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Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

ABSTRACT: The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.

SUBMITTER: Cooke RJ

PROVIDER: S-EPMC1137239 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Project description:The hepatotoxicity of bromobenzene (BB) derives from its reactive metabolites (epoxides and quinones), which arylate cellular proteins. Application of proteomic methods to liver proteins from rats treated with a hepatotoxic dose of [14C]-BB has identified more than 40 target proteins, but no adducted peptides have yet been observed. Because such proteins are known to contain bromophenyl- and bromodihydroxyphenyl derivatives of cysteine, histidine, and lysine, the failure to observe modified peptides has been attributed to the low level of total covalent binding and to the "dilution" effect of multiple metabolites reacting at multiple sites on multiple proteins. In this work glutathione S-transferase (GST), a well-known and abundant BB-target protein, was isolated from liver cytosol of rats treated with 14C-BB by use of a glutathione (GSH)-agarose affinity column and further resolved by reverse-phase high-performance liquid chromatography (HPLC) into subunits M1, M2, A1, A2 and A3. The subunits were identified by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whole-molecule mass spectrometry, and peptide mass mapping and found to contain radioactivity corresponding to 0.01-0.05 adduct per molecule of protein. Examination of tryptic digests of these subunits by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization mass spectrometry (ESI-MS) failed to reveal any apparent adducted peptides despite observed sequence coverages up to 87%. However, use of HPLC-linear ion-trap quadrupole Fourier transform mass spectrometry (LTQ-FTMS) to search for predicted modified tryptic peptides revealed peaks corresponding, with a high degree of mass accuracy, to a bromobenzoquinone adduct of peptide 89-119 in both GSTA1 and A2. The identity of these adducts and their location at Cys-111 was confirmed by tandem mass spectrometry (MS-MS). No evidence for the presence of any putative BB-adducts in GST M1, M2, or A3 was obtained. This work highlights the challenges involved in the unambiguous identification of reactive metabolite adducts formed in vivo.

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Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

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