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 (clostridiopeptidase A, EC 3.4.24.3) was noted. This collagenase inhibitor may be a factor in the regulation of extracellular connective tissue catabolism.     

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D: -Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.     

Vacuolization of HeLa cells by a partially purified Clostridium histolyticum cytotoxin

Clostridium histolyticum vacuolating cytotoxin was partially purified from culture broth using ammonium sulfate precipitation, gel filtration and hydrophobic interaction chromatography. The toxin caused vacuolization of HeLa cells visible under a light microscope after 2 h and distinct after 8 h. Transmission electron microscopy revealed the presence of numerous vacuoles, condensation of the mitochondrial matrix, increased cytoplasm density and increased amounts of heterochromatin. Apoptosis was not detected either by electron microscopy or by an apoptosis/necrosis discrimination assay with fluorescein-labeled annexin V and propidium iodide, or DNA fragmentation assay. Calcium ion influx was detected by flow cytometry after labeling cells with Fluo-4 AM. Vacuolation of HeLa cells by C. histolyticum cytotoxin was inhibited by bafilomycin A1, suggesting involvement of H+ -ATPase in the formation of vacuoles.     

New Culture Conditions for Clostridium histolyticum leading to Production of Collagenase of High Specific Activity

S ummary : New culture conditions are described for the growth of Clostridium histolyticum allowing for the production of collagenase (E.C.3.4.4.19) in a highly purified form, free of general protease activity. The conditions include the use of a mixture of sodium bisulphite-oxidized sodium thioglycollate to provide the reducing environment within an otherwise standard culture medium. Also, new modifications in the preparation of collagenase A are reported.     

Characterization of a Human Serum Inhibitor of Clostridium histolyticum Proteinase(s)

Normal animal sera inhibit at least one Clostridium histolyticum proteinase. An assay procedure based on immune hemolysis was developed for the estimation of this inhibition. This inhibitory activity occurs in various levels in the sera of different animal species. The highest titers have been obtained with rat sera. The inhibitory activity from human serum was isolated and purified 16- to 27-fold by Sephadex G-200 gel filtration and diethylaminoethyl cellulose or hydroxylapatite chromatography. The properties of the human serum inhibitor of the clostridial proteinase were compared with a trypsin inhibiting factor found in the partially purified preparations. Identical behavior of the two inhibitory factors was observed when measured by heat inactivation, beta-mercaptoethanol sensitivity, pH stability, and sucrose gradient centrifugation. The inhibitory factor has an approximate sedimentation coefficient (S(20,w)) of 17. Goat anti-alpha-2-macroglobulin specifically precipitated the clostridial proteinase inhibitor from a partially purified preparation.     

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 sedimentation coefficient values of approx. 112 000 and 5.3 S, respectively.     

A novel aminopeptidase from Clostridium histolyticum

An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (K_m and V_max) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn⁺⁺ or Co⁺⁺ is essential for its activity. Only slight activation was observed with Ni⁺⁺ and Cd⁺⁺, while Zn⁺⁺ and Cu⁺⁺ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS).The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase^1–5. In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro)_n, (Pro-Gly-Pro)_n-OMe, α,DNP-(Pro-Gly-Pro)_n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro)_n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.     

嗜杀酿酒酵母毒素蛋白及其杀伤质粒的研究

Killer toxin from \%Saccharomyces cerevisiae\% SK was isolated by ultrafiltration of culture supernatants and purified by poly(ethylene glycol). The toxin migrates as one single protein band on SDS-PAGE and its molecular weight is 15kD. The SK toxin has the greatest lethal effect on the sensitive yeast strain in the lat lag phase. Extraction and purification of killer heretity factor(dsRNA) from SK found that M dsRNA plasmid and L dsRNA plasmid have different molecular lengths being 1.7kb and 4.0kb.     

Investigation of common and specific components of Cl. septicum and Cl. histolyticum toxins

Interrelations between the common and specific components in the toxins of several strains of Cl. septicum and Cl. histolyticum were investigated. The method of tissue culture, which yields more stable results than biological tests on animals, was used. It has been demonstrated that native toxins of Cl. seticum (7 strains) and Cl. histolyticum (7 strains) cause cytotoxic changes in chick embryo fibroblasts. These changes are similar to each other and identical with changes occurring under the effect of concentrated toxins of the mentioned microorganisms. Cross reactions of neutralization with antitoxic and species-specific sera against Cl. septicum and Cl. histolyticum have shown that the strains of Cl. septicum and Cl. histolyticum synthesize toxins with components possessing common antigenic properties. The strains of Cl. histolyticum synthesize a greater amount of components common with Cl. septicum than the strains of Cl. septicum in which the amount of heterologous antigens varies.     

High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

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 latter lies within the region cleaved by the eukaryotic collagenases.     

Complement-inactivating Proteinase(s) from Clostridium histolyticum

A proteinase fraction inhibiting the hemolytic activity of guinea pig complement was obtained from supernatant fluids of Clostridium histolyticum cultures and purified 150- to 350-fold by ammonium sulfate precipitation, Sephadex G-75 gel filtration, and diethylaminoethyl cellulose chromatography. An assay was developed based on the inactivation of hemolytic complement. Partially purified anticomplementary preparations were active against casein and were capable of "solubilizing" Escherichia coli endotoxin. Two components were found by differential heat inactivation, with complement and casein as substrates, but only one of these components was active against endotoxin. The more heat-stable activity, showing 50% inactivation at about 47 C, was characterized as to pH and ionic strength optima and sensitivity to reagents such as cysteine, beta-mercaptoethanol, ethylenediaminetetraacetate, and heavy metals.     

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-fold decrease in the molar concentration of N-terminal residues and a considerable reduction in their number was obtained by using the former enzyme preparation.     

Cloning of a novel collagenase gene from the gram-negative bacterium Grimontia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis

The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed < 20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ~ 60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ~ 4-fold greater activity than that of C. histolyticum collagenase.     

Partial Purification of a Species-Specific Lethal Factor from Amoeba indica1

The species-specific lethal factor obtained from Amoeba indica, a freeliving mononucleate amoeba, can be partially purified by differential centrifugation, gel exclusion chromatography, and elution from Cibacron blue-agarose. The factor is unstable in the partially purified form, but activity is correlated with a heat-labile protein having an apparent molecular weight of 186,000. The factor causes inhibition of division and death when injected into A. proteus. Purification of the lethal factor indicates that aggregation of the factor with amoebal proteins is not a hindrance to further investigation. The natural role of the factor is discussed.     

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.     

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 bovine ligamentum nuchae by the same method.     

In vitro strategies for selection of eye-spot resistant sugarcane lines using toxins of Helminthosporium sacchari

In vitro strategies were applied for the selection of eye-spot resistant variants from susceptible sugarcane cultivar Co 419 Different selective units (callus and leaf) of the susceptible cultivar were subjected to sub-lethal to lethal doses of toxins (culture filtrate and partially purified toxin) of H. sacchari, with the objective of improving the efficacy of in vitro selection protocols. All the selective units gave more or less similar response with culture filtrate, but a distinct response was observed when leaf was subjected to partially purified toxin treatment. The response was characterised by the degree of resistance exhibited by the regenerated seedlings.