Inactivation of dopamine β-monooxygenase by hydrogen peroxide and by ascorbate

The inactivation of the water-soluble form of bovine adrenal dopamine β-monooxygenase by H₂O₂ and by ascorbate was studied. Inactivation by H₂O₂ was slow for the copper-free apoenzyme, but addition of copper gave a rapid inactivation. The results presented indicate that the enzyme-bound copper during this inactivation catalyzes partial destruction of its own binding site. The reaction orders for the inactivation by H₂O₂ seem to be 1.0 with respect to the enzyme and in the range 0.6 to 0.8 with respect to H₂O₂. The rate of inactivation obtained in the presence of ascorbate increases with addition of copper and is faster than that obtained by similar concentrations of H₂O₂. The data could not, however, be used to decide whether the inactivation by ascorbate was catalyzed by the enzymebound copper. The inactivation reaction in the presence of ascorbate seems to be of first order with respect to ascorbate at ascorbate concentrations less than 40 μm and decreases toward zero as the ascorbate concentration is increased. Experiments with the Cu(I)-chelator, bathocuproine disulfonate, revealed that inactivation led to weaker binding of copper to the protein, and this effect was more pronounced with H₂O₂ than with ascorbate.     

Photodynamic Inactivation of Antigenic Determinants of Single-Stranded DNA Bacteriophage φX174

Bacteriophage phiX174 when photodynamically inactivated (i.e., when rendered unable to produce plaques as a result of exposure to visible light in air in the presence of proflavine) progressively lost their capacity to bind efficiently with homologous antiserum. Such loss of serum-blocking power was evident with heat-inactivated but not with UV-irradiated phage. The ability of the phages to adsorb to host cells, however, remained practically unaltered even after photodynamic inactivation. It thus appears that photodynamic damages in the so-called "jacket" component of the phiX174 coat proteins are partly responsible for the loss of plaque-forming ability, whereas the "spikes" are either poor antigens or insensitive to photodynamic treatment.     

Reactivation of Photoinactivated Single-Stranded DNA Bacteriophage φX174 by UV-Irradiated Escherichia coli Cells

Reactivation of single-stranded DNA phage, photodynamically inactivated in the presence of proflavine sulfate, by three isogenic Escherichia coli strains having different DNA repair capabilities has been studied. It was found that reactivation of photoinactivated phiX174 was possible only if the host cells were recombination proficient (recA(+)) and had been lightly irradiated with UV light prior to infection; the presence of the uvrA(+) gene was not essential. Only a small part of the proflavine-mediated photodynamic damage in phiX174 could be repaired in this fashion. Burst sizes of reactivated phages were, however, comparable to those of normal unirradiated phages.     

Inactivation of Bacteriophages by ε-Poly-l-lysine Produced by Streptomyces

Degradation of a β-O-4lignin substructure model dimer by a white rot fungus, Phanerochaete chrysosporium, was investigated using a culture containing H₂¹⁸O, and the following conclusions were made. a) The direct hydrolysis at C_β of the β-O-4 dimer was not involved in formation of arylglycerol. b) About half of the oxygen at the benzyl (C_α) position of the glycerol was derived from H₂O (H₂¹⁸O) and the other half was from the oxygen at the benzyl (C_α) position of the substrate β-O-4 dimer. c) But, the oxygen at the C_α position of the substrate β-O-4 dimer did not migrate to the C_α position of the aryglycerol.     

Inactivation and structural alteration of α-amylase by low-pressure carbon dioxide microbubbles

The efficiency of low-pressure carbon dioxide microbubbles (CO₂MB) to inactivate α-amylase was analysed kinetically, and structural alteration of α-amylase by CO₂MB was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence analysis of tryptophan (Trp) residues. Activity and Trp fluorescence intensity of α-amylase treated by CO₂MB decreased with increasing temperature, pressure and exposure time, and lowering the initial buffer pH, respectively. In the kinetic analysis, it was confirmed that the decreased temperature-dependency and increased activation energy associated with the inactivation of α-amylase by CO₂MB were induced by pressurizing the mixing vessel and that the decreased pressure-dependency and increased activation volume concomitant to the inactivation of α-amylase by CO₂MB was induced by increasing the temperature in the heating coil. In SDS-PAGE, CO₂MB was suggested to induce the structural alteration of α-amylase because the band density decreased after CO₂MB treatment, although this phenomenon was not related to the inactivation efficiency. However, Trp fluorescence analysis showed that the alteration of the tertiary structure of α-amylase by CO₂MB was related to the inactivation efficiency. Therefore, CO₂MB was more effective than thermal treatment in inactivating α-amylase, and the inactivation efficiency was suggested to be related to the alteration of the enzyme’s tertiary structure.     

Inactivation of red beet β-glucan synthase by native and oxidized phenolic compounds

The effects of phenolic compounds on glucan synthase, a membrane-bound enzyme from red beet root, were examined. Different classes of phenolic compounds were screened in the absence and presence of polyphenoloxidase (PPO). At levels less than 1 mM, inactivation occurred with many of the compounds tested. However, in most cases oxidation by PPO was required. Coumarin, previously demonstrated to interfere with cell wall polysaccharide biosynthesis, was not inhibitory. Mechanistic studies utilizing catechol showed that phenolic inactivation could be protected against by PPO inhibitors and thiol protective reagents. However, once inactivation occurred, it could not be reversed. Ommission of thiols and polyvinylpyrolidone from homogenization buffers did not reduce glucan synthase levels of microsomal preparations. It appears that glucan synthase, a membrane-bound enzyme, is as susceptible to phenolic effects as cytosolic enzymes and in situ inactivation is a function of the availability of both endogenous phenolics and PPO.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

Inhibition by α-Amanitin of Adenovirus 12 Replication in Human Embryo Kidney Cells and of Adenovirus Transformation of Hamster Cells

ADENOVIRUS infection of human embryonic kidney (HEK) cultures seems to induce cellular RNA synthesis, which is preceded by a transient increase in the activities of the Mg²⁺-activated and Mn²⁺-(NH₄)₂SO₄-activated DNA dependent RNA polymerases and in the rate of histone acetylation¹. The two polymerase reactions, assayed in isolated cell nuclei, apparently reflect the activities of distinct nucleolar and nucleo-plasmic RNA polymerases^2,3. We were therefore prompted to test the effect of a specific inhibitor of the mammalian DNA-dependent RNA polymerase function, α-amanitin, on the multiplication of adenovirus. α-Amanitin is a bicyclic octapeptide isolated from the poisonous mushroom Amanita phalloides⁴ and which blocks RNA synthesis in intact animals^5,6. Nuclei isolated from the livers of such animals show a reduced activity of the RNA polymerases associated with nucleoplasm^5,6 and the nucleolus⁶.     

Inactivation of rat ovarian 20α-hydroxysteroid dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20α-hydroxysteroid dehydrogenase at pH 7.7, 25 °C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the K_d values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min^?1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20α-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a pK_a near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme.     

Inactivation of Bacteriophage ϕX174 by d-Fructose 6-Phosphate

The inactivation of bacteriophage ?X174 by d-fructose 6-phosphate was investigated. This inactivation was inhibited by EDTA or reducing agents, and stimulated by Cu²⁺ but other metal ions could not be substituted for Cu²⁺. The reaction was also inhibited by superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and various free radical scavengers.

No detectable changes were observed in adsorption capacity of phage and in the conformation of the virion. The viral DNA in the virion was, however, found to be cleaved. This strand scission was also enhanced by Cu²⁺ and protected by catalase. Similar results were obtained when ?X174 DNA was directly treated with d-fructose 6-phosphate.

It is concluded that the inactivation of ?X174 is due to DNA strand scission in the virion by the free radical of d-fructose 6-phosphate or oxygen radicals generated during autoxidation of d-fructose 6-phosphate.     

Inactivation of rat brain acetylcholinesterase by pyridoxal 5′-phosphate

Effects of pyridoxal 5′-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5′-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5′-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5′-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5′-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5′-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5′-phosphate could be reversed upon exhaustive dialysis of the pyridoxan 5′-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5′-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.     

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O₂-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.     

Mode of Host Cell Penetration by Bacteriophage φX174

Bacteriophage phiX174 is an icosahedral phage which attaches to host cells without the aid of a complex tail assembly. When phiX174 was mixed with cell walls isolated from the bacterial host, the virions attached to the wall fragments and the phage deoxyribonucleic acid (DNA) was released. Attachment was prevented if the cell walls were treated with chloroform. Release of phage DNA, but not viral attachment, was prevented if the cell walls were incubated with lysozyme or if the virions were inactivated with formaldehyde. Treatment of the cell walls with lysozyme released structures which were of uniform size (6.5 by 25 nm). These structures attached phiX174 at the tip of one of its 12 vertices, but the viral DNA was not released. The virions attached to these structures were oriented with their fivefold axis of symmetry normal to the long axis of the structure. No virions were attached to these structures by more than one vertex. Freeze-etch preparations of phiX174 adsorbed to intact bacteria showed that the virions were submerged to one half their diameter into the host cell wall, and the fivefold axis of symmetry was normal to the cell surface. A second cell could not be attached to the outwardly facing vertex of the adsorbed phage and thus the phage could not cross-link two cells. When the virions were labeled with (3)H-leucine, purified, and adsorbed to Escherichia coli cells, about 15% of the radioactivity was recovered as low-molecular-weight material from spheroplasts formed by lysozyme-ethylenediaminetetraacetic acid. Other experiments revealed that about 7% of the total parental virus protein label could be recovered in newly formed progeny virus.     

The Inactivation of Arx in Pancreatic α-Cells Triggers Their Neogenesis and Conversion into Functional β-Like Cells

Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing β-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in α-cells is sufficient to promote the conversion of adult α-cells into β-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon⁺ cells thereby generated being subsequently converted into β-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of α-cell-mediated β-like cell neogenesis. Importantly, the loss of Arx in α-cells is sufficient to regenerate a functional β-cell mass and thereby reverse diabetes following toxin-induced β-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.     

Isolation and Properties of Bacillus subtilis Strains Lysogenized by a Clear Plaque Mutant of Bacteriophage φ105

A clear plaque mutant of the temperate Bacillus phage phi105 lysogenized a small fraction of infected cells forming an integrated prophage at or near the normal phi105 insertion site. These lysogens exhibited a spontaneous induction rate approximately 1,000-fold lower than wild type and were noninducible (ind(-)) by mitomycin C. Prophage was induced, however, when competent cultures were incubated with transforming DNA. The ind(-) phenotype could not be attributed solely to the clear plaque mutation and appears to involve a cell-specific factor. Lysogenization by the clear plaque mutant, in contrast to wild-type phage, did not cause a marked reduction in transformation efficiency.     

In vitro Antioxidant and Cytotoxic Activities of Extracts of Endemic Tanacetum erzincanense Together with Phenolic Content by LC-ESI-QTOF-MS

In this study, phenolic composition, and in vitro biological activities of ethyl acetate (EAE) and methanol (ME) extracts obtained from the aerial parts of endemic Tanacetum erzincanense were investigated. Total phenolic and flavonoid content of the extracts were determined by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. Antioxidant capacity of the extracts was evaluated over radical scavenging (DPPH and ABTS) and metal ion reducing power (FRAP and CUPRAC) tests. Individual phenolic compounds in ME was analyzed by high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS). Cell inhibitory potential of the extracts was tested against colorectal adenocarcinoma (HT-29), breast adenocarcinoma (MCF-7), and hepatocarcinoma (HepG2) cells by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. The results showed that ME contains higher TPC (64.4 mg GAE/g) and TFC (62.2 mg QE/g) than those of EAE (41.5 mg GAE/g and 40.0 mg QE/g). LC-ESI-QTOF/MS analysis revealed that ME is rich in phenolic compounds, namely, chlorogenic acid, apigenin, quercetin, luteolin, and diosmetin. Antioxidant assay results indicated that ME possess stronger activity than EAE and a power that competes with synthetic antioxidants. XTT assay results demonstrated that although both extracts displayed a considerable cytotoxicity against the tested cancer cell lines in a time and dose-dependent manner, ME expressed its selective inhibitory action towards MCF-7 cells with an IC₅₀ value of 20.4 μg/mL for 72 h. These results may serve as a basis for further in vivo studies to examine the potential applications of T. erzincanense in food and pharmaceutical industries.