Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).     

Measurement of drug-metabolizing systems in Salmonella typhimurium strains G46, TA1535, TA100, TA1538 and TA98

Salmonella typhimurium strains which are commonly used in the Ames test for screening potential carcinogens were examined for a number of drug-metabolizing systems. Neither cytochrome P-450 itself nor two activities catalyzed by the cytochrome P-450 system in mammalian cells, i.e., benzpyrene monooxygenase and ethoxycoumarin O-deethylation, could be detected. Nor do these bacterial strains demonstrate any ability to detoxify epoxides by hydrating them or to conjugate p-nitrophenol with glucuronic acid.On the other hand, S. typhimurium strains G46, TA1535, TA100, TA1538 and TA98 contain considerable amounts of acid-soluble thiols, approx. 5–10% of which is glutathione. These bacteria can also enzymatically conjugate glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) and can reduce oxidized glutathione using NADPH as cofactor.Thus, enzymatic and non-enzymatic reaction of immediate carcinogens with thiol groups in S. typhimurium may have a significant effect on the outcome of the Ames test in certain cases.     

Localization of the amino phospholipids in sarcoplasmic reticulum membranes revealed by trinitrobenzene-sulfonate and fluorodinitrobenzene

The chemical probes for amino compounds 2,4,6-trinitrobenzenesulfonate (TNBS) and 1-fluoro-2,4-dinitrobenzene (FDNB) were utilized to determine the localization of the amino phospholipids in the sarcoplasmic reticulum membranes. At low concentrations (<1 mM), TNBS does not penetrate the sarcoplasmic reticulum membrane, while FDNB readily penetrates it. The results show that about 70% of the total phosphatidylethanolamine is located on the external surface of the membrane, about 20% is on the internal surface and 10% is probably strongly interacting with the proteins since it is not accessible to the probes. In contrast, most of the phosphatidylserine is located on the inner surface of the membrane. This molecular distribution of the amino phospholipids supports a structural assymmetry of the sarcoplasmic reticulum membrane.     

Organization of thiol groups of electric-eel electric-organ sodium-plus-potassium ion-stimulated adenosine triphosphatase studied with bifunctional reagents.

The reactions of three bifunctional thiol-blocking reagents of differing cross-linking spans and two monofunctional thiol-blocking reagents with the Na+ + K+-stimulated ATPase of the electric-eel electric organ were examined. 1,5-Difluoro-2,4-dinitrobenzene with a cross-linking span of 0.3--0.5 nm (3--5 A) and high solubility in non-polar solvent was the most efficient inhibitor of enzyme activity; thus essential thiol groups exist in a non-polar environment and are approx. 0.3--0.5 nm (3--5 A) from their nearest thiol-group neighbours. Ligands promoting phosphorylation of the Na+ + K+-stimulated ATPase decreased the number of thiol groups bridged by 1,5-difluoro-2,4-dinitrobenzene and by 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone [0.7--1.0 nm (7--10 A) span]. Phosphorylation is associated with a conformational change in the enzyme.     

Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol--20mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C(18) column kept at 40 degrees C. A good linearity was found in the range of 0.5-250 microg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from -0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 microl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.     

Reactivity of sarcoplasmic reticulum from rabbit cardiac muscle with 1-fluoro- and 1,5-difluoro- 2,4-dinitrobenzene

Sarcoplasmic reticulum preparations from rabbit cardiac and fast skeletal muscle react differentially with low concentrations of 1-fluoro- and 1,5-difluoro-2,4-dinitrobenzene. Dinitrophenylation of cardiac sarcoplasmic reticulum by 1-fluoro-2,4-dinitrobenzene is not affected by Ca2+ and is limited to the lipoprotein-lipid region. This contrasts sharply with the predominant Ca2+-dependent dinitrophenylation of the ATPase protein of rabbit skeletal sarcoplasmic reticulum by this reagent. Formation of non-serial high mol. wt. oligomers by 1,5-difluoro-2,4-dinitrobenzene is significantly greater in cardiac than in skeletal vesicles. Substrate MgATP2- does not protect rabbit cardiac sarcoplasmic reticulum ATPase activity or Ca2+ uptake from dinitrophenylation when monofunctional and bifunctional reagents are used. Chemical differences in the overall structure of the two kinds of membrane preparations can be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the reactivity of these reagents.     

Optical biosensor consisting of glutathione-S-transferase for detection of captan

The optical biosensor consisting of a glutathione-S-transferase (GST)-immobilized gel film was developed to detect captan in contaminated water. The sensing scheme was based on the decrease of yellow product, s-(2,4-dinitrobenzene) glutathione, produced from substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), due to the inhibition of GST reaction by captan. Absorbance of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme reactor of the sensor system was fabricated by the gel entrapment technique for the immobilized GST film. The immobilized GST had the maximum activity at pH 6.5. The optimal concentrations of substrates were determined with 1 mM for both of CDNB and GSH. The optimum concentration of enzyme was also determined with 100 μg/ml. The activity of immobilized enzyme was fairly sustained during 30 days. The proposed biosensor could successfully detect the captan up to 2 ppm and the response time to steady signal was about 15 min.     

The effects of anion transport inhibitors on structural transitions in erythrocyte membranes

Red blood cell membranes have been labeled with several covalent and non-covalent inhibitors of anion transport and their heat capacity profiles determined as a function of temperature. Covalent inhibitors include the amino reactive agents 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, pyridoxal phosphate and 1-fluoro-2,4-dinitro benzene. The non-covalent inhibitors include several well known local anesthetics. The study was undertaken in order to identify regions of the membrane involved in anion transport. Covalent modification in all cases resulted in a large upward shift of the C transition, which is believed to involved a localized phospholipid region. Evidence is presented which indicates that Band III protein and this phospholipid region are in close physical proximity on the membrane. Addition of non-covalent inhibitors affects the membrane in either or both of two ways. In some cases, a lowering and broadening of the C transition occurs; in other the B₁ and B₂ transitions are altered. These latter transitions are believed to involve both phospholipid and protein, including Band III. These results may indicate that the non-covalent inhibitors produce their inhibitory effect on anion transport at least in part by interacting with membrane phospholipid.     

Determination of radioactive proline and hydroxyproline by a simple paper-chromatographic procedure

A simple method to separate and determine radioactive proline and hydroxyproline by paper chromatography is described. The localization of the imino acids separation is achieved by direct nondestructing staining with 1-fluoro-2,4-dinitrobenzene dye. The imino acids are quantitated directly by liquid scintillation counting in the presence of paper strips. The method was applied to bone cultures with good reproducibility, sensitivity and linearity over a wide range of radioactivity. The procedure was also tested in fibroblast cultures. The results for hydroxyproline were in good correlation with the widely used method of Juva and Prockop (Juva, K. and Prockop, D.J. (1966) Anal. Biochem. 15, 77–83), in which hydroxyproline is oxidized to pyrrole, and then extracted and purified by column chromatography before counting radioactivity.     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

Xenobiotica-metabolizing enzymes in Drosophila melanogaster: activities of epoxide hydratase and glutathione S-transferase compared with similar activities in rat liver.

Activities of epoxide hydratase and glutathione (GSH) S-transferase were investigated in subcellular fractions of Drosophila melanogaster, and these activities were compared with analogous enzymic activities in extracts from rat liver. Microsomes of Drosophila were active in the hydratation of styrene oxide catalyzed by epoxide hydratase. The post-microsomal supernatant of Drosophila catalyzed the conjugation of GSH with 1-chloro-2,4-dinitrobenzene. However, GSH S-transferase activity with styrene oxide as the electrophilic substrate was not measurable. The respective specific activities of epoxide hydratase (per mg microsomal protein) and GSH S-transferase (per mg cytosolic protein) were factors of 5- and 10-fold lower than the corresponding activities in rat liver. However, when expressed per gram body weight, activities of both epoxide hydratase and GSH S-transferase were 3 times higher for Drosophila enzymes. The apparent Km values for the two Drosophila enzymes were higher, whereas the apparent Km values were lower, than the values found for the rat-liver enzymes. Among 3 different Drosophila strains (a wild-type, a white eye-color carrying mutant strain and a DDT-resistant strain), preliminary experiments showed no differences as far as these two enzymic activities were concerned. It is concluded that the results obtained in genetic toxicology testing with Drosophila are probably relevant to effects to be expected in mammalian systems with compounds requiring metabolic processes involving the enzymes investigated here.     

A comparison of catecholamine-induced internalization of beta-adrenergic receptors and receptor-mediated endocytosis of epidermal growth factor in human astrocytoma cells. Inhibition by phenylarsine oxide

The ligand-induced internalization of beta-adrenergic receptors and the receptor-mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta-adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups.     

Pyruvate flux into resealed ghosts from human erythrocytes

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ranged from 15.0 mM at $\text{μ = 0.05}$ to 3.7 mM at $\text{μ = 0.01}$, while the $\text{V}$ went from $\text{0.611 · 10}{}^{-15}$ to $\text{0.137 · 10}{}^{-15}\text{mol}\text{·}\text{min}{}^{-1}\text{·}\text{ghost}{}^{-1}$. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO₄ mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.     

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k_cat) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.     

Glutathione and glutathione S-transferases in the salmonella mammalian-microsome mutagenicity test

Levels of the tripeptide glutathione (GSH) and the activity of glutathione S-transferases were investigated in S9 fractions of rats and mice and in Salmonella typhymurium tester strains TA1535, TA100, TA1538 and TA98. The S9 and Salmonella typhimurium tester strains had high levels of glutathione. Compared with S9, the activity of GSH S-transferases was lower in the bacteria. However, electrophiles such as 1-chloro-2,4-dinitrobenzene (CDNB), diethyl maleate and styrene oxide were effectively bound to bacterial GSH.

The mutagenicity of the direct mutagen CDNB was drastically lowered in presence of S9 fractions but not in presence of microsomes. A comparable decrease was obtained when microsomal supernatant, which contains GSH and GSH S-transferases, was added to the microsomes. Addition of GSH in excess completely abolished mutagenicity of CDNB. These results demonstrate that the conjugation of electrophiles with GSH mediated by the S9 fraction or the bacterial tester strains represents an important detoxication mechanism which may influence the results obtained with the Salmonella typhimurium mammalian-microsome mutagenicity test.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.     

Sensitization of D-glucuronic acid transport system of E. coli to protein group reagents in presence of substrate or absence of energy source

N-ethylmaleimide and 1-fluoro-2,4-dinitrobenzene inactivate D-glucuronic acid transport in $\text{E. coli}$ K12. The inactivation is highly enhanced by the two substrates of the transport system, D-glucuronate and D-galacturonate, or by inhibitors of respiratory energy production. The significance of these results is discussed in the framework of a model of a mobile carrier which can exist in two or more distinct conformational states.     

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg²⁺ and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [³H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [³H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.     

Transport of glutamate and aspartate across the membranes of rat liver mitochondria

Chromatographic analyses have indicated that aspartate and glutamate constitute from 50–70% of the total free amino acids in freshly isolated mitochondria. Radioactive tracer studies indicate that while the l-isomers of glutamate and aspartate are rapidly accumulated by mitochondria, the d-isomers of these amino acids do not penetrate the mitochondrial membrane. The action of two inhibitory compounds, 1-fluoro-2,4-dinitrobenzene (Sanger's reagent) and tannic acid, on the transport of l-glutamate and l-aspartate has been examined. A marked inhibition of l-glutamate transfer by 1-fluoro-2,4-dinitrobenzene is observed. A corresponding effect on the transport of either l-aspartate or the anionic substrate, succinate has not been found. Tannic acid, an agent previously known to inhibit certain carrier-mediated solute fluxes in mitochondria, is shown also to inhibit the uptake of both l-glutamate and l-aspartate. These findings are consistent with the view that the mitochondrial membranes of rat liver cells contain distinct, stereospecific transport mechanisms for aspartate and glutamate.     

Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis

The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis. The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C. jejuni and Agrobacterium tumefaciens. The monofunctional enzymes (2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations. We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.