Mycobacterium leprae and phenoloxidase activity.

Our earlier studies indicated that the enzyme o-diphenoloxidase was absent in Mycobacterium leprae separated from depromatous human tissues. At that time the bacilli were not available from any other source. The existence or absence of this enzyme in M. leprae recovered from infected armadillo tissues were reinvestigated. The intact cells which were metabolically active, failed to oxidize DOPA. Likewise, DOPA and its derivatives were not oxidized by the enzymatically active cell-free preparations from M. leprae. Upon incubation of DOPA for more than 2 h with whole cell suspensions or particulate fractions, there was no development of colour with an absorption maximum of 540 nm as has been reported for an intermediate of DOPA oxidation. However, DOPA and several phenolic compounds were very actively oxidized by mushroom tyrosinase. The results suggested that M. leprae is deficient in o-diphenoloxidase, and this enzyme is not an intrinsic characteristic of this mycobacterium.     

Uptake of radioactive DOPA by Mycobacterium leprae in vitro.

Our previous studies demonstrated that Mycobacterium leprae contains a characteristic o-diphenoloxidase which converts a variety of phenolic compounds to quinones in vitro. This enzyme was not present in any other mycobacteria tested. The results reported here deal with the uptake and binding of radioactive DOPA by M. leprae. The leprosy bacilli incubated with tritium-labelled DOPA, readily took up the substrate. The binding of DOPA by the bacilli was markedly inhibited by diethyldithiocarbamate. The organisms also bound tritiated norepinephrine. Mycobacterium phlei which does not oxidize phenolic substrates failed to bind DOPA. Cultures of melanocytes which contain o-diphenoloxidase took up tritiated DOPA. Catecholamine metabolism is known to be important in myocardial cells. Cultures of turtle-heart cells did not oxidize DOPA to quinone; however, these cells bound the labelled substrate. A cell line of fibroblasts derived from armadillo skin neither oxidized nor took up DOPA. The results indicate that, like melanocytes and turtle-heart cells, M. leprae probably possesses specific receptor sites for the binding and subsequent metabolism of phenolic substrates.     

Oxidation of Phenolic Compounds by Mycobacterium leprae and Inhibition of Phenolase by Substrate Analogues and Copper Chelators

Experiments were conducted on the substrate specificity of phenoloxidase in Mycobacterium leprae, by using various phenolic compounds. Comparative studies were carried out with the enzyme from mammalian and plant sources. The phenolase of M. leprae was found to be similar to the enzyme of plant origin in oxidizing a variety of substrates; it was different from the mammalian enzyme, which has a limited substrate specificity. The findings confirmed that phenoloxidase is a specific property of M. leprae and is not a result of adsorption of host-tissue enzymes. The method used in separation of bacilli from infected tissues was evaluated for its effect on the viability of the organisms. This was tested by using M. lepraemurium as a model. The preparative procedure was found to have no adverse effect on the ability of the organisms to multiply in the mouse foot-pad. Several inhibitors of phenoloxidase have been tested-both substrate analogues and compounds which bind copper in the enzyme. Substances binding copper were found to be more effective. Since phenolase has been found to be a characteristic metabolic activity in M. leprae, nontoxic inhibitors of the enzyme offer possibilities of developing a rational chemotherapy of leprosy.     

Effect of Inhibitors on Phenoloxidase of Mycobacterium leprae

Previous results had shown that the human leprosy bacilli possess a phenoloxidase, which, when compared with the enzyme from mammalian and plant sources, seemed unique in the range of substrates utilized and in the nature of the products formed. The effect of several inhibitors on the enzyme in Mycobacterium leprae was tested. Compounds which bind copper were found to be more effective than substrate analogues. Diethyldithiocarbamate penetrated the bacillus and completely suppressed its phenolase activity. Diasone (a derivative of diaminodiphenylsulfone used in the treatment of leprosy) proved to be a potent inhibitor of phenoloxidase of mammalian and plant origin. However, it was less efficient in the case of M. leprae. A biochemical peculiarity of M. leprae was observed in its ability to metabolize mimosine and penicillamine. These compounds produced total inhibition of tyrosinase in melanoma extract and of mushroom tyrosinase. Nontoxic inhibitors of phenoloxidase in the leprosy bacilli may be of value in developing a rational approach to chemotherapy of the disease.     

Nonphysiological redox-agents are reduced at the binding center of NADP(H) glutathione reductase]

Studies of the acceptor reductase reaction of yeast glutathione reductase (EC 1.6.4.2) revealed that the competitive inhibitors for NADPH, 2',5'-ADP and Br- decrease the rate constants for the enzyme oxidation by ferricyanide, phenanthrene quinone, and juglone. A similar effect is observed when NADH which does not bind to the reduced enzyme is used as substrate. These observations support the hypothesis that non-physiological redox agents are reduced at the NADP(H)-binding center of glutathione reductase and that NADP(H) binding stimulates the reaction by displacing tyrosine-197 which protects FAD from the solvent.     

Temperature-induced changes in viability, diphenoloxidase and permeability of Mycobacterium leprae

Among mycobacteria secretion of the enzyme diphenoloxidase has been established as a property of Mycobacterium leprae. The antileprosy drug dapsone (DDS), which completely inhibits the enzyme from plant and mammalian sources, does not readily penetrate intact M. leprae. When the drug is complexed with polylysine, it easily permeates the bacteria and produces 100% inhibition of its diphenoloxidase, suggesting a permeability barrier of the cytoplasmic membrane of M. leprae to dapsone. In this study: (1) when the organisms, purified from fresh tissues of experimentally infected armadillos, were treated with dilute alkali or exposed to warmer temperatures, DDS penetrated the bacteria and inhibited the diphenoloxidase. Washing with trypsin had no effect. Dapsone easily permeated the bacilli, purified from tissues stored at 0 degrees C or at -80 degrees C. (2) Diphenoloxidase of freshly-prepared M. leprae was stimulated when the bacteria were exposed to 50 degrees C for 10 min; at 60 degrees C the activity decreased, and at 100 degrees C the enzyme was completely inactivated. When the enzyme was assayed at temperatures below 37 degrees C, the activity was considerably lower, indicating that M. leprae may not be a psychrophilic organism in this respect. (3) The bacteria exposed to 50 degrees C failed to multiply in mouse footpads. M. leprae remained viable in tissues stored at 0 degrees C or -80 degrees C; but when the bacteria purified from these tissues were frozen, they lost their viability. On the other hand, the organisms separated from fresh tissues remained viable when frozen at -80 degrees C. The inhibition of diphenoloxidase of M. leprae by dapsone could serve as an indirect method to assess the integrity of the bacterial cell membrane and to predict whether the bacteria would retain their viability on freezing.     

alpha,beta-Dehydro-3,4-dihydroxyphenylalanine derivatives: potential schlerotization intermediates in natural composite materials.

Proteins containing the post-translationally modified amino acid L-3,4-dihydroxyphenylalanine (DOPA) undergo autosclerotization as a means of assuring cohesive resilience in many structural matrices found in nature. To explore the chemical mechanism of sclerotization, we examined the oxidation products of relatively simple analogs of a peptidyl DOPA residue, such as N-acetylDOPA ethyl ester and N-acetyldopamide, together with those of several oligopeptides. Oxidation, induced by either of two catecholoxidases or by sodium periodate, resulted in the Lewis base catalyzed formation of derivatives of the unusual amino acid 3,4-dihydroxy-alpha,beta-dehydroDOPA (delta DOPA). The N-acetyl delta DOPA ethyl ester representative of this group of derivatives was characterized by NMR and uv spectroscopy. A variety of peptides developed analogous uv spectra upon oxidation. A similar reaction was observed upon oxidation of 3,4-dihydroxyphenylpropanoic (dihydrocaffeic) acid, but not after oxidation of N-acetyldopamine. Evidence is presented that this conversion is the result of a rearrangement of the DOPA quinone moiety to its delta DOPA tautomer, and that this tautomerization can be a dominant fate for peptidyl DOPA quinone, provided a Lewis base catalyst is available and competing reactions are minimized. Formation of delta DOPA in natural or synthetic polymers would increase the variety of crosslinks available to sclerotizing matrices. delta DOPA has been found in naturally occurring oligopeptides isolated by other workers from several marine species.     

Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Effect of 3,4-dihydroxyphenylalanine on Cu2+-induced Inactivation of HDL-associated Paraoxonase1 and Oxidation of HDL; Inactivation of Paraoxonase1 Activity Independent of HDL Lipid Oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Mechanism of Inactivation of Bacteriophage J1 by Glutathione

Mechanism of inactivation of a double-stranded DNA phage, phage Jl of Lactobacillus casei, by reduced form of glutathione (GSH) was studied.

Air (oxygen) bubbling, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by GSH. Partial oxidation of GSH resulted in a more rapid rate of inactivation. In contrast, nitrogen bubbling, reducing agents, chelating agents and radical scavengers prevented the inactivation. Fully oxidized GSH had no phagocidal effect. These results indicate that the inactivating effect of GSH requires the presence of molecular oxygen and is caused by free radical involved in the mechanism of GSH oxidation.

The target of GSH in the phage particle was not the tail protein but DNA. GSH reacted with phage DNA and caused single-strand scissions in the DNA, as exhibited by alkaline sucrose gradient centrifugation; thus inactivating phage.     

Tyrosinase-catalyzed unusual oxidative dimerization of 1,2-dehydro-N-acetyldopamine

Tyrosinase, which usually catalyzes the conversion of o-diphenols to o-benzoquinones, catalyzed an unusual oxidative dimerization of 1,2-dehydro-N-acetyl-dopamine to a benzodioxan derivative. The identity of the product was confirmed by UV, IR spectra, and NMR studies. During the oxidation, generation of a transient reactive intermediate could be witnessed by its characteristic visible absorption spectrum. Typical phenoloxidase inhibitors such as phenylthiourea, potassium cyanide, sodium azide, and sodium fluoride drastically inhibited the above reaction. Mimosine, a known competitive inhibitor of o-diphenoloxidase activity, also inhibited the new reaction competitively, suggesting that both the observed oxidative dimerization and the conventional quinone production are catalyzed by the same active site copper of tyrosinase. Based on our earlier findings (Sugumaran, M., and Lipke, H. (1983) FEBS Lett. 155, 65-68; Sugumaran, M. (1986) Biochemistry 25, 4489-4492) that phenoloxidases can produce quinone methides from certain 4-alkylcatechols, possible mechanisms for this new reaction are presented.     

Inhibition of glucose transport by benzoquinone and the addition product of benzoquinone and dithiothreitol

Hydroxylated benzene derivatives inhibited transport of d-glucose into calf-thymocyte plasma-membrane vesicles. The relative effectiveness of these was pyrogallol >hydroquinone catechol phloroglucinol. The most thoroughly studied of these agents, hydroquinone, produced weak, immediate inhibition when first added to membranes (K_i > 10 mM). This was followed by a gradual, time-dependent inhibition of the residual transport activity. The instantaneous inhibition could not be prevented by any agent tested, whereas the time-dependent phase was affected by reducing agents and superoxide dismutase. Several reducing agents (dithiothreitol, glutathione, NADH, ascorbate, bisulfite but not cysteine) prevented, while superoxide dismutase and cysteine potentiated time-dependent inhibition when added to the membrane suspension simultaneously with hydroquinone. NADH and ascorbate also prevented, whereas dithiothreitol potentiated, further time-dependent inhibition when added to membranes 2 h after hydroquinone. In contrast, all three reducing agents arrested time-dependent inhibition when added 2 h after pyrogallol. Numerous agents had no effect on time-dependent hydroquinone inhibition: oxidants (H₂O₂), metal chelators (EDTA, bathophenanthroline disulfonate, Desferral), radical scavengers (benzoate, ethanol), anti-oxidants (butylated hydroxytoluene) and catalase. Benzoquinone, an oxidation product of hydroquinone, was a much more potent inhibitor (K_i 1 mM) than hydroquinone. Several reducing agents (ascorbate, NADH, bisulfite) prevented this effect, while cysteine and dithiothreitol potentiated it. Below 300 μM, benzoquinone had little or no effect on sugar transport with or without glutathione or cysteine. Addition of dithiothreitol to benzoquinone (10–300 μM) resulted in potent inhibition of sugar transport (K_i 50 μM). Maximal inhibition occurred with a 1 : 1 mol ratio of these agents or with excess dithiothreitol. The inhibitory agent from benzoquinone and dithiothreitol lost potency in the presence of air and membranes, but was stable for hours in the presence of either of these alone. dl-threo-1,4-bis(2,5-dihydroxyphenylthio)-2,3-butanediol was obtained from the reaction of equimolar quantities of dithiothreitol and benzoquinone in ethanol. The structure of this adduct was established by spectroscopic and chemical methods. This compound exhibited all of the properties of the inhibitor which had been formed from benzoquinone and dithiothreitol in aqueous solution.     

Free and peptide-bound DOPA can inhibit initiation of low density lipoprotein oxidation

Hydroxyl radicals have been shown to convert free tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) which has reducing properties. During protein or peptide oxidation such reducing species are also formed from tyrosine residues. Free DOPA or peptide-bound DOPA (PB-DOPA) is able to promote radical-generating events, facilitating the damage of biomolecules such as nucleic acids. Radical induced lipid oxidation in low density lipoprotein (LDL) transforms the lipoprotein into an atherogenic particle. As PB-DOPA has been found in atherosclerotic plaques, we tested the ability of free and PB-DOPA to influence LDL oxidation. Free DOPA, in contrast to tyrosine had strong inhibitory action on both, the copper-ion initiated and metal ion independent (AAPH-induced) lipid oxidation. Free DOPA also inhibited LDL oxidation induced by the copper transport protein ceruloplasmin. To test if PB-DOPA was also able to inhibit LDL oxidation, DOPA residues were generated enzymatically in the model peptides insulin and tyr-tyr-tyr, respectively. PB-DOPA formation substantially increased the ability of both molecules to inhibit LDL oxidation by copper or AAPH. We hypothesize that DOPA-peptides and -proteins may have the potential to act as efficacious antioxidants in the atherosclerotic plaque.     

Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase

This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (.NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and.NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg(2+), and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by.NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to.NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions.     

Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level

The effect of treatment with benzothiadiazole (BTH) or l -2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.     

Chemoattractant efficacy: oxidation of stimulus by responding cells

Although all human neutrophils have receptors for formyl-methionyl-leucyl-phenylalanine, only 20-30% migrate in vitro at optimal attractant concentrations. Since this attractant can be oxidized by myeloperoxidase from stimulated neutrophils, we determined if its efficacy was increased by protection from oxidation. Efficacy was increased by reducing agents and by molecules with oxidizable sulfur, 10(-5) M methionine or 1.5 X 10(-6) M albumin. The antioxidant effect was on the attractant, not the cells: albumin in the cell compartment did not increase responses. Furthermore, antioxidants had no effect on efficacy of fNle-Leu-Phe-Nle-Tyr-Lys, an attractant that also stimulated superoxide release but had no oxidizable sulfur.     

Protoporphyrinogen oxidation in chloroplasts and plant mitochondria,a step in heme and chlorophyll synthesis

The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.     

Shear stress induces apoptosis in vascular smooth muscle cells via an autocrine Fas/FasL pathway

Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.     

Enhanced NAD(P)H:Quinone Reductase Activity Prevents Glutamate Toxicity Produced by Oxidative Stress

Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.     

Mechanism of Inactivation of Bacteriophage J 1 by Dithiothreitol,Cysteine, Mercaptoethanol,or Thioglycollate

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacilluscasei, by reducing agents containing thiol group(s) other than glutathione was studied mainly with dithiothreitol (DTT).

Air bubbling, oxidizing agents, and transition metal ions enhanced the rate of phage inactivation by DTT. Partial oxidation of DTT resulted in a more rapid rate of phage inactivation. In contrast, nitrogen bubbling, reducing agents including high concentrations of DTT itself, chelating agents, and radical scavengers prevented phage inactivation. Fully oxidized DTT had no phagocidal effect. These results indicate that the inactivating effect of DTT requires the presence of molecular oxygen and is indirectly caused by free radicals involved in the mechanism of DTT oxidation. The target attacked by DTT in phage particle was not protein but DNA; DTT reacted with DNA to produce single-strand scissions in DNA, which were the cause of inactivation of phage.

This was true also for L-cysteine, 2-mercaptoethanol, and thioglycollate.

Possible mechanisms by which these thiols fail to inactivate phage at high thiol concentrations are also discussed.