Purification Of Glutathione S-transferase Research Articles

Escherichia coli and Sf9 Contaminant Databases to Increase Efficiency of Tandem Mass Spectrometry Peptide Identification in Structural Mass Spectrometry Experiments.

Filtering of nonspecifically binding contaminant proteins from affinity purification mass spectrometry (AP-MS) data is a well-established strategy to improve statistical confidence in identified proteins. The CRAPome (contaminant repository for affinity purification) describes the contaminating background content present in many purification strategies. However, full contaminant lists for nickel-nitrilotriacetic acid (NiNTA) and glutathione S-transferase (GST) affinity matrices are lacking. Similarly, no Spodoptera frugiperda (Sf9) contaminants are available, and only the FLAG-purified contaminants are described for Escherichia coli. For MS experiments that use recombinant protein, such as structural mass spectrometry experiments (hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking, and radical foot-printing), failing to include these contaminants in the search database during the initial tandem MS (MS/MS) identification stage can result in complications in peptide identification. We have created contaminant FASTA databases for Sf9 and E. coli NiNTA or GST purification strategies and show that the use of these databases can effectively improve HDX-MS protein coverage, fragment count, and confidence in peptide identification. This approach provides a robust strategy toward the design of contaminant databases for any purification approach that will expand the complexity of systems able to be interrogated by HDX-MS.

In vitro NLK Kinase Assay.

This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. KM values were 1.59 and 0.53 mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. Vmax values for CDNB and GSH were also determined as 5.58 and 1.88 EU/mL, respectively. In addition, inhibition effects of Ag+, Cu2+, Cd2+, Fe3+, Pb2+, Cr2+, Co2+ and Zn2+ metal ions were investigated on the enzyme activity and IC50, Ki values were calculated for these metal ions.

Biochemical Changes of Glutathione S-Transferase Activity During the Germination of Trifolium alexandrinum

The key objective of this study was to investigate the effect of germination on biochemical and enzymatic antioxidant activities of Trifolium alexandrinum seeds. The T. alexandrinum was chosen for GST purification and characterization due to its highest GST activity and antioxidant capacity, beside its economical importance. Seeds of T. alexandrinum were germinated for 6 days. Changes in glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) in germinating and dormant seeds of T. alexandrinum were evaluated. Also the changes in the total phenolic and flavonoid contents as well as the antioxidant capacities were monitored for 6 days of germination using spectrophotometeric methods. Simple reproducible procedures for the purification of T. alexandrinum GST from both dormant and 6 days germinated seeds were established using DEAE-Sepharose and Sephadex G-100 columns chromatography. The results showed the presence of three isoenzymes for both dormant and germinated seeds designated as GST1, GST2 and GST3. The major GST of the dormant seed was GST2 while for the germinated one was GST3. Dormant GST2 was expressed as a heterodimer with molecular weight of 27.5 and 28.5 KDa while germinated GST3 was expressed as a homodimer with molecular weight of 27 KDa. Characterization of both dormant GST1 & GST2 and germinated GST2 and GST3 including optimum pH, kinetic parameters [kmGSH of 1.17±0.39, 1.22±0.13, 0.84±0.19, 0.96±0.24mM, respectively and kmCDNB of 0.65±0.095, 0.57±0.11, 0.59±0.14, 1.02±0.18 mM, respectively]. The substrate selectivity using different electrophilic compounds and inhibitor effects were carried out. In opposite to other isoenzymes, germinated GST3 exhibited enzymatic activity towards ethacrynic acid with specific activity of 0.073±0.005 µmol/min/mg protein while cibacron blue was the most potent inhibitor for these isoenzymes.

Purification of Histidine‐tagged and GST tagged proteins using ÄKTA™ start (LB264)

Expression of recombinant proteins using affinity tags is a commonly used purification strategy. Histidine and glutathione‐S transferase (GST) are the most common and versatile of these tags. Histidine tags are small and therefore less disruptive to the protein structures and functions. GST tags enhance solubility of the target protein, hence improving the yield.Manual purification of tagged proteins often face issues with time to purify, low reproducibility and low yield etc. In addition a researcher who is purifying manually needs to spend time in setting‐up the run, sample loading, collection and measuring the final protein concentration.We describe here the purification Histidine‐tagged and GST‐tagged protein using ÄKTA start with Frac30 fraction collector and UNICORNTM start system control software.We purified an N‐terminal Histidine tagged protein expressed in E.coli on a pre‐packed HisTrapTM FF 1mL column. The purification method was completely automated for unattended operation by applying large volume of sample using pump and collecting peak fractions using Frac30 fraction collector. We have consistently obtained high levels of purity (> 95%) of the tagged protein, which is 1.6 fold higher when compared to a conventional manual purification method.In the second example, we describe the purification of GST tagged protein expressed in E.coli on a pre‐packed GSTrapTM FF 1ml column using ÄKTA start with Frac30 fraction collector. The target protein was purified using pre‐defined method workflow available from the instrument display. The result file that was stored on USB memory stick is imported for viewing and report generation using UNICORN start software.ÄKTATM start is the newly developed laboratory scale protein purification system that can address the challenges faced during manual purification. Automated purification of tagged proteins has showed higher yields and purity with high consistency.

A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins

Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100–500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.

A Strategy for Purifying Glutathione S-transferase in the Presence of Sodium Dodecyl Sulfate

Glutathione S-transferase (GST) is widely used to prepare and purify GSTtagged fusion proteins. Although GST improves protein solubility, detergents must often be used to achieve protein solubilization from bacterial lysates. However, purification of GST by affinity chromatography cannot be achieved in the presence of even low concentrations of the detergent sodium dodecyl sulfate (SDS). Here we show that 2-methyl-2,4-pentanediol (MPD) can prevent SDS from interfering with purification of GST, thus enabling purification of proteins that require SDS to improve their solubility.

Characterization of the Purified Glutathione S-transferases from Two Psocids Liposcelis bostrychophila and L. entomophila

Glutathione S-transferases (GSTs) from Liposcelis bostrychophila Badonnel and L. entomophila (Enderlein) (Psocoptera: Liposcelididae) were purified by glutathione-agarose affinity chromatography, and characterized subsequently by their Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH), respectively. The specific activity of the purified GST toward CDNB was 2.3-fold higher in L. bostrychophila than in L. entomophila. Though the specific activities of purified enzymes varied between the two species, the purification yields were similar. SDS-PAGE revealed one band at 23 kDa for both the species. GSTs of L. entomophila exhibited higher Michaelis-Menten constants ( K m) but lower maximal velocity ( V max) values than those of L. bostrychophila. The optimum pH for CDNB conjugation of L. bostrychophila and L. entomophila GSTs was 7.0 and 7.5, and optimum temperature was 35 and 40°C, respectively. Inhibition kinetics showed that cibacron blue, curcumin, bromosulfalein, ethacrynic acid, and carbosulfan had excellent inhibitory effects on GSTs in both species, but the inhibitory effects of beta-cypermethrin, fenpropathrin, tetraethylthiuram disulfide, and diethyl maleate were not significant.

Adult Drosophila melanogaster glutathione S-transferases: Effects of acute treatment with methyl parathion

The effect of acute treatment of methyl parathion (MP) on the expression of BSP/GSH-agarose purified glutathione S-transferases (GSTs) in Drosophila melanogaster was investigated. Using 2-D gel analysis of the identified Epsilon-class, only DmGSTE6 (100%) and DmGSTE7 (72%) demonstrated significant increases in expression, suggesting the possibility that both may be involved in MP metabolism. A smaller percentage increase was also observed in DmGSTD1 (18%), a known DDT dehydrochlorinase, DmGSTE3 and DmGSTE9 and a putative Epsilon-class GST, CG16936, were shown not to be responsive to the challenge. Our finding demonstrates that not all member of the Epsilon-class GST, which are known for their role in insecticide resistance, are immediately responsive to this toxic challenge.

Characterization of the Molecular Basis of Group II Intron RNA Recognition by CRS1-CRM Domains

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Arsenic Trioxide Augments Chk2/p53-mediated Apoptosis by Inhibiting Oncogenic Wip1 Phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.

Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

The study aims to detect the subcellular localization of ERF (ethylene-responsive element binding factor) transcription factor W17 protein, the interaction between W17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22°C in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST (glutathione-S-transferase) in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 (DE3). IPTG (0.5 mmol L −1) was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32P-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses (e.g., salt-) induced signaling pathway.

Biological Activities of Purified Harpin<sub>Xoo</sub> and Harpin<sub>Xoo</sub> Detection in Transgenic Plants Using Its Polyclonal Antibody

Many harpins have been found in plant pathogen bacteria that can elicit disease and insect resistance in plants, and promote plant growth. In this work, we overexpressed and purified Xanthomonas oryzae pv. oryzae harpin, harpin(Xoo), in Escherichia coli BL21/pGEX-hpa1. Harpin(Xoo) was fused to the C-terminus of glutathione S-transferase (GST) and purified using the Bulk GST purification module and thrombin cleavage capture kit. Purified harpin(Xoo) protein was sensitive to protease K and stable to heat treatment, and could not induce a hypersensitive response after treatment with various plant metabolic inhibitors; these characteristics were similar to harpin(Ea) of Erwinia amylovora. The purified harpin(Xoo) showed a similar ability to induce tobacco mosaic virus resistance in tobacco as harpin(Ea). Its antibody worked well in detecting the purified harpin(Xoo), harpin(Xoo) in the total protein of E. coli BL21/pGEX-hpa1 and an hpa1 transgenic rice.

A Novel Diacylglycerol-lactone Shows Marked Selectivity in Vitro among C1 Domains of Protein Kinase C (PKC) Isoforms α and δ as Well as Selectivity for RasGRP Compared with PKCα

Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms alpha, beta, gamma, delta, and epsilon, with apparent Ki values ranging from 340 nm for PKCalpha to 29 nm for PKCepsilon. When binding to isolated C1 domains of PKCalpha and -delta, 130C037 showed good affinity (Ki= 1.78 nm) only for deltaC1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of deltaC1b. 130C037 bound intact RasGRP1 and RasGRP3 with Ki values of 3.5 and 3.8 nm, respectively, reflecting 8- and 90-fold selectivity relative to PKCepsilon and PKCalpha. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED50 = 286 nm), partial reduction of PKCepsilon (ED50 > 10 microm), and no effect on PKCalpha. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKCepsilon, and no translocation of PKCalpha. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets.

Expression and evaluation of IgE-binding capacity of recombinant Pacific mackerel parvalbumin

ABSTRACT Background Parvalbumin is the major and cross-reactive allergen in fish. Sufficient amounts of IgE-reactive recombinant fish parvalbumin are needed for diagnosis and immunotherapy of fish allergy. Methods A DNA fragment corresponding to parvalbumin of the Pacific mackerel Scomber japonicus was synthesized and cloned into the expression vector pGEX-6p-3 to produce glutathione S-transferase (GST)-fusion parvalbumin in Escherichia coli. The GST-free recombinant parvalbumin was purified using the RediPack GST Purification Module (Amersham Pharmacia Biotech, Buckinghamshire, UK). Parvalbumins of seven species of fish (Japanese eel, horse mackerel, red sea bream, Pacific mackerel, skipjack, bigeye tuna and Japanese flounder) were purified by gel filtration and reverse-phase HPLC. The IgE-binding capacity was examined by ELISA and antigenic cross-reactivity by inhibition ELISA. Results The GST-free recombinant Pacific mackerel parvalbumin was obtained in an electrophoretically pure state. Data from ELISA and inhibition ELISA revealed that the recombinant parvalbumin contains most of the IgE-binding epitopes of the natural counterpart. In addition, the recombinant parvalbumin inhibited the IgE reactivities of the pooled patient serum to parvalbumins purified from six species of fish in almost the same magnitude as the natural Pacific mackerel parvalbumin. Conclusions Because the recombinant Pacific mackerel parvalbumin bearing the IgE-binding capacity of the natural counterpart is cross-reactive with various fish parvalbumins, it can be a useful tool for the diagnosis and immunotherapy of fish allergy.

A Bacterially Produced Virus Enhancing Factor from an Entomopoxvirus Enhances Nucleopolyhedrovirus Infection in Armyworm Larvae

Using an Escherichia coli expression system, pGEX-2T, that expresses foreign sequences as fusion proteins with a glutathione S-transferase (GST) carrier, we have expressed a virus enhancing factor (EF) from Pseudaletia separata entomopoxvirus, which enhances P. unipuncta multi nucleopolyhedrovirus (PsunMNPV) infection in larvae of the armyworm, P. separata. The lysates of transformed E. coli cells, which were not active in enhancing PsunMNPV infection, became active when treated with either trypsin or thrombin. The GST–EF fusion protein in a lysate was purified with a bulk GST purification module and cleaved into the EF and GST moieties with thrombin. Removal of the GST moiety with glutathione–Sepharose 4B resulted in a highly purified EF preparation, which enhanced PsunMNPV infection in armyworm larvae and PsunMNPV fusion with an armyworm cell line, SIE-MSH-805-F.

Purification, characterization and comparison of glutathioneS-transferases from black-grass (Alopecurus myosuroidesHuds) biotypes

In the UK biotypes of black-grass (Alopecurus myosuroides Huds) showing resistance to both chlorotoluron (CTU) and aryloxyphenoxypropionate graminicides are increasingly being observed. Although the precise mechanisms involved in this resistance have yet to be identified, increased herbicide metabolism has been implicated as being involved in at least some cases of resistance. Glutathione S-transferases (GSTs) are a group of enzymes which have been demonstrated to metabolise herbicides in some plants, and the resistant black-grass biotype Peldon contains approximately double the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) of susceptible biotypes. To investigate further the possible role of GSTs in herbicide resistance in black-grass, a purification procedure has been developed for these enzymes. A 27.5 kDa polypeptide possessing GST activity was purified from the susceptible biotype Herbiseed. Purification of GSTs from the resistant biotype Peldon also identified this polypeptide along with an additional 30 kDa polypeptide. An in-vitro kinetic study of both crude and purified GST extracts, and western blot analysis using antisera raised against the 27.5 kDa polypeptide, suggest that the 30 kDa polypeptide may possess GST activity, and is not a precursor of the 27.5 kDa polypeptide. These results are discussed and compared to GST profiles for other weeds and crops demonstrating herbicide resistance or tolerance.

Conjugation of isoprene monoepoxides with glutathione, catalyzed by α, μ, π and θ-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the α, μ, π and θ-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [ 35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the μ and θ class glutathione S-transferases were much more efficient than the α and π class glutathione S-transferases, both for rat and man. Because the μ- and θ-class glutathione S-transferases are expressed in about 50 and 40–90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glutathione S-transferases: rat GST Tl-1 showed about 2.1–6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2–14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50–2000 μM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25–50-fold lower rate of GSH conjugation.

Stereoselective formation of glutathione S-conjugates from halovinylmercapturate sulphoxides

1. The glutathione S-transferase catalysed formation of glutathione S-conjugates from halovinylmercapturate sulphoxides was investigated in rat liver and kidney cytosol, with purified glutathione S-transferases and in rat in vivo. 2. The two diastereomers of the sulphoxides of N-acetyl-S-(1,2-dichlorovinyl)-Lcysteine, N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine show different reactivities with glutathione and glutathione S-transferases. Rat liver and kidney cytosol catalyses the formation of a 1:1 mixture of two diastereomers of (E)-N-acetyl-S-(2-glutathione-S-yl-2-chlorovinyl)-L-cysteine sulphoxide from N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulphoxide and of (E)-N-acetyl-S-(2-glutathione-S-yl-1,2-dichlorovinyl)-L-cysteine sulphoxide from N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulphoxide. In contrast, only one diastereomer of the Z-isomers was formed. 3. N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine sulphoxide reacted spontaneously with glutathione at high rates, a 1:1 mixture of both diastereomers of N-acetyl-S-(2-glutathione-S-yl-1-chlorovinyl)-L-cysteine sulphoxide was formed. 4. Metabolism of N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine sulfoxide and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine sulfoxide under by α-class glutathione S-transferases yielded identical products as observed with the cytosolic enzymes. No reaction was observed in the presence of rat liver μ class glutathione S-transferases or human glutathione S-transferase M1. 5. Formation of these glutathione conjugates was also observed in the bile of rat after i.p. administration of the mercapturic acid sulphoxides. The results obtained show that stereochemical aspects may govern the regioselectivity and substrate specificity in glutathione S-transferase-catalysed reactions.

Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants.

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.