Inhibition of RNA polymerase from Bacillus thuringiensis and Escherichia coli by beta-exotoxin.

The characteristics of exotoxin inhibition of deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase isolated from Escherichia coli and Bacillus thuringiensis were investigated. RNA polymerase isolated from a variety of growth stages was partially purified and assayed using several different native and synthetic DNA templates, and exotoxin inhibition patterns were recorded for each. Although 8 to 20-h RNA polymerase extracts of E. coli retained normal sensitivity to exotoxin (50% inhibition at a concentration of 7.5 X 10(-6) M exotoxin), RNA polymerase isolated from late exponential and ensuing stationary-phase cultures of B. thuringiensis were nearly 50% less sensitive than exponential RNA polymerase activity. Inhibition patterns relating culture age at the time of RNA polymerase extraction to exotoxin inhibition suggested a direct correlation between diminishing exotoxin sensitivity and sporulation. Escherichia coli RNA polymerase could be made to mimic the B. thuringiensis exotoxin inhibition pattern by removal of sigma from the holoenzyme. After passage through phosphocellulose, exotoxin inhibition of the core polymerase was 30% less than the corresponding inhibition of E. coli holoenzyme. Heterologous enzyme reconstruction and assay were not possible due to loss of activity from the B. thuringiensis preparation during phosphocellulose chromatography, apparently from the removal of magnesium. In enzyme velocity studies, inhibition with exotoxin was noncompetitive with respect to the DNA template in the RNA polymerase reaction.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis. The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei

The effects of the exotoxin from Bacillus thuringiensis on DNA-dependent RNA polymerases from rat liver were examined. The exotoxin inhibits all RNA polymerase activity at both low and high ionic strength in intact nuclei, and soluble enzymes are similarly affected. This inhibition is relieved by ATP. Dephosphorylated exotoxin did not inhibit the soluble enzymes. Nucleolar and nucleoplasmic RNA polymerases respond to different concentration ranges of exotoxin, and the compound can be used in intact nuclei to isolate the nucleoplasmic activity.     

Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.     

Characterization of Competitive Inhibitors for the Transferase Activity of Pseudomonas aeruginosa Exotoxin A

A series of small, nonpolar compounds were tested for their ability to inhibit the ADP-ribosyl transferase activity of Pseudomonas aeruginosa exotoxin A. The IC 50 values for the compounds tested ranged from 87 nM to 484 μM for NAP and CMP12, respectively. It was demonstrated that NAP was a competitive inhibitor of the ADPRT reaction for the NAD + substrate with a K i of 45 ± 5 nM, which was in good agreement with the dissociation constant determined independently (K D =56 ± 6 nM). The IC 50 value for NAP was 87 ± 12 nM, which strongly correlated with the K i and K D values. Furthermore, NAP was shown to noncovalently associate with the exotoxin A active site using exhaustive dialysis, NMR, and electrospray mass spectrometry. Finally, a computer molecular model using the X-ray structure of the substrate-bound toxin was generated with NAP bound to the active site of exotoxin A at the nicotinamide-binding site. This model is consistent with the X-ray structure of the catalytic domain of poly-ADP-ribose polymerase complexed with 4-amino-naphthalimide (Compound 4) that was included in this study.     

A comparative study of mouse liver and mouse blastocyst DNA-dependent RNA polymerases.

DNA-dependent RNA polymerase has been studied in adult mouse liver and mouse blastocysts. The enzyme from mouse liver was resolved into three enzyme forms by DEAE-Sephadex chromatography. Two of the forms, IA and IB, are insensitive to α-amanitin, have low $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratios, and are optimally active at low ionic strength. Form II is inhibited by α-amanitin, has a higher $\text{Mn}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ activity ratio, and is most active at high ionic strength. An optimal reaction temperature of 37 ° C was found for all enzyme forms. All of the isolated enzyme forms are inhibited by the exotoxin from Bacillus thuringiensis and the inhibition can be partially reversed by increased ATP levels. Forms IA and IB are most active with native template while form II prefers denatured DNA.The blastocyst RNA polymerase activity exhibits similar requirements for divalent metal ions and ionic strength to the purified liver enzymes. The maximum inhibition of blastocyst RNA polymerase obtained with α-amanitin and exotoxin differs from that observed for purified liver enzymes but is similar to the inhibition of liver homogenate. However, the concentrations of inhibitor required for maximum inhibition by α-amanitin and exotoxin is different for the blastocyst and liver homogenate enzymes.     

Evaluation of the toxic effects of preventive and therapeutic preparations in transplantable cell cultures from different species and of different origins. I. Demonstration of the toxic properties of commercial batches of adsorbed DPT vaccine in cell cultures

Continuous cell cultures are sensitive test systems used not only for the determination of the toxicity of diphtheria exotoxin, but also for revealing the toxic properties of adsorbed DPT vaccine. Morphological changes induced by the action of diphtheria exotoxin and adsorbed DPT vaccine in the cultures of L929, HeLa, FL, L132 and monkey tonsil cells are similar in character. The effect produced by the diphtheria exotoxin and adsorbed DPT vaccine on cell cultures may be characterized as toxic, subtoxic and minimal. Nevertheless, even in the cultures treated with minimal concentrations the pathological state of the cells can be detected in the next 2 or 3 serial subcultures.     

The superantigenic activity of streptococcal pyrogenic exotoxin B is independent of the protease activity

The nature of the mitogenic activity of pyrogenic streptococcal exotoxin B, also known as streptococcal cysteine protease, has been debated in the literature. Streptococcal exotoxin B has been shown to cleave interleukin-1beta precursor and create biologically active interleukin-1beta, a major cytokine mediating inflammation and shock. This activity could mimic the mitogenicity and cytokine release induced by superantigens in lymphocyte stimulating experiments. In this study, the protease activity of streptococcal exotoxin B was irreversibly inhibited by covalent binding of a tripeptide and the superantigenic properties of streptococcal exotoxin B were found not to be influenced by this inactivation. Native as well as protease-inactivated streptococcal exotoxin B was shown to stimulate T-cell proliferation without a need of metabolically active antigen presenting cells. Furthermore, streptococcal exotoxin B-induced T-cell proliferation was shown to require HLA-DQ since addition of HLA-DQ monoclonal antibodies totally inhibited the mitogenic activity of streptococcal exotoxin B, indicating that streptococcal exotoxin B, as other superantigens, makes direct contact with the T-cell receptor via HLA class II. The aim of this study was to characterize the relationship between the proteolytic and superantigenic properties of streptococcal exotoxin B.     

DNA-dependent RNA polymerase from Spirochaeta aurantia

Abstract A DNA-dependent RNA polymerase was isolated from Spirochaeta aurantia . The M _r values of the holoenzyme subunits are 164000, 142000, 84000, and 44500. The RNA polymerase activity was sensitive to heparin, streptolydigin, and actinomycin D, while rifampicin and streptovaricin did not inhibit activity.     

Detection and characterization of DNA polymerase from Trypanosoma brucei.

The predominant DNA polymerase activity has been isolated from the parasitic flagellated protozoan, Trypanosoma brucei. Like mammalian DNA polymerase-alpha the trypanosome DNA polymerase is of large molecular weight (S, 6--8), is resistant to thermal denaturation, is sensitive to N-ethylmaleimide, and is inhibited by high ionic strength. However, specific antisera that cross-react with mammalian DNA polymerase-alpha from different species fail to cross-react with the trypanosome polymerase.     

The assessment of the toxigenicity of clinical strains of Pseudomonas aeruginosa by an immunoenzyme method using nitrocellulose filters

The enzyme immunoassay (EIA) on nitrocellulose filters was adapted for the detection of exotoxin A in 104 P. aeruginosa strains isolated from patients and the environment in surgical wards in hospitals of Moscow and Alma-Ata. The method was shown to be highly sensitive: it permitted the detection of 5.ng of P. aeruginosa exotoxin A. The screening of 104 P. aeruginosa clinical strains by means of EIA on nitrocellulose filters revealed that these strains exotoxin A in 88.5% of cases.     

An alpha-like DNA polymerase from Halobacterium halobium

Two DNA polymerases have been isolated from extracts of Halobacterium halobium, one having a sedimentation coefficient of 11 S, designated as alpha-like polymerase and possessing the following characteristics. It is sensitive to both aphidicolin and N-ethylmaleimide but indifferent to the presence of a dideoxynucleoside triphosphate. Therefore this polymerase is very similar to the alpha DNA polymerase of eukaryotes. The enzyme requires 5 M NaCl for maximum activity. The other polymerase has a sedimentation coefficient of 4.4 S and is resistant to both aphidicolin and N-ethylmaleimide. However, it is inhibited by a dideoxynucleoside triphosphate.     

DNA primase activity found in an alpha-like DNA polymerase obtained from Halobacterium halobium

An aphidicolin-sensitive DNA polymerase was purified from extracts of Halobacterium halobium. The analysis of this alpha-like DNA polymerase on polyacrylamide gels under denaturing conditions revealed two peptides with molecular masses of 70 kDa and 60 kDa in equal amounts. Like the DNA polymerase alpha isolated from eukaryotes, the alpha-like DNA polymerase possesses primase activity using UTP and polydeoxyadenylate as template. The primase activity was sensitive to aphidicolin and inhibited by an antiserum against the alpha-like DNA polymerase of H. halobium. The primase activity was dependent on the presence of high salt concentrations.     

Preliminary characterisation of inhibitors of DNA polymerase isolated from Xenopus laevis early embryos

Inhibitors of DNA polymerase have been detected in Xenopus laevis ovary and egg extracts. The characteristics of the inhibitors differ between the two extracts. In ovary preparations, the inhibitor is retained by dialysis tubing and is heat sensitive, whereas in egg extracts it is diffusable and heat stable. In both extracts, the activity co-elutes with DNA polymerase after ion exchange chromatography. Chromatography of ovary extracts renders the inhibitor diffusable and heat stable. Preliminary characterisation of inhibitory activity from eggs shows that the substance is sensitive to pronase digestion and has an approx. 300-500 molecular weight. Kinetic studies demonstrate that the inhibitor is uncompetitive with the DNA template and show mixed inhibitory kinetics with respect to the deoxynucleotides.     

Co-fractionation of an endonuclease activity during the purification of DNA polymerase-alpha from regenerating rat liver. Properties and separation from DNA polymerase.

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high-molecular-weight DNA polymerase-alpha from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co-fractionated with DNA polymerase in a great variety of purification procedures involving ion-exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA-cellulose. The endonuclease acted on native or denatured DNA by introducing single-strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.     

Elimination of residual RNase activity from purified preparations of RNA-directed DNA polymerase.

Preparations of RNA-directed DNA polymerase purified from RNA tumor viruses by standard methods generally contain trace amounts of single-stranded RNA endonucleolytic activity detectable only by relatively sensitive methods. This contaminating RNase activity has been found to be completely inhibited when RNA-directed DNA polymerase reactions are carried out in the presence of low concentrations of bentonite. Under these conditions, only minimal inhibition of the DNA polymerase and RNase H activities of the RNA-directed DNA polymerase was observed.     

Cytotoxic activity of a recombinant chimaeric protein between Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin

A segment of the exotoxin A gene of Pseudomonas aeruginosa, coding for the N-terminal end of domain I and domain II of the toxin (ETA), was genetically fused to the diphtheria toxin gene of Corynebacterium diphtheriae, coding for the N-terminal end of A fragment of diphtheria toxin (DT). The resulting hybrid protein (termed CED1) was produced in large amounts and exported to the periplasm in Escherichia coli. This chimaeric protein reacted with both anti-ETA and anti-DT antisera. Furthermore, the chimaeric protein displayed ADP-ribosylation activity and exhibited cytotoxicity to mouse 3T6 fibroblasts. These results demonstrated that the chimaeric protein is cytotoxic, and that the toxic potential of DTA can be selectively internalized and translocated via domains I and II of exotoxin A, which are thus sufficient to direct and translocate an enzymatically active heterologous polypeptide segment into the cytosol of sensitive cells.     

Effect of the exotoxin of Bacillus thuringiensis on normal and ecdysone-stimulated ribonucleic acid polymerase activity in intact nuclei from the fat body of Sarcophaga bullata larvae

A nuclear preparation from the fat-body of Sarcophaga bullata was obtained which incorporates nucleotides at a steady rate. Two activities, differing in their response to alpha-amanitin, are present. The activities are not separated by changes in the concentration of (NH(4))(2)SO(4) as effectively as in mammalian nuclei. The activity resistant to alpha-amanitin is stimulated by ecdysone, and both normal and the ecdysone-stimulated activities are inhibited by the exotoxin. The amanitin-sensitive enzyme is also inhibited by exotoxin, but higher concentrations are required.     

Mitochondrial DNA-directed RNA polymerase from Saccharomyces cerevisae mitochondria

A DNA-directed RNA polymerase activity has been detected in yeast mitochondrial extracts which is sensitive to rifampicin. This activity is distinct from that found in the nucleus and cytoplasm, and is possibly coded for, or under the control of the mitochondrial genome.     

Preliminary characterisation of inhibitors of DNA polymerase isolated from Xenopus laevis early embryos

Inhibitors of DNA polymerase have been detected in Xenopus laevis ovary and egg extracts. The characteristics of the inhibitors differ between the two extracts. In ovary preparations, the inhibitor is retained by dialysis tubing and is heat sensitive, whereas in egg extracts it is diffusable and heat stable. In both extracts, the activity co-elutes with DNA polymerase after ion exchange chromatography. Chromatography of ovary extracts renders the inhibitor diffusable and heat stable. Preliminary characterisation of inhibitory activity from eggs shows that the substance is sensitive to pronase digestion and has an approx. 300–500 molecular weight. Kinetic studies demonstrate that the inhibitor is uncompetitive with the DNA template and show mixed inhibitory kinetics with respect to the deoxynucleotides.