboxylate group decreased the potency of the inhibitors by two orders of mag ...19882832171
near stoichiometric, irreversible inactivation of bacterial collagenases by o-chloranil (3,4,5,6-tetrachloro-1,2-benzoquinone).the hydrogen-abstracting quinone derivative 3,4,5,6-tetrachloro-1,2-benzoquinone (o-chloranil) caused a strong, near stoichiometric, irreversible inactivation of the collagenases from bacillus cereus, clostridium histolyticum and achromobacter iophagus. p-chloranil was a weaker inactivator. o-chloranil reacted rapidly with a site that affected substrate binding. amino acid analyses of native and totally inactivated enzymes, and the ph-profile of inactivation suggest that the dissociated form of ...19882837215
preparation by direct metal exchange and kinetic study of active site metal substituted class i and class ii clostridium histolyticum collagenases.active site metal substitutions for both gamma- and zeta-collagenases from clostridium histolyticum have been made by direct metal exchange. the incubation of co(ii), cu(ii), ni(ii), cd(ii), and hg(ii) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. for both collagenases, the exchange reactions with co(ii) and cu(ii) are complete within 1 min. however, the changes in activity obs ...19882849992
ketone-substrate analogues of clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors.a series of ketone-substrate analogues has been synthesized for the two classes of collagenases from clostridium histolyticum and shown to be competitive inhibitors. these compounds have sequences that match those of specific peptide substrates for these enzymes. the best inhibitor is the ketone analogue of cinnamoyl-leu-gly-pro-pro, which has a ki value of 18 nm for epsilon-collagenase, a class ii enzyme. this is the tightest binding inhibitor reported for any collagenase to date. plots of log ...19882844227
preparation and reconstitution with divalent metal ions of class i and class ii clostridium histolyticum apocollagenases.both gamma- and zeta-collagenases from clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at ph 7.5 in the presence of 10 mm cacl2 with ki values of 0.11 and 0.040 mm, respectively. the inhibition is caused by removal of the single, active-site zn(ii) present in each of these enzymes. the nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme. dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis ...19882849991
enzyme-antibody histochemistry. a method for detection of collagens collectively.different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. the common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. however, all these collagens are substrates for the collagenase of clostridium histolyticum. in this report we describe a method that allows the visualization of collagens, collectively, in a tissue preparation. the method is based on the affinity b ...19872820910
purification and properties of collagenase from a streptomyces species.a collagenase active against native, insoluble collagen was isolated from the culture filtrate of a streptomycete which has been designated streptomyces sp. c-51. collagenase was produced by growing this strain in media containing gelatin. purification by ammonium sulfate fractionation and chromatographies on deae-toyopearl and deae-cellulofine columns produced active enzyme which was free of contaminating proteins, including nonspecific proteinases, but which contained two active subspecies (i ...19872822678
purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium bacillus cereus (strain soc 67).the major collagenolytic proteinase present in the culture filtrate of bacillus cereus (strain soc 67, isolated from the human oral cavity) has been purified to homogeneity by a procedure that comprised concentration of ultrafiltered growth medium on a millipore pttk00005 membrane, precipitation with ammonium sulfate, gel permeation chromatography, chromatofocusing, fast protein liquid chromatography on an anion-exchange column, and finally fast protein liquid chromatography on a gel column. the ...19873040751
[inhibitory action of methysergide bimaleate on the collagenase of clostridium histolyticum].inhibitory effects of methysergide bimaleate on the collagenase of the clostridium histolyticum have been established. having previously shown the inhibitory action of serotonin on this bacterial collagenase, the authors have tested methysergide bimaleate as another inhibitor. after injection in the peritoneum of the rats of methysergide bimaleate and collagenase together, lesions are minimal or absent, in contrast with the dramatic effects of collagenase alone. this shows the antagonist role of ...19872830001
collagenase chemonucleolysis via epidural injection. a review of 252 cases.since 1975, collagenase has been injected into 252 herniated intervertebral discs for treatment of sciatica. highly purified preparations of chinese collagenase were extracted from clostridium histolyticum ch 1093 in lyophylized form, which had a specific collagenolytic activity. it had a wide margin of safety as proven by repeated experiments in dogs by means of intradiscal, extradural, intradural, and intravenous injections. one hundred to two hundred units of collagenase dissolved in 5 ml of ...19873026711
limited proteolysis of type i collagen at hyperreactive sites by class i and ii clostridium histolyticum collagenases: complementary digestion patterns.the initial proteolytic events in the hydrolysis of rat tendon type i collagen by the class i and ii collagenases from clostridium histolyticum have been investigated at 15 degrees c. sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been used to detect the initial cleavage fragments of both the alpha 1(i) and alpha 2 chains, which migrate at different rates in the buffer system employed. experiments with the class i collagenases indicate that the first cleavage occurs across all thr ...19873032235
purification of human collagenases with a hydroxamic acid affinity column.human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling pro-leu-gly-nhoh to agarose. following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield. importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated. the column was specific for the human enzy ...19863021211
purification and characterization of a streptomycete collagenase.a soil streptomycete designated as streptomyces sp. a8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-l-prolyl-l-leucyl-glycyl-l-prolyl-d-a rginine into two tripeptides. the enzyme was purified by diethyl aminoethyl cellulose chromatography and sephadex g-150 gel filtration. the purified enzyme had an apparent molecular wei ...19863023269
effect of proteolytic enzymes on transepithelial solute transport.the effects of proteases on air-space clearance (ac) of small ([14c]sucrose, 342 daltons) and large (125i-neutral dextran, 70,000 daltons) solutes were studied in isolated, fluid-filled hamster lungs that were perfused in a nonrecirculating system. when instilled into the air spaces, porcine pancreatic elastase (0.1-0.4 mg/ml) and bovine pancreatic trypsin (bpt) (0.5-2.0 mg/ml), but neither clostridium histolyticum collagenase (5.0 mg/ml) nor phenylmethylsulfonyl fluoride-inactivated bpt caused ...19862430929
use of specific collagenases for the isolation of rat liver cells with preserved lipase activities.the heparin-releasable neutral lipase (ec 3.1.1.3) from rat liver is inactivated by the common preparations of collagenases (ec 3.4.24.3) used for the isolation of liver cells. we show that two collagenases purified from clostridium histolyticum allow both the complete preservation of this lipolytic activity and a good viability of liver cells isolated by the usual perfusion protocol.19863010771
tenderization of beef with bacterial collagenase.the feasibility of using a purified collagenase produced by clostridium histolyticum as a meat tenderizer was studied. experiments were conducted with enzymes in model systems to compare collagenase with the currently used plant proteinases, papain, bromelain and ficin. collagenase was shown to have a greater activity in hydrolyzing insoluble collagen than salt-soluble-protein (ssp) and highest activity between 40° and 60°c, with little to no activity above 60°c. collagenase was added to raw ste ...198622055648
aldehyde and ketone substrate analogues inhibit the collagenase of clostridium histolyticum.the collagenase from clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. this enzyme catalyzes the cleavage of the x-gly peptide bond in the repeating sequence of collagen: -gly-pro-x-gly-pro-x-. thus the s3, s2, and s1 subsites on the enzyme appear to be occupied by the sequence -gly-pro-x- and the s1', s2', and s3' subsites also by -gly-pro-x-. short peptides up to and including n alpha-acyltetrapeptides containing the repeat sequence do not d ...19853002433
gas chromatography/mass spectrometry of bacterial amines.bacterial amines were examined by gas chromatography/mass spectrometry. under electron impact all trifluoroacetamides exhibited peaks at m/z 69 due to [cf3]+. many trifluoroacetamides also showed peaks at m/z 97 corresponding to the [cocf3]+ ion fragment. the spectra of n-alkyl and aralkyl trifluoroacetamides were consistent with the spectra and their interpretations in the earlier literature. molecular ions were of low abundance for all alkyl trifluoroacetamides having alkyl chains longer than ...19852931124
mode of hydrolysis of collagen-like peptides by class i and class ii clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities.the action of three class i (beta, gamma, and eta) and three class ii (delta, epsilon, and zeta) collagenases from clostridium histolyticum on two series of peptides with collagen-like sequences has been examined. the peptides in the first series all contain 4-nitrophenylalanyl-gly-pro-ala in subsites p1 through p3', but each is successively lengthened in the n-terminal direction by addition of an appropriate residue until subsite p5 is occupied. the second group of peptides all have cinnamoyl-l ...19853002446
substrate specificity of beta-collagenase from clostridium histolyticum.the substrate specificity of beta-collagenase from clostridium histolyticum has been investigated by measuring the rate of hydrolysis of more than 50 tri-, tetra-, penta-, and hexapeptides covering the p3 to p3' subsites of the substrate. the choice of peptides was patterned after sequences found in the alpha 1 and alpha 2 chains of type i collagen. each peptide contained either a 2-furanacryloyl (fa) or cinnamoyl (cn) group in subsite p2 or the 4-nitrophenylalanine (nph) residue in subsite p1. ...19852982835
purification of nonspecific protease-free collagenase from clostridium histolyticum.the collagenase (ec 3.4.24.3) produced by the bacterium clostridium histolyticum has been purified free from nonspecific protease contaminants by a two-step procedure. the crude culture medium is chromatographed over heparin-sepharose and sephacryl s-200, and the resulting preparation has no activity versus noncollagenous proteins or n alpha-benzoyl-l-arginine ethyl ester, yet cleaves native thermally reconstituted collagen fibrils quite efficiently (specific activity, 3000 units/mg). the purifi ...19852990251
inhibition of collagenase activity by extracts of bovine ocular tissues.bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity. although lens extract was not inhibitory, the cornea and vitreous both contained inhibitors of collagenase. more inhibition was present in the filtrate of the vitreous than in the retentate, whereas the total amount of inhibition in the cornea was distributed almost equally between the two fr ...19852992886
complementary substrate specificities of class i and class ii collagenases from clostridium histolyticum.the substrate specificities of three class i (beta, gamma, and eta) and three class ii (sigma, epsilon, and zeta) collagenases from clostridium histolyticum have been investigated by quantitating the kcat/km values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the p3 through p3 subsites of the substrate. for both classes of collagenases, there is a strong preference for gly in subsites p1' and p3. all six enzymes also prefer substrates that contain pro and ala ...19853002445
clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors.a new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme clostridium histolyticum collagenase. the hydrolysis of the depsipeptide substrate was followed on a ph stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. the best thio ester substrate, boc-abz-gly-pro-leu-sch2co-pro-nba, had a kcat/km of 63 000 m-1 s-1, while several shorter thio ester sequences were ina ...19852992578
purification and separation of individual collagenases of clostridium histolyticum using red dye ligand chromatography.six collagenases present in the culture filtrate of clostridium histolyticum have been purified to homogeneity. chromatography over hydroxylapatite, sephacryl s-200, and l-arginine-affi-gel 202 removes the brown pigment and the great majority of the contaminating proteinases active against casein, benzoyl-l-arginine ethyl ester, and elastin. reactive red 120 dye ligand chromatography subdivides the collagenases, which have very similar physicochemical properties, among four fractions. the final ...19846087887
purification and characterization of three forms of collagenase from clostridium histolyticum.three collagenases from clostridium histolyticum, designated c1, c2, and c3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified. peptide maps of the enzymes prepared by digestion with staphylococcus aureus v-8 protease were found to be similar. cleavage of native c1 with alpha-chymotrypsin or v-8 protease yielded c2 and c3. this suggested that proteolysis of the mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes. previ ...19846095892
characterization of the individual collagenases from clostridium histolyticum.the six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) from clostridium histolyticum isolated in the preceding paper [bond, m. d., & van wart, h. e. (1984) biochemistry (first paper of three in this issue)] have been characterized in detail. the molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68 000 to 125 000. isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range. ...19846087888
relationship between the individual collagenases of clostridium histolyticum: evidence for evolution by gene duplication.the relationship between the six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) isolated and characterized in the preceding papers [bond, m.d., & van wart, h.e. (1984) biochemistry (preceding two papers in this issue)] has been investigated. chemical modification reactions establish that all six enzymes contain essential carboxyl, tyrosine, and lysine residues. circular dichroism spectra of the peptide bond region show that the secondary structures of the collagenases are very simil ...19846087889
isolation and ultrastructure of rabbit ovarian mesothelium (surface epithelium).this study evaluated the efficacy of various dissociating procedures for isolating mesothelial or surface epithelial cells from rabbit ovaries. the procedure which provided the highest and most homogenous cell yield involved the following sequence: (a) incubation of whole ovaries for 60 min in clostridium histolyticum collagenase, type i (300 u/ml); (b) vortexing of ovaries at 30 rpm for 60 s in medium 199; (c) removal of partially detached surface epithelium by gentle scraping with a microdisse ...19846392121
characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures.primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. all the enzyme present in the culture medium is latent and binds to columns of heparin-sepharose. the yield of activity from the heparin-sepharose columns (measured after organomercurial treatment) is approximately 300-1000% depending on the chondrocyte culture batch. recombination of column fractions shows that the increase in acti ...19836313063
enzymatic isolation of cells from neonatal calvaria using two purified enzymes from clostridium histolyticum.the enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. the use of chromatographically purified collagenase alone is often ineffective or very slow to release cells ...19836315459
enhancement of eosinophil effector function by soluble factors released by s. mansoni: role of proteases.schistosomulum-released products (srp) have been shown to enhance both expression of rat and human eosinophil fc receptors and igg-dependent cytotoxicity. the present work provides additional evidence of the secretion of eosinophil-enhancing factors by schistosomula and other developmental stages of schistosomes, including adult worms. the heat lability, as well as the strong inhibition of the stimulating activity of srp by the protease inhibitor trasylol, suggest that thermolabile proteases sec ...19836190922
degradation of basement membrane collagen by proteinases from human gingiva, leukocytes and bacterial plaque.basement membranes in human gingiva are found in dento-epithelial junction, epithelial-connective tissue junction and endothelium-connective-tissue junction where they have important attachment and filtering functions. the ability of plaque and gingiva-derived proteinases to degrade type iv collagen, the major protein of basement membranes, was examined in vitro. the basement membrane collagen (type iv) isolated from bovine lens capsules was incubated in the presence of enzyme samples. the degra ...19836315910
characterization of a large fragment from annelid cuticle collagen and its relationship to the intact molecule.digestion of the cuticle collagen from the annelid nereis virens with clostridium histolyticum collagenase yields a native, collagenase-resistant fragment (ccrf) of the molecule with an mr of about 900,000 (kimura and tanzer, j. biol. chem. 252: 8018, 1977). we have produced 940 nm long, sls-crystallites from the collagenase-resistant fragment; the sls pattern matches a region at one end of the 2,400 nm sls obtained from intact cuticle collagen. upon denaturation, the fragment yields two subunit ...19836321095
inhibition of collagenase from clostridium histolyticum by phosphoric and phosphonic amides.di- and tripeptides with sequences present in collagen that are known to occupy the s1' through s3' subsites at the active site of the collagenase from clostridium histolyticum do not themselves inhibit this zinc protease. thus glycylproline, glycylprolylalanine, and their c-terminal amides are not inhibitors. n alpha-phosphorylglycylproline, n alpha-phosphorylglycyl-l-prolyl-l-alanine, and their c-terminal amides are weak inhibitors with ic50's (concentration causing half-maximal inhibition) of ...19836313042
[probable inhibition of a bacterial collagenase by serotonin].we have previously demonstrated that intraperitoneal injections in rats of collagenase (from clostridium histolyticum) are followed by severe lesions. here we show that the injection of collagenase together with serotonin has no or only small effects. it appears to be partly due to serotonin. creatinin and sulfate which form an injectable complex with serotonin have no influence on this phenomenon. the part of ph 4,4 of the injected solution is discussed. these facts suggest that serotonin may p ...19826290002
bacterial collagenases and their clinical applications.clostridium histolyticum cultures secrete a specific collagenase which can be precipitated from the medium in relatively large amounts. the enzyme is a metalloproteinase capable of cleaving native collagen types i, ii, iii, iv and v. in addition to being a valuable tool in the laboratory the bacterial collagenase has found clinical applications in the treatment of third degree burns and decubitus, diabetic or arterial ulcers. several cases of successful topical use of the crude enzyme in an oint ...19826295413
[neutral and basic compounds present in gases produced by clostridium histolyticum, clostridium hastiforme and clostridium ghoni cultured under vacuum in sodium thioglycolate glucose broth].the study of gases produced by clostridium histolyticum, c. hastiforme and c. ghoni cultured under vacuum in sodium thioglycolate glucose broth makes it possible to detect compounds not described before for these bacteria, and confirms the production of methane in anaerobic spore-forming bacteria.19826817857
chemical characterization of the homogeneous collagenase from clostridium histolyticum.pure collagenase (clostridiopeptidase a, ec 3.4.24.3) having a molecular weight of 70 000 was obtained from the culture medium of clostridium histolyticym by a combination of ultrafiltrations, molecular sieve, affinity and hydrophobic chromatography. the value of its specific activity is the highest of those described previously but 6-times lower than that of the collagenase from achromobacter iophagus (ec 3.4.24.8). its amino acid composition differs from previous data, namely by the presence o ...19816266487
enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase.the enzymatic isolation of cells from fetal rat calvaria is most effectively achieved with crude clostridium histolyticum collagenase. however, this bacterial collagenase damages the cells during the digestion of the tissue. we have used cell density, as measured by isopycnic centrifugation on polysucrose gradients, as an indicator of cell damage. there are at least two enzymes in crude bacterial collagenase capable of damaging the cells in this tissue. one of these is clostripain that has been ...19816263099
a continuous spectrophotometric assay for clostridium histolyticum collagenase. 19816269461
identification of essential amino acid residues in clostridium histolyticum collagenase using chemical modification reactions. 19816272790
production of collagenase inhibitor by the growth cartilage of embryonic chick bone: isolation and partial characterization.production of collagenase and collagenase inhibitors by the explants of epiphyseal, metaphyseal and diaphyseal regions of embryonic chick limbs during development has been investigated. collagenase-inhibitory activity was first detected in the culture medium of the diaphyseal region of limbs at stage 36 where cartilage matrix erosion began to occur. however, neither active nor latent collagenase was detected in the media of any regions examined. at stage 38 and later, the maximum production of t ...19816286227
oxygen-dependent metronidazole-resistance of clostridium histolyticum.sensitivity determinations with 6 strains of clostridium histolyticum showed that the inhibitory action of metronidazole was highly dependent on the oxygen concentration of the environment. under anaerobic conditions they were sensitive but at increased oxygen concentrations moderately sensitive or resistant. the flexible resistance of this aerotolerant anaerobe against metronidazole may interfere with results of sensitivity determinations, estimation of blood levels and it may influence the eff ...19817269847
[degradation of collagen by the cariogenic bacteria, streptococcus mutans].the behaviour of cariogenic bacteria (streptococcus mutans) is studied with regard to collagen, which represents 90% of the dentine organic matrix. collagenase activity of cariogenic bacteria is measured with radioactive precursors and gel electrophoresis and compared to reference bacterial collagenase (clostridium histolyticum). labelled collagen substrate has been prepared with two different methods: extraction by 0,5 m acetic acid from young rat skin, previously labelled with l-proline 14c, o ...19806248261
circular dichroism comparative studies of two bacterial collagenases and thermolysin.the recently isolated and purified collagenase produced by achromobacter iophagus, the collagenase from clostridium histolyticum, and thermolysin, three enzymes having common properties, were studied by circular dichroism. from the spectra of the aqueous solutions obtained in the peptide region, the fraction of alpha helix, beta sheet and aperiodic segments in the three proteins could be estimated. good similarity was found between achromobacter collagenase and thermolysin, which both contain a ...19806250633
[isolation and properties of 3 clostridium histolyticum collagenases].the collagenases (i, ii and iii) have been obtained in a highly purified state from fresh cultural medium of clostridium histolyticum. the collagenases were similar in their properties to clostridiopeptidase a. the three enzymes differed in their molecular weights, isoelectric points and in some chemical properties. collagenase ii exhibited the most potent hydrolytic activity. its collagenolytic activity was two-fold higher and the peptidase activity was twenty-fold higher as compared with that ...19806252691
inhibitor of human collagenase from cultures of human tendon.a potent inhibitor of human collagenases, released from human tendon explants in culture, has been purified and partially characterized. the tendon inhibitor has an estimated molecular weight of 25,000. it is relatively heat-stable but undergoes loss of activity following exposure to trypsin. it inhibits trypsin-activated rheumatoid synovial collagenase as well as the enzyme obtained from polymorphonuclear leukocytes. no inhibition of collagenase from clostridium histolyticum (clostridiopeptidas ...1979218961
some newly characterized collagenases from procaryotes and lower eucaryotes.chemical and enzymatic properties of four collagenases newly isolated from anaerobic clostridium histolyticum, aerobic achromobacter iophagus, and from two lower eucaryotes, the fungus entomophthora coronata and the insect hypoderma lineatum are reviewed. the problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in achromobacter by their macromolecular substrates are discussed. the two bacterial collagenases are zn-metallo-enzymes; th ...1979220520
differences in the degradation of native collagen by two microbial collagenases.the early stages of degradation of native collagen by two bacterial collagenases were studied by electron microscopy and by automatic edman degradation. the purified collagenase from clostridium histolyticum was shown to cleave native collagen at several sites, but not progressively from the n-terminus, as had been previously suggested. the homogeneous collagenase from achromobacter iophagus cleaves native collagen preferentially at two sites corresponding to the interbands 33-34 and 41-42. the ...1979224860
evaluation of a rapid, sensitive and specific assay for the determination of collagenolytic activity in biological samples.several methods for the determination of collagenolytic activity were compared from the point of view of sensitivity, selectivity, simplicity and practical value for large numbers of biological samples. a labelled collagen substrate was prepared using [3h]acetic anhydride. the specificity of the assay as well as conditions allowing an optimum detection limit were investigated. the influence of low temperatures, lyophilisation and salt concentration on clostridium histolyticum collagenase have be ...1979226285
immunofluorescent study of the spore antigens of proteolytic strains of clostridium botulinum.by means of the spore fluorescent antibody technique 31 strains of clostridium botulinum types a (18 strains), b (10 strains) and f (3 strains) were found to belong to the same homogeneous group irrespective of their toxigenic types. some strains of this species also cross-reacted with certain strains of clostridium sporogenes types i, ii and iii and clostridium histolyticum type ii. by spore antigenic analysis it was found that clostridium parabotulinum contained two components designated l and ...1979379206
clindamycin-associated enterocolitis in guinea pigs: evidence for a bacterial toxin.experimental enterocolitis was induced in guinea pigs by intraperitoneal injection of clindamycin. specimens of feces were collected daily in phosphate-buffered saline (ph 7.0) and pooled every second day. the pooled samples were centrifuged to remove solids, and the supernatant was sterilized by membrane filtration. the sterile fecal supernatants were then dialyzed for 48 h against two 15-liter changes of phosphate-buffered saline and subsequently tested for toxicity in cultured monolayers of m ...1979422233
alpha-clostripain. chemical characterization, activity, and thiol content of the highly active form of clostripain.a highly active form of clostripain, composed of two polypeptide chains (mr = 43,000 and 12,500), was isolated by hydrophobic chromatography from the culture medium of clostridium histolyticum. it differs in amino acid composition, namely in the value for cyst(e)ine, from that previously reported. the analyses of the separated chains are given. activity is related to the number of free cysteine residues and full activity is obtained only after complete reduction of the disulfide bonds. specific ...1979762145
specific cleavage of reduced and s-carboxamidomethylated neurophysin ii by the collagenase of clostridium histolyticum.purified collagenase of clostridium histolyticum was shown to cleave reduced and s-carboxamidomethylated bovine neurophysin between cys-13 and gly-14. the scission resulted in formation of two separable fragments: a smaller peptide arising from residues 1 through 13, and a larger peptide comprising the remainder of the residues of the protein. by dansylation procedures, the smaller peptide was shown to have amino-terminal alanine as expected from the sequence of neurophysin ii, and the larger pe ...1978207339
effects of gentamicin on trypsin, chymotrypsin, and collagenase.the effects of gentamicin on three proteolytic enzymes were studied. gentamicin was tested at concentrations of 0.5-500 microgram/ml. trypsin was tested at 0.5 microgram/ml using p-tosyl-l-arginine methyl ester and at 0.1 and 0.5 microgram/ml using azocoll as the substrate. chymotrypsin was tested at 0.1 and 0.5 microgram/ml with azocoll. a soluble [14c]collagen assay was used to measure activity of collagenase derived from clostridium histolyticum. the profiles of proteolytic activity vs. genta ...1978210240
age changes in collagen characteristics of bone and skin of a short-lived species of reptile.decalcified bone collagen of older male garden lizards, calotes versicolor, was less susceptible to digestion by collagenase from clostridium histolyticum than that from younger individuals. in aged skin the percentage solubility and the soluble/insoluble collagen ratio decreased, with a concomitant rise in insoluble and total collagen. collagen/unit area increased with advancing age in both dorsal and ventral skin. these results from a non-mammalian poikilothermic vertebrate provide additional ...1978212937
preparation of crude clostridium histolyticum collagenase for therapeutic purposes.the production of a freeze-dried enzymatic preparation from the category of crude collagenases has been described. the method is based on the utilization of a highly proteolytic clostridium histolyticum strain whose products have more advantageous properties for therapeutic purposes than the products of the strain commonly used as yet.1978214494
[detection of collagenase in passive haemagglutination using collagen-coated erythrocytes (author's transl)].human rheumatoid arthritis (ra) collagenase and bacterial collagenase were shown to agglutinate collagen-coated erythrocytes. native collagens of type i and type ii reacted equally well, while denatured collagens showed less distinct agglutination activity. the sensitivity of the method for the detection of purified bacterial collagenase from clostridium histolyticum is very high (100 pg). it is, however, low for human ra collagenase. the agglutination reaction is not inhibited by concentrations ...1978213904
purification and characterization of a marine bacterial collagenase.a true collagenase was isolated from the culture fluid of a marine bacterium which has been designated vibrio b-30 (atcc 21250). collagenase production was obtained only in media containing collagen or certain degradation products of collagen. partial purification on deae-cellulose and sephadex g-200 columns produced active enzyme which was free of nonspecific proteases but which contained two collagenases. the two collagenases have the same apparent molecular size, and evidence is presented to ...1978210785
the interaction of collagenase with hydroxyapatite and related materials and enzymatic properties of the adsorbed enzyme.the commercial collagenase from clostridium histolyticum was adsorbed on hydroxyapatite, on bovine femur shaft and on enamel and dentin powders. the substrate specificity of the adsorbed enzyme tested, with chromophore substrate, azocoll and native collagen, differed from that obtained with the soluble enzyme. the adsorption and the substrate specificity was also dependent on the adsorbent used. a pretreatment of hydroxyapatite with chondroitin sulphate, hyaluronate and dna lowered the adsorptio ...1978208720
proteolytic activation of rat liver adenylate cyclase by a contaminant of crude collagenase from clostridium histolyticum.treatment of rat liver plasma membranes with various commercial preparations of crude collagenase from clostridium histolyticum at concentrations as low as 1 mug/ml, resulted in activation of the adenylate cyclase system. maximal activation occurred at 50 to 100 mug/ml of collagenase, and promoted a 2- to 3-fold increase in the basal activity as well as in the activities stimulated by catecholamines, glucagon, fluoride, or gtp. this was due to an increase in the maximal velocity of the cyclizing ...1977191449
the proteolytic action of bacterial proteases from clostridium histolyticum on bovine fibrinogen.bacterial proteases from clostridium histolyticum bring about the polymerization of fibrinogen into structured fibres, the period being 9 nm when the ionic strength (i) of the solution is between 0.1 and 0.2. at i=0.3 no polymerization occurs, but the successive actions of bacterial proteases and thrombin induce polymerization, the fibres obtained showing a periodic structure: the major period is 23 nm with two minor striations. the striation pattern is different from that obtained with fibrin ...1977193574
change in the stability of tendon collagen during ageing in the reptile, calotes versicolor. a brief note.age related changes in the digestibility of tendon collagen of the garden lizard, calotes versicolor, were traced using type i collagenase from clostridium histolyticum. collagen from older lizards showed less susceptibility for collagenase digestion than the younger lizards suggesting increased number of cross-linkages comparable with mammals.1976186668
collagenase enzymes from clostridium: characterization of individual enzymes.four collagenases have been purified to apparent homogeneity from extracts of clostridium histolyticum and partially characterized. the four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen. the enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase i = 5.50, collagenase ii = 5.65, and collagenases i ...1976184822
haemorrhagic and inflammatory properties of collagenase from c. histolyticum.collagenase from clostridium histolyticum induced haemorrhages when applied to the surface of dog lung; it exerted a similar effect on mouse lung when injected intrathoracically. injected into rat paws, bacterial collagenase induced haemorrhage and oedema. effects of collagenase were prevented by several procedures that inhibit collagenolytic activity (heating at various temperatures and incubation with metal-complexing agents such as edta, penicillamine and dithiothreitol). protein protease inh ...1976184702
chemical characterization and study of the autodigestion of pure collagenase from achromobacter iophagus.only one collagenase (ec 3.4.24.3) is produced by the non-pathogenic achromobacter iophagus strain. the chromatography of the crude enzyme on de-32 cellulose followed by gel filtration on sephadex g-100 in the presence of 1 m sodium chloride led to the isolation of a homogeneous enzyme. its specific activity (1.642 mukat/mg) represents the highest value ever obtained for a bacterial collagenase. the amino acid composition of a. iophagus collagenase differs from that of clostridium histolyticum m ...1976177067
an extracellular aminopeptidase from clostridium histolyticum.an aminopeptidase was isolated from the culture filtrate of clostridium histolyticum and purified to homogeneity. absence of endopeptidase activity in the purified preparation was demonstrated. gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component ...19764318
extracellular aminopeptidase from clostridium histolyticum. 197613266
consecutive use of omega-aminoalkylagaroses. resolution and purification of clostripain and collagenase from clostridium histolyticum. 1976178310
the effect of calcium chloride on the activity and inhibition of bacterial collagenase.the enzymatic behavior and inhibition patterns of collagenase of clostridium histolyticum in the presence of 0.5 m and 3.4 mm cacl2 have been examined viscosimetrically. the more concentrated salt was found to enhance the rate of digestion of calfskin collagen when either measured viscosimetrically or colorimetrically by trinitrobenzenesulfonate. however, the rate of digestion of calfskin gelatin is unaffected by 0.5 m cacl2 as determined colorimetrically. calcium chloride also proved to have a ...1976178615
cleavage of beta-casein by collagenases from achromobacter iophagus and clostridium histolyticum. 1976182537
responses of clostridium botulinum type b and e progenitor toxins to some clostridial sulfhydryl-dependent proteases.sulfhydryl-dependent proteases produced by clostridium botulinum types a, b, and f, clostridium histolyticum, clostridium sporogenes and clostridium perfringens activate preferentially type e over type b progenitor toxin but less efficiently than trypsin. the results explain why activable toxin is demonstrable in culture of a strongly proteolytic type b strain.19751104932
rabbit corneal damage produced by pseudomonas aeruginosa infection.gross, light microscopic, and electron microscopic examination of the rabbit corneal destruction produced by experimental pseudomonas aeruginosa infections revealed a combination of acute inflammation and liquefaction necrosis of the cornea. degeneration of the epithelial cells and the start of polymorphonuclear leukocyte infiltration of the cornea occurred initially. these changes were followed by loss of the epithelium, degeneration and loss of the keratocytes and endothelium, loss of the char ...1975169202
action of bacterial collagenase on ascaris cuticle collagen.the collagen from the cuticle of ascaris lumbricoides was digested by clostridium histolyticum collagenase [ec 3.4.24.3] in the presence and absence of cacl2. about 1.2 mumoles of amino groups per mg collagen was liberated when the digestion was performed in the presence of 5 mm cacl2, whereas about 0.5 mumole of amino groups per mg collagen was liberated by digestion in the absence of cacl2. in contrast, cacl2 influenced the extent of hydrolysis of rat tail tendon collagen only slightly. the re ...1975175053
the application of collagenase, purified by affintiy chromatography, to the isolation of insoluble elastin from bovine aorta.clostridium histolyticum collagenase (clostridiopeptidase a, ec 3.4.4.16), purified by affinity chromatography, was applied to the isolation of insoluble elastin from bovine aorta. the extremely low level of n-terminal residues (1.6 mol per 10(6) g of protein) present in this preparation indicated the almost complete lack of hydrolytic damage caused by the isolation procedure. the amino acid profile of the aortic elastin was found to be almost identical to that of insoluble elastin prepared from ...1975167862
collagenase production by achromobacter iophagus.achromobacter iophagus produced collagenase (ec 3.4.24.3) when cultured aerobically in buffer containing 5% peptone. the bacterium is non-pathogenic and tests on rabbits indicated that the culture medium was atoxic. the collagenase, which hydrolyzed insoluble and soluble native collagen, was purified by (nh4)2 so4 precipitation, starch block electrophoresis, and gel filtration. it was shown to be serologically distinct from clostridium histolyticum collagenase and to have molecular weight and se ...1975165833
application of affinity chromatography to the purification of collagenase for the isolation of insoluble elastin.clostridium histolyticum collagenase (clostridiopeptidase a, ec 3.4.4.19) was purified by batchwise separation with deae-cellulose followed by affinity chromatography on a column of alkali-treated elastin. the n-terminal amino acid profile of elastin isolated from bovine ligamentum nuchae using this enzyme preparation was compared with that of a duplicate sample purified with a mixture of collagenase i and ii (yoshida, e, and noda, h. (1965) biochim. biopsys. acta 105, 562-574). an approx. three ...1975164935
[composition of gases emitted by clostridium histolyticum cultivated on sodium thioglycolate media in a vaccuum, after alkalinisation of the medium at the end of culturing]. 19744216432
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