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.     

Mechanism of Inactivation of Bacteriophage J1 by Ascorbic Acid

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacillus casei, by ascorbic acid was investigated.

Bubbling air, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by ascorbic acid. In contrast, bubbling nitrogen gas, other reducing agents and radical scavengers prevented the inactivation. The results indicated that the inactivating effect of ascorbic acid was oxygen dependent and caused by free radicals formed during the autoxidation of ascorbic acid.

The target of ascorbic acid in the phage particle was not the tail protein but DNA. Ascorbic acid caused single-strand scissions in phage DNA, as exhibited by alkaline sucrose density gradient centrifugation analysis, and caused a slight decrease in the viscosity of DNA.     

Low N-ethylmaleimide concentrations activate ryanodine receptors by a reversible interaction, not an alkylation of critical thiols

Previous studies proposed that N-ethylmaleimide (NEM) alkylates 3 classes of thiols on skeletal muscle ryanodine receptors (RyRs) producing 3 phases of channel modification, as function of time and concentration. NEM (5 mm) decreased, increased, and then decreased the open probability (P(o)) of the channel by thiol alkylation, a reaction not reversed by reducing agents. We now show that low NEM concentrations (20-200 microm) elicit Ca(2+) release from sarcoplasmic reticulum (SR) vesicles, but contrary to expectations, the effect was fully reversed by reducing agents or by washing SR vesicles. In bilayers, NEM (0.2 mm) increased P(o) of RyRs within seconds when added to the cis (not trans) side, and dithiothreitol (DTT; 1 mm) decreased P(o) in seconds. High (5 mm) NEM concentrations elicited SR Ca(2+) release that was not reversed by DTT, as expected for an alkylation reaction. A non-sulfhydryl reagent structurally related to NEM, N-ethylsuccinimide (0.1-0.5 mm), also elicited SR Ca(2+) release that was not reversed by DTT (1 mm). Other alkylating agents elicited SR Ca(2+) release, which was fully (N-methylmaleimide) or partially (iodoacetic acid) reversed by DTT and inhibited by ruthenium red. Nitric oxide (NO) donors at concentrations that did not activate RyRs inhibited NEM-induced Ca(2+) release, most likely by an interaction of NO with NEM rather than an inactivation of RyRs by NO. Thus, at low concentrations, NEM does not act as a selective thiol reagent and activates RyRs without alkylating critical thiols indicating that the multiple phases of ryanodine binding are unrelated to RyR activity or to NEM alkylation of RyRs.     

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.     

Oxygen protection of bacteriophage T1 against ionizing radiations

Bacteriophage T1 was suspended in distilled water and in phosphate buffer, saturated with oxygen, nitrogen, hydrogen, and carbon monoxide, and irradiated with gamma rays and x-rays. Under the same conditions phage was exposed to hydrogen peroxide. Oxygen acted as a protective agent against both irradiation and hydrogen peroxide inactivation. As a protective agent against irradiation, oxygen was more efficient in distilled water than in buffer. The phage was much more sensitive to irradiation in the presence of hydrogen or nitrogen than in the presence of oxygen. Survivals of phage irradiated in suspensions saturated with hydrogen and with nitrogen did not differ significantly. From this it was concluded that oxygen did not protect T1 by removing atomic hydrogen from the irradiated medium, since the hydrogen-saturated medium increased the yield of atomic hydrogen but did not increase the yield of inactivated phage. It was presumed, therefore, that phage is sensitive to OH radicals and this was confirmed by irradiating phage with UV in the presence of hydrogen peroxide and comparing this survival with the survivals obtained from hydrogen peroxide alone and from UV alone. The combined effect of hydrogen peroxide and UV acting simultaneously was greater than the effect attributable to hydrogen peroxide and UV acting separately. Evidence for sensitivity to HO₂ radicals was considered, and the effect was attributed chiefly to an oxidizing action since phage sensitivity is greater at higher hydrogen ion concentrations, which favor oxidation by HO₂ radicals. Since the OH radical is a more efficient oxidizing agent than O^-, the former being favored in an acid medium, the latter in an alkaline medium, and since the phage is more sensitive in the first situation than in the second, the present tests proved the importance of oxidation as the mechanism of inactivation. Since some inactivation was encountered when phage was exposed to reducing agents, independently of irradiation, it was concluded that phage is somewhat sensitive to reducing agents, but the inactivation attributable to ionizing radiations is due chiefly to oxidation, against which these reducing agents are very efficient protectors. Under no circumstances did hydrogen peroxide protect T1, whether produced by irradiation in the medium or added beforehand to the medium to be irradiated. The first point was investigated by irradiating T1 in the presence of hydrogen and oxygen combined; this produced a higher yield of hydrogen peroxide but a lower survival of T1. In all these tests phage survival under irradiation was directly correlated with oxygen content of the medium rather than with production of hydrogen peroxide. It is proposed that the protective effect of oxygen is due to a reaction between the phage and oxygen, and this complex confers stability upon the phage.     

The Mechanism of Inactivation of Bacteriophage ϕX174 by Autoxidizable Synthetic Polysaccharides

Autoxidizable synthetic polysaccharides prepared by polycondensation of reducing aldose or ketose in dimethyl sulfoxide containing pohsphorus pentaoxide [Polymer, 13, 190 (1972)] inactivated phage ?X174. Another autoxidizable polysaccharides obtained by oxidation of natural glucans with the same oxidant also inactivated ?X174. The ?X174 inactivation was due to strand scission of viral DNA in the virion. The inactivation reaction was stimulated by Cu²⁺ and inhibited by EDTA, Superoxide dismutase, catalase and several radical scavengers. These results suggest that oxygen radicals produced during autoxidation of polysaccharides are responsible for ?X174 inactivation.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Antiphage properties of many kinds of chemicals such as antibiotics, surface-active agents and chelating agents were examined on Brevibacterium lactofermentum No. 2256—phage P465 system using double-layer agar method, as a part of the basic study, for preventing phage infection in the industrial fermentation.

A great majority of inhibitors which were selected were usually nonspecific and inhibited also bacterial growth. Among about 200 chemicals tested, 5 antibiotics such as chloramphenicol and tetracycline, 6 chelating agents such as phytic acid and 19 surface- active agents such as PEG monoester and POE alkyl ether showed the selective inhibitions for phage infection at the concentrations which did not affect bacterial growth, or at the subbactericidal concentrations that suppressed bacterial growth slightly.

Of the above chemicals which showed selective inhibitions for phage infection, a possible mechanism of chelating agents chiefly of phytic acid was investigated. When 0.1 to 0.2% of phytic acid was present in the medium, the effect of inhibition was most remarkable; this could be applied to the actual phage-infected l-glutamic acid fermentation. Phytic acid had no direct phagocidal action, nor did it inhibit the late step of the phage multiplication; but it prevented the adsorption of phages, which required inorganic cofactors such as Mg²⁺ or Ca²⁺ in this step, to the host bacteria. Moreover, a part of the infected bacteria was made incapable of forming plaques in the presence of phytic acid. These results suggested that the chelation between Mg²⁺ or Ca²⁺ and phytic acid would remove the metal ions essential for phage adsorption and prevent the phage adsorption and infection of phage DNA, consequently, the phage infection.

The effect of the non-ionic surface-active agents (SAA) on the infection of phage P465 of Br. lactofermentum was examined by adsorption and one-step growth experiments as a part of the basic study on the prevention of phage-infection in the industrial fermentation. Among various SAA tested, polyoxyethylene stearyl ether (POE-SE), polyethylene glycol monooleate (PEG-MO) and polyoxyethylene sorbitan monostearate (Tween 60) had remarkably demonstrated the selective inhibition of phage infection.

The effect of the above three SAA was apparently restricted to the initial adsorption step of phage infection, for the phage already adsorbed would not be affected by exposure to SAA. However, the results of one-step growth experiment indicated that Tween 60 inhibited not only the phage-adsorption, but also the maturation of phage already adsorbed in the host cells. The rate of the inhibition was found to be directly related to the concentration of agent. And, the most effective adsorption-inhibition was exhibited at the critical micelle concentration of SAA. The concentration as used in our experiments did not affect the viability of either phages or the host cells.

The results also indicated that the inhibition of phage-adsorption was due to the action of SAA on the surface of the bacterial cells rather than on the phage. This is supported by the observation that preincubation of phage with SAA did not affect either the subsequent adsorption rate or the plaque-forming ability of the phage. In contrast with above, a short-term exposure of bacterial cells to SAA caused an apparent change to the cell surface which was only partially restored by washing repeatedly. Moreover, the inhibitory effect of SAA on phage-adsorption appears quite specific in the phage-host system.     

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.     

Partially irreversible inactivation of mitochondrial aldehyde dehydrogenase by nitroglycerin

Mitochondrial aldehyde dehydrogenase (ALDH2) may be involved in the biotransformation of glyceryl trinitrate (GTN), and the inactivation of ALDH2 by GTN may contribute to the phenomenon of nitrate tolerance. We studied the GTN-induced inactivation of ALDH2 by UV/visible absorption spectroscopy. Dehydrogenation of acetaldehyde and hydrolysis of p-nitrophenylacetate (p-NPA) were both inhibited by GTN. The rate of inhibition increased with the GTN concentration and decreased with the substrate concentration, indicative of competition between GTN and the substrates. Inactivation of p-NPA hydrolysis was greatly enhanced in the presence of NAD(+), and, to a lesser extent, in the presence of NADH. In the presence of dithiothreitol (DTT) inactivation of ALDH2 was much slower. Dihydrolipoic acid (LPA-H(2)) was less effective than DTT, whereas glutathione, cysteine, and ascorbate did not protect against inactivation. When DTT was added after complete inactivation, dehydrogenase reactivation was quite modest (< or =16%). The restored dehydrogenase activity correlated inversely with the GTN concentration but was hardly affected by the concentrations of acetaldehyde or DTT. Partial reactivation of dehydrogenation was also accomplished by LPA-H(2) but not by GSH. We conclude that, in addition to the previously documented reversible inhibition by GTN that can be ascribed to the oxidation of the active site thiol, there is an irreversible component to ALDH inactivation. Importantly, ALDH2-catalyzed GTN reduction was partly inactivated by preincubation with GTN, suggesting that the inactivation of GTN reduction is also partly irreversible. These observations are consistent with a significant role for irreversible inactivation of ALDH2 in the development of nitrate tolerance.     

Phage-inactivating Effect of Iron(II)-Ascorbate Complex

The effect of iron(II)-ascorbate complex on various phages was investigated. At 10- ⁶ M, the complex inactivated all nine phages examined. The mechanism of the inactivation was studied with phage J1, the most sensitive to the complex. The addition of H₂O₂ or Cu²⁺ to the reaction mixture increased the inactivation. Bubbling of nitrogen through the reaction mixture and the addition of Fe³⁺, a reducing agent, a chelating agent, or a radical scavenger prevented inactivation. These findings suggest the involvement of oxygen radicals in the inactivation. The complex had no effects on the SDS-PAGE pattern or amino acid composition of bovine serum albumin, or the structural protein of phage J1. The complex nicked the supercoiled form of pUC18 DNA, giving first single-stranded breaks (the open circular form) and then double-stranded breaks (the linear form). Strands of M13mp8 DNA, λDNA, and J1 DNA were also broken. The breaks could account for the inactivation.     

The nature of inhibition of 3-hydroxy-3-methylglutaryl CoA reductase by garlic-derived diallyl disulfide

A concentration dependent inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase was found on preincubation of microsomal preparations with diallyl disulfide, a component of garlic oil. This inhibited state was only partially reversed even with high concentrations of DTT. Glutathione, a naturally occurring reducing thiol agent, was ineffective. The substrate, HMG CoA, but not NADPH, was able to give partial protection for the DTT-dependent, but not glutathione-dependent activity. The garlic-derived diallyl disulfide is the most effective among the sulfides tested for inhibition of HMG CoA reductase. Formation of protein internal disulfides, inaccessible for reduction by thiol agents, but not of protein dimer, is likely to be the cause of this inactivation.     

Probable involvement of sulfhydryl groups and a metal as essential components of the cellulase of Clostridium thermocellum

The crude extracellular cellulase from Clostridium thermocellum was oxidatively inactivated by air and inhibited by sulfhydryl reagents. Activity-loss was prevented and reversed by the addition of a high concentration (10 mM) dithiothreitol (DDT) at zero time and up to 24 h respectively. In the presence of a low concentration (0.4 mM) of DTT, the enzyme was more rapidly inactivated than in air alone. This was probably due to autoxidation of the low DTT concentration to H₂O₂ as shown by its prevention by a high DTT concentration, exclusion of air, or catalase; and by the oxidative inactivation of the enzyme by H₂O₂. The inactivation by H₂O₂ could be prevented by a high concentration of DTT but not by air exclusion. EDTA protected the enzyme from inactivation in air by a low concentration of DTT or by H₂O₂. This is presumably due to the role of metals in oxidation of SH groups. Furthermore, copper (5 M) also caused inactivation and this was prevented by the presence of a high DTT concentration. Even in the protective atmosphere of a high DTT concentration, cellulase was inactivated by certain apolar chelating agents such as o-phenanthroline and -¹-dipyridyl, such inactivation being preventable by the prior incubation of the chelator with a mixture of Fe²⁺ and Fe³⁺. These data suggest that the clostridial cellulase, unlike the enzyme from aerobic fungi, contains essential sulfhydryl groups and is stimulated by iron. The endo--glucanase component of the cellulase complex was not susceptible to oxidative inactivation.Abbreviations DTT dithiothreitol - CMC carboxymethylcellulose - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - p-CMB p-chloromercuribenzoic acid     

Human erythrocyte delta-aminolevulinate dehydratase inhibition by monosaccharides is not mediated by oxidation of enzyme sulfhydryl groups

The heme pathway enzyme delta-aminolevulinate dehydratase is a good marker for oxidative stress and metal intoxication. This sulfhydryl enzyme is inhibited in such oxidative pathologies as lead, mercury and aluminum intoxication, exposure to selenium organic species and diabetes. Oxidative stress is a complicating factor in diabetes, inducing non-enzymatic glucose-mediated reactions that change protein structures and impair enzyme functions. We have studied the effects of high glucose, fructose and ribose concentrations on delta-ALA-D activity in vitro. These reducing sugars inhibited delta-ALA-D with efficacies in the order fructose=ribose>glucose. The possible mechanism of glucose inhibition was investigated using lysine, DTT, and t-butylamine. Oxidation of the enzyme's critical sulfhydryl groups was not involved because DTT had no effect. We concluded that high concentrations of reducing sugars or their autoxidation products inhibit delta-ALA-D by a mechanism not related to thiol oxidation. Also, we are not able to demonstrate that the formation of a Schiff base with the critical lysine residue of the enzyme is involved in the inhibition of delta-ALA-D by hexoses.     

Rat liver cytosol contains NADPH- and GSH-dependent factors able to restore ornithine decarboxylase inactivated by removal of thiol reducing agents.

Removal of dithiothreitol (DTT) from partially purified ornithine decarboxylase (ODC) led to an almost complete inhibition of enzymic activity. The inactivation was reversed by addition of millimolar concentrations of DTT, whereas natural reductants such as NADPH or NADH were ineffective, and GSH had only a limited effect. Addition of rat liver cytosol to the incubation mixture resulted in a noticeable re-activation of ODC; however, dialysed cytosol had little effect unless NADPH or GSH was present. Fractionation of rat liver cytosol by gel filtration on Sephadex G-75 yielded two fractions involved in the NADPH- and GSH-dependent re-activation of ODC: one designated 'A', eluted near the void volume (Mr greater than or equal to 60,000), and the other designated 'B', eluted later (Mr approx. 12,000). The NADPH-dependent mechanism required both fractions A and B for maximal ODC re-activation; the most effective concentration of NADPH was 0.15 mM, although a significant effect was observed at a concentration more than 10-fold lower. The GSH-dependent mechanism involved the mediation of Fraction B only, and operated at millimolar concentrations of GSH. These results suggest the existence of reducing systems in the cytosol, which may play a role in maintaining, and potentially in regulating, ODC activity by modulation of its thiol status.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence,reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Molecular characterization of a 2-Cys peroxiredoxin induced by abiotic stress in mungbean

A mungbean low temperature-inducible VrPrx1 encoding 2-Cys peroxiredoxin (2-Cys Prx) was cloned by subtractive suppression hybridization. The deduced VrPrx1 amino acid sequence showed highest sequence homology to 2-Cys Prxs of Phaseolus vulgaris (95%), Pisum sativum (89%), and Arabidopsis thaliana (87%). VrPrx1 RNA and protein levels were increased by low temperature, hydrogen peroxide (H₂O₂), and wounding but decreased by high salinity, drought, and exogenous abscisic acid. Recombinant His-tagged VrPrx1 recombinant protein protected DNA and glutamine synthetase activity from degradation via the thiol/Fe(III) oxygen mixed-function oxidation system, and exhibited peroxidase activity to H₂O₂ in the presence of the reducing agent dithiothreitol (DTT) in vitro. The oxidized dimers and oligomers of the VrPrx1 recombinant protein were reduced to monomers by DTT or thioredoxin. Subcellular localization studies confirmed that VrPrx1-GFP was targeted to the plastid. To evaluate the function of VrPrx1 in planta, the antioxidant activities and photosynthetic efficiency were investigated in VrPrx1-overexpressing Arabidopsis plants. VrPrx1 ectopic expression conferred improved photosynthetic efficiency under oxidative stress conditions. Hence, mungbean VrPrx1 may play an important role in protecting the photosynthetic apparatus against oxidative and abiotic stress conditions.     

Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity

The extraction of high-quality genomic DNA for PCR amplification from sunflower (Helianthus annuus) and cotton (Gossypium spp.) is challenging because of the presence of polysaccharides, secondary metabolites, and polyphenolics in the tissues. A high-throughput DNA extraction protocol was needed in our laboratory for simple sequence repeats (SSR)-marker screening and other molecular analyses that do not require organic extraction steps of phenol or chloroform. Here we describe 2 improved highthroughput protocols for DNA extraction and in-PCR modification that result in successful PCR amplification of sunflower and cotton. While the sunflower DNA extraction protocol uses reducing agents such as sodium metabisulfite and dithiothreitol (DTT), the cotton protocol uses polyvinylpyrrolidone (PVP) in PCR reactions and reducing agents in the DNA extraction procedure.     

Alteration of sperm thiol-disulfide status and capacitation in the guinea pig

Epididymal spermatozoa of the guinea pig were incubated under conditions known to promote a rapid synchronous capacitation in a large proportion of the spermatozoa (Ca²⁺-free medium with lysophosphatidylcholine, LC) or in Ca ²⁺-free medium without LC. To study the effects of altered thiol-disulfide status and content, incubations were conducted with reagents that maintain and increase thiol groups (DTT, GSH), maintain and increase disulfide groups (diamide, GSSG), or which irreversibly block thiol groups by alkylation (NEM). The permeable DTT inhibited LC-induced capacitation and at high concentrations diminished the percentage of acrosome reactions in capacitated spermatozoa. The permeable diamide exhibited a stimulatory effect upon capacitation. The largely impermeable GSH and GSSG exhibited effects similar to their respective permeable counterparts but their effects were moderate and required extremely high concentrations. The DTT inhibition of LC-induced capacitation was reversible by washing and a further 1 hr incubation. In this final incubation after removal of DTT by washing, LC was absent too so its stimulatory effect must have been accomplished prior to washing and in the presence of DTT. NEM-alkylation of the existing thiol population did not affect LC-induced capacitation but alkylation of the increased thiol population after prior DTT treatment was inhibitory of capacitation. These results suggest that the maintenance and/or formation of disulfide groups on enzymes or structural proteins may be a component of the capacitation process. In contrast, the formation and maintenance by alkylation of increased thiol groups but not the maintenance of existing thiol groups, is inhibitory of capacitation. The relevance of these findings to a role for a thiol-sensitive proteinase in capacitation is discussed.     

Double mode of inhibition-inducing interactions of 1,4-naphthoquinone with urease: arylation versus oxidation of enzyme thiols

In their inhibition-inducing interactions with enzymes, quinones primarily utilize two mechanisms, arylation and oxidation of enzyme thiol groups. In this work, we investigated the interactions of 1,4-naphthoquinone with urease in an effort to estimate the contribution of the two mechanisms in the enzyme inhibition. Jack bean urease, a homohexamer, contains 15 thiols per enzyme subunit, six accessible under non-denaturing conditions, of which Cys592 proximal to the active site indirectly participates in the enzyme catalysis. Unlike by 1,4-benzoquinone, a thiol arylator, the inactivation of urease by 1,4-naphthoquinone under aerobic conditions was found to be biphasic, time- and concentration-dependent with a non-linear residual activity-modified thiols dependence. DTT protection studies and thiol titration with DTNB suggest that thiols are the sites of enzyme interactions with the quinone. The inactivated enzyme had approximately 40% of its activity restored by excess DTT supporting the presence of sulfenic acid resulting from the oxidation of enzyme thiols by ROS. Furthermore, the aerobic inactivation was prevented in approximately 30% by catalase, proving the involvement of hydrogen peroxide in the process. When H2O2 was directly applied to urease, the enzyme showed susceptibility to this inactivation in a time- and concentration-dependent manner with the inhibition constant of H2O2 Ki = 3.24 mM. Additionally, anaerobic inactivation of urease was performed and was found to be weaker than aerobic. The results obtained are consistent with a double mode of 1,4-naphthoquinone inhibitory action on urease, namely through the arylation of the enzyme thiol groups and ROS generation, notably H2O2, resulting in the oxidation of the groups.