Monobromobimane: a substrate for the fluorimetric assay of glutathione transferase

Hulbert, Peter B.; Yakubu, Sani Ibn

doi:10.1111/j.2042-7158.1983.tb02962.x

Cited by 45 publications

(22 citation statements)

References 8 publications

(3 reference statements)

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“…MBCl has been previously used to determine intracellular glutathione levels in intact, cultured neurons (Nieminen et al, 1990;Stabel-Burow et al, 1997). MBCl reacts rapidly and specifically with GSH to form a highly fluorescent adduct, whereas unbound MBCl has no fluorescence (Hulbert and Yakubu, 1983). In this study we used MBCl to measure the intracellular GSH content in neurons from hippocampal diploid and Ts16 cultures.…”

Section: Gsh Level In Cultured Diploid and Ts16 Neuronsmentioning

confidence: 98%

THIS ARTICLE HAS BEEN RETRACTED: Diminished Glutathione Levels Cause Spontaneous and Mitochondria‐Mediated Cell Death in Neurons from Trisomy 16 Mice:

Schuchmann

Heinemann

2000

Journal of Neurochemistry

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It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid and Ts16 cultures. Addition of the GSH precursors cysteine and cystine and the antioxidant tocopherol to the culture medium increased the GSH concentration up to 126.0% in diploid and up to 111.9% in Ts16 neurons. Moreover, we observed a reduced spontaneous neuronal death rate in diploid and Ts16 cultures. Following the application of 50 -100 M glutamate to culture medium, we found a GSH increase in the presence of cysteine, cystine, tocopherol, and cyclosporin A, an inhibitor of mitochondrial permeability transition (diploid, 105.8 -110.8%; Ts16, 83.1-96.3%). However, only tocopherol and cyclosporin A had a protective effect on glutamate-induced neuronal death. The results suggest that reduced GSH levels affect the increase of a spontaneous and a mitochondria-mediated, cyclosporin A-sensitive type of neuronal cell death. Therefore, elevating intracellular GSH concentration may have neuroprotective effects in Down's syndrome and Alzheimer's disease.

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Section: Gsh Level In Cultured Diploid and Ts16 Neuronsmentioning

confidence: 98%

THIS ARTICLE HAS BEEN RETRACTED: Diminished Glutathione Levels Cause Spontaneous and Mitochondria‐Mediated Cell Death in Neurons from Trisomy 16 Mice:

Schuchmann

Heinemann

2000

Journal of Neurochemistry

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“…For mBBr, the enzymatic activity was measured in a total volume of 0.9 ml using a PerkinElmer Life Sciences MPF-3 fluorescence spectrophotometer (excitation at 395 nm and emission at 480 nm) by monitoring the formation of the conjugate of mBBr (100 M) and glutathione (600 M) in 0.1 M potassium phosphate buffer (pH 6.5) at 25°C, according to the method of Hulbert and Yakubu (33). A known amount of glutathione-bimane was used as a fluorescence standard to calibrate and calculate the product formation.…”

Section: Methodsmentioning

confidence: 99%

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Ralat

Colman

2004

Journal of Biological Chemistry

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Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (K I ) of hGST P1-1 on [BITC] is hyperbolic, with K I ‫؍‬ 66 ؎ 7 M. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione ؉ ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr 103 and Cys 47 are modified equally. S-Methylglutathione reduces modification of Cys 47 , indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr 103 , suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in K m for BITC and binds ANS poorly, whereas Y103F has a normal K m for BITC and K d for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.

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“…The conjugate of glutathione and CDNB absorbs at 340 nm with Atj4onm = 9.6 mM-l cm-l (Habig et al, 1974) and the conjugate of glutathione and mBBr has a fluorescence emission maximum at 480 nm, when excited at 395 nm (Hulbert & Yakubu, 1983). The abilities of the modified and control enzymes to catalyze the reaction of glutathione with CDNB and mBBr were, respectively, measured spectrophotometrically or fluorometrically in either 0.1 M potassium phosphate buffer or 0.1 M Pipes, pH 6.5, at 25 "C. The ability of modified and control enzyme to catalyze the isomerization of As-androstene-3, 17-dione to A4-androstene-3, 17-dione was determined spectrophotometrically from the change in A248nm ( A E~~~, , , , , = 16.3 mM" cm-') (Benson et al, 1977) in 25 mM Tris/phosphate buffer, pH 8.5, or in 0.1 M potassium phosphate buffer, pH 6.5, at 25 "C in the presence of 0.1 mM glutathione and 0.1 mM DTT.…”

Section: Kinetic Characterization Of Modified Enzymementioning

confidence: 99%

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S‐transferase, 1–1

1996

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Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with I-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a K, of 0.78 mM and k,,, of 0.01 1 min". S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,S3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pK,, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pK, of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.

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Monobromobimane: a substrate for the fluorimetric assay of glutathione transferase

Cited by 45 publications

References 8 publications

THIS ARTICLE HAS BEEN RETRACTED: Diminished Glutathione Levels Cause Spontaneous and Mitochondria‐Mediated Cell Death in Neurons from Trisomy 16 Mice:

THIS ARTICLE HAS BEEN RETRACTED: Diminished Glutathione Levels Cause Spontaneous and Mitochondria‐Mediated Cell Death in Neurons from Trisomy 16 Mice:

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S‐transferase, 1–1

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