Glutathione transferase from human erythrocytes: Nonidentity with the enzymes from liver

doi:10.1016/S0003-9861(78)80011-3

Archives of Biochemistry and Biophysics

Volume 188, Issue 2, June 1978, Pages 287-293

https://doi.org/10.1016/S0003-9861(78)80011-3 Get rights and content

Glutathione transferase ϱ has been purified to homogeneity from human erythrocytes. The enzyme has a molecular weight of 47,500 and is composed of two subunits of the same apparent molecular weight. The enzyme is active in catalyzing the reaction of glutathione, as a nucleophile, with a variety of compounds bearing an electrophilic center. Thioether formation with 1-chloro-2,4-dinitrobenzene and with ethacrynic acid are among the most rapid reactions under standard assay conditions. The erythrocyte enzyme has a different amino acid composition from that of glutathione transferases from human liver, whereas all of the liver enzymes appear to have the same composition. The erythrocyte transferase also differs from the liver transferases in its low isoelectric point of 4.5, its lack of reactivity with antibody produced against a liver glutathione transferase, and its more limited spectrum of substrates. The enzyme is present at a concentration of about 1.2 mg/100 ml of packed human erythrocytes.

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Cited by (170)

Biotransformation of organic nitrates by glutathione S-transferases and other enzymes: An appraisal of the pioneering work by William B. Jakoby
2022, Analytical Biochemistry
Citation Excerpt :
GST ρ requires as the second substrate GSH [13], which is present in human RBCs at concentrations of about 2 mM. However, the activity of GST ρ towards GTN and erythrityl tetranitrate was near the detection limit of the method [13]. Although it cannot be excluded, it is unlikely that GST ρ in RBCs can catalyze the conversion of organic nitrates to GSNO2 which could form nitrite and nitrate.
Organic nitrates (R–ONO₂; R, organic residue) such as nitroglycerin are used as drugs in part for more than a century. Their pharmacological use is associated with clinically relevant tolerance which is reportedly known since 1888. The underlying mechanisms of both, the mechanisms of action and the main pharmacological effect, which is vasodilatation and reduction of blood pressure, and the development of tolerance, which means increasing need of drug amount in sustained long-term therapy, are still incompletely understood. William B. Jakoby and associates were the first to report the biotransformation of organic nitrates, notably including nitroglycerin (i.e., glycerol trinitrate; GTN), by glutathione S-transferase (GST)-catalyzed conjugation of glutathione (GSH) to the nitrogen atom of one of the three nitrate groups of GTN to generate glutathione sulfenyl nitrite (glutathione thionitrate, S-nitroglutathione; GSNO₂). Jakoby's group was also the first to suggest that GSNO₂ reacts with a second GSH molecule to produce inorganic nitrite (ONO⁻) and glutathione disulfide (GSSG) without the catalytic involvement of GST. This mechanism has been adopted by others to the biotransformation of GTN by mitochondrial aldehyde dehydrogenase (mtALDH-(CysSH)₂) which does not require GSH as a substrate. The main difference between these reactions is that mtALDH forms an internal thionitrate (mtALDH-(CysSH)-CysSNO₂) which releases inorganic nitrite upon intra-molecular reaction to form mtALDH disulfide (mtALDH-(CysS)₂). Subsequently, ONO⁻ and GSNO₂ are reduced by several proteins and enzymes to nitric oxide (NO) which is a very potent activator of soluble guanylyl cyclase to finally relax the smooth muscles thus dilating the vasculature. GSNO₂ is considered to rearrange to GSONO which undergoes further reactions including GSNO and GSSG formation. The present article is an appraisal of the pioneering work of William B. Jakoby in the area of the biotransformation of organic nitrates by GST. The two above mentioned enzymatic reactions are discussed in the context of tolerance development to organic nitrates, still a clinically relevant pharmacological concern.
Measurement of the tripeptides glutathione and ophthalmic acid by gas chromatography-mass spectrometry
2022, Analytical Biochemistry
Glutathione (GSH, $γ$ -glutamyl-cysteinyl-glycine) is the most abundant intracellular dicarboxylic tripeptide with multiple physiological roles. GSH and its symmetric disulfide GSSG co-exist in biological samples. The GSH-to-GSSG molar ratio is high within cells and low in extracellular compartments. It is widely used as a biomarker of oxidative stress. Here, we report on a novel two-step derivatization procedure which allows for the specific quantitative measurement of GSH by gas chromatography-mass spectrometry (GC-MS) in the electron-capture negative-ion chemical ionization mode. The method is based on the derivatization of GSH first with pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C) and subsequently with 2 M HCl/CH₃OH (30 min, 80 °C). Esterification in 2 M HCl/CD₃OD is used for the in situ synthesis of deuterium-labelled GSH for use as internal standard. Quantification is performed by selected-ion monitoring of m/z 557 for GSH and m/z 560 for the internal standard (1 mM). Linear regression analysis between the peak area ratio of m/z 557 to m/z 560 (y) and the GSH concentration (x; range, 0–1 mM) resulted in the regression equations y = 0.02 + 0.62x (r² = 0.976) in the absence and y = 0.02 + 0.63x (r² = 0.997) in the presence of GSSG (range, 0–1 mM), underlying the linearity of the method for GSH and suggesting lack of interference by GSSG. γ-Glu-Cys and Cys-Gly also did not interfere. The GC-MS method demonstrated that in human hemolysate, nitrite at toxicological concentrations (range, 0–15 mM) depletes erythrocytic GSH. For the GC-MS analysis of the GSH tripeptide analogue ophthalmic acid, which lacks a Cys moiety, the derivatization order was HCl/CH₃OH followed by pentafluoropropionic anhydride in ethyl acetate.
Hidden allostery in human glutathione transferase P1-1 unveiled by unnatural amino acid substitutions and inhibition studies
2013, Journal of Molecular Biology
Citation Excerpt :
Support for this interpretation was subsequently derived from equilibrium binding studies, which showed hyperbolic binding isotherms rather than cooperativity,4 but the true nature of the underlying mechanism remains unresolved. The human GST P1-1, first isolated from erythrocytes and placenta,5,6 was later studied and reported as showing Michaelis–Menten behavior7 but also as allosteric if the assay temperature was varied.8 In the present investigation, the elusive kinetic behavior of human GST P1-1 has been further probed by site-directed mutagenesis, insertion of unnatural amino acids, and inhibition studies.
Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis–Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased k_cat and 300-fold increased K_M^GSH values, displays an apparent Hill coefficient of 0.82 ± 0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency k_cat/K_M^GSH to near the wild-type value but still yields Hill coefficients of 0.61 ± 0.08 and 0.86 ± 0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99 ± 0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.
FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1
2013, Chemico-Biological Interactions
Citation Excerpt :
In human cells there are seven classes of soluble cytosolic GSTs (termed alpha, mu, pi, sigma, zeta, omega, and theta) occurring in dimeric forms [6]. One of the enzymes, GST P1-1, first isolated from erythrocytes [7] and placenta [8,9] is of particular interest since it is expressed at high levels in many tumor cell lines [10,11]. Its presence in drug-resistant tumors such as human malignant melanoma [12] and murine osteosarcoma [13] suggested that GST P1-1 could contribute to the resistance phenotype.
Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted.
We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro.
We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC₅₀ values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme.
In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.
Differential expression of glutathione-S-transferase isoenzymes in various types of anemia in Taiwan
2007, Clinica Chimica Acta
Published reports concerning the expression of GST in various anemias including aplastic, hemolytic, iron deficiency and thalassemia anemia has been insufficient. We improved the conventional GST assay by incorporating a chloroform treatment to remove the interference of hemoglobin and evaluated the altered expression of GSTs in various anemias in Taiwan.
We incorporated a chloroform treatment to eliminate the interference of hemoglobin. Erythrocyte total GST and isoenzymes activities from 35 control subjects and 125 subjects of various anemias, including aplastic, hemolytic, iron deficiency and thalassemia anemias were measured spectrophotometrically.
Chloroform treatment did not significantly affect GST activities in erythrocytes of control subjects while the activities of erythrocyte total GST and α-GST were significantly increased in all anemic patients (P < 0.001). The expression of μ-GST was significantly decreased, although at a less extent, in cases of aplastic, iron deficiency and thalassemia anemia (P < 0.05), but π-GST was not physiologically different in various types of anemia.
The determination of changes in erythrocyte GST activity is a promising indicator of oxidative stress conditions that occur in various types of anemia. Measurement of GST activity might be useful for the evaluation of prophylactic treatment in trials of antioxidant strategies.
Free radical scavengers in susceptible/resistant Biomphalaria alexandrina snails before and after infection
2004, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
The activities of catalase (Cat), superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione transferase (GST), glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde3-phosphate dehydrogenase (G3PD) were studied in tissue and hemolymph of susceptible (S) (EgBS₂) and resistant (R) (EgBR₂) Biomphalaria alexandrina snails. The results showed that CAT and GST were higher in the hemolymph of snails susceptible to Schistosoma mansoni than in that of snails resistant to infestation, while SOD and G3PD were lower in the susceptible snails. The role of these enzymes as free radical scavengers was traced 1 and 24 h after infection of the two snail lines with S. mansoni. Moreover, the activities of SOD and G3PD were also measured 2 and 4 weeks post infection. The results revealed that the overall enzymatic activities were higher in susceptible than in resistant snail tissues. After 1 h of infection, all enzymes were increased in R and S snails except GST and G6PD which decreased in S snails. After 24 h of infection, GST increased in S snails and G3PD decreased in both S and R snails while other enzymes reached normal levels.

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¹: Permanent address: Department of Biochemistry, The University of Tennessee, Center for the Health Sciences, Memphis, Tennessee 38163. This work was carried out at the National Institutes of Health under an Intergovernmental Personnel agreement with the University of Tennessee.

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Glutathione transferase from human erythrocytes: Nonidentity with the enzymes from liver

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochem. Pharmacol.