A high throughput screen identifies chemical modulators of the laminin-induced clustering of dystroglycan and aquaporin-4 in primary astrocytes.
(10/17)
Studies on the active site of rat glutathione S-transferase isoenzyme 4-4. Chemical modification by tetrachloro-1,4-benzoquinone and its glutathione conjugate.
(14/17)
The active site of glutathione S-transferase isoenzyme 4-4, purified from rat liver, was studied by chemical modification. Tetrachloro-1,4-benzoquinone, a compound previously shown to inactivate glutathione S-transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro-1,4-benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue. Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro-1,4-benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate. The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro-1,4-benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue. Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro-1,4-benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70-80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S-transferase 4 subunit. (+info)
Active site-directed irreversible inhibition of glutathione S-transferases by the glutathione conjugate of tetrachloro-1,4-benzoquinone.
(15/17)
Purified glutathione S-transferase from rat liver cytosol are irreversibly inhibited by the glutathione conjugate of tetrachloro-1,4-benzoquinone, 2-S-glutathionyl-3,5,6-trichloro-1,4-benzoquinone. The inhibition is due to covalent binding in or near the active site, resulting in modification of a single amino acid residue/subunit, presumably a cysteine residue. The amount of inhibition is related to the molar ratio of the inhibitor and the enzyme and is independent of the enzyme concentration. A 70-80% inhibition is obtained on incubating the enzyme with a 5-fold molar excess of the conjugate. Complete 100% inhibition is never reached. The derivative bound to the enzyme still possesses a quinone structure and is able to react with thiol-containing compounds. Reduction of the enzyme-bound quinone abolishes its reactivity but does not decrease the inhibition. At 0 degrees C, the glutathione conjugate of tetrachloro-1,4-benzoquinone inhibits the glutathione S-transferases at a much higher rate than the corresponding beta-mercaptoethanol conjugate, indicating a distinct targetting effect of the glutathione moiety. However, the parent compound, tetrachloro-1,4-benzoquinone, also has a considerable affinity for the enzymes. Although it does not react as fast as the glutathione conjugate, it reacts with the same amino acid residue. Protection from inhibition by the substrate analog S-hexylglutathione also indicates an active site-directed modification. Small but significant differences exist between the different rat liver transferase isoenzymes; using a 20-fold molar excess the inhibition ranges from 78 to 98% for the conjugate, and from 72 to 93% for the quinone, with isoenzyme 1-1 being the most and isoenzyme 2-2 the least inhibited forms. (+info)
Modification of glutathione S-transferase 3-3 mutants with 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone. Identification of the C-terminal tryptic fragment as part of the H-site and evidence that 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone is not specific for cysteine labelling.
(16/17)
A triple mutant of rat liver glutathione S-transferase 3-3 that has all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were reacted with 2-(S-glutathionyl)-3,5,6-trichloro-1,4-benzoquinone (GS-1,4-TCBQ). The modified proteins were analysed on a triple-quadrupole mass spectrometer equipped with an electrospray ionization source. At an enzyme: GS-1,4-TCBQ ratio of 1:10, the enzymes were modified at multiple sites. Covalent attachment of a single inhibitor on to the protein was achieved by lowering the enzyme: GS-1,4-TCBQ ratio to 1:1. Results from m.s. analyses suggest that the inhibitor on the CallSY115F mutant exists as a glutathionyl dichlorobenzoquinone derivative. The modifiers of the CallS mutants are glutathionyl monochlorobenzoquinone derivatives. Therefore, GS-1,4-TCBQ reacts at a single site on CallSY115F, but probably cross-links two regions on wild-type and CallS mutant. To confirm our observation, CallS was modified with 1-chloro2,4-dinitrobenzene, which specifically labels Tyr-115, before reacting with GS-1,4-TCBQ. The inhibitor formed a glutathionyl dichlorobenzoquinone adduct on the dinitrophenyl-CallS mutant. In addition, the benzoquinone derivative on the protein can be partially removed by 1-chloro-2,4-dinitrobenzene. Peptide mapping and sequencing analysis of the GS-1,4-TCBQ-modified CallS mutant revealed that the C-terminal 16-amino-acid fragment is labelled. Molecular modelling suggests the C(5) and C(6) on the benzoquinone ring of the inhibitor interact with the oxygen atoms of Tyr-115 and Ser-209 respectively. (+info)