Trypanothione S-Transferase Activity in a Trypanosomatid Ribosomal Elongation Factor 1B
2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês
10.1074/jbc.m311039200
ISSN1083-351X
AutoresTim J. Vickers, Alan H. Fairlamb,
Tópico(s)Research on Leishmaniasis Studies
ResumoTrypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the α, β, and γ subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the γ subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bγ genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bγ had no S-transferase activity, suggesting that eEF1Bγ is unstable in the absence of the other subunits. Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the α, β, and γ subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the γ subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bγ genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bγ had no S-transferase activity, suggesting that eEF1Bγ is unstable in the absence of the other subunits. Infections with parasitic protozoa of the order Kinetoplastida are a common cause of serious illness and death in the tropics. Trypanosoma brucei sp. cause sleeping sickness in Africa, Trypanosoma cruzi is the cause of Chagas' disease in South America, and infections with Leishmania sp. produce a variety of pathologies termed the leishmaniases in Asia, Africa, South America, and Europe. In general, the chemotherapy of these diseases is poor, with the available drugs suffering various drawbacks such as toxicity, limited efficacy, and drug resistance (1Fairlamb A.H. Trends Parasitol. 2003; 19: 488-494Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Croft S.L. Coombs G.H. Trends Parasitol. 2003; 19: 502-508Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar). For instance, the majority of cases of visceral leishmaniasis in India do not respond to antimonials, which are the first-line drugs (3Zilberstein D. Ephros M. Black S.J. Seed J.R. World Class Parasites (Vol. 4): Leishmania. Kluwer Academic Press, London2002: 115-136Google Scholar). A promising target for the design of new drugs to treat these illnesses involves thiol metabolism. In these parasites, this depends upon trypanothione (T[SH]2 1The abbreviations used are: T[SH]2 and T[S]2, trypanothione and trypanothione disulfide, respectively; GSH and GSSG, glutathione and glutathione disulfide, respectively; GspdSH, glutathionylspermidine; CDNB, 1-chloro-2,4-dinitrobenzene; T[SDNB]2, trypanothione bis-dinitrobenzene; BS3, bis(sulfosuccinimidyl)suberate; GST, glutathione S-transferase; TST, trypanothione S-transferase; eEF1A, eukaryotic elongation factor 1A (formerly eEF-1α); eEF1B, the eukaryotic elongation factor 1B complex (formerly eEF-1βγδ) (eEF1Bα was formerly eEF-1β, eEF1Bβ was formerly eEF-1δ, and eEF1Bγ was formerly eEF-1γ); ARS, aminoacyl-tRNA synthetase; RT, reverse transcription; DTT, dithiothreitol; BSA, bovine serum albumin; PEG, polyethylene glycol; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; NEM, N-ethylmaleimide; MALDI-TOF, matrix-assisted laser desorption time of flight; ValRS, valyl-tRNA synthetase. or N1,N8-bis(glutathionyl)spermidine) (4Fairlamb A.H. Blackburn P. Ulrich P. Chait B.T. Cerami A. Science. 1985; 227: 1485-1487Crossref PubMed Scopus (597) Google Scholar), in contrast to most other organisms (including their mammalian hosts), which use glutathione as the unmodified tripeptide (Fig. 1) (5Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (691) Google Scholar). The main function of trypanothione is the maintenance of cellular redox state, and consequently, studies have concentrated on its role as an antioxidant (6Flohé L. Hecht H.J. Steinert P. Free Radic. Biol. Med. 1999; 27: 966-984Crossref PubMed Scopus (184) Google Scholar). Another major function for cellular thiols is the detoxification of xenobiotics, i.e. chemicals that are foreign to a particular organism. These include naturally occurring compounds, industrial chemicals, drugs, herbicides, and pesticides. Hydrophobic xenobiotics can readily diffuse into cells and are usually eliminated by Phase I and Phase II biotransformation reactions. These produce generally less reactive and more polar compounds, which can be actively extruded from the cytosol. An important group of Phase II reactions are catalyzed by glutathione S-transferases (GSTs) and involve conjugation with glutathione (7Sies H. Ketterer B. Glutathione Conjugation: Mechanisms and Biological Significance. Academic Press, London1988Google Scholar). GSTs have been purified from a wide range of organisms and are almost invariably dimers of subunits with masses of ∼25 kDa. In any one organism, many different GST isozymes are usually expressed simultaneously, with these isozymes having wide and overlapping substrate specificities. Such a broad specificity system assists an organism in the metabolism of the multitude of different reactive xenobiotics to which it may be exposed. In addition, GSTs are also important in the detoxification of endogenous reactive chemical species produced during oxidative stress, such as lipid hydroperoxides (8Hurst R. Bao Y.P. Jemth P. Mannervik B. Williamson G. Biochem. J. 1998; 332: 97-100Crossref PubMed Scopus (128) Google Scholar) or reactive aldehydes (9Berhane K. Widersten M. Engstrom A. Kozarich J.W. Mannervik B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1480-1484Crossref PubMed Scopus (386) Google Scholar). The GSTs therefore have a central role in detoxification metabolism, and the overexpression of these enzymes is a common mechanism of drug resistance (10Hayes J.D. Wolf C.R. Sies H. Ketterer B. Glutathione Conjugation: Mechanisms and Biological Significance. Academic Press Limited, London1988: 316-355Google Scholar). Although the classical GSTs have been well characterized, the GST fold is also found in functionally unrelated proteins such as plant stress-induced proteins (11Czarnecka E. Nagao R.T. Key J.L. Gurley W.B. Mol. Cell. Biol. 1988; 8: 1113-1122Crossref PubMed Scopus (127) Google Scholar), β-etherases (12Masai E. Katayama Y. Kubota S. Kawai S. Yamasaki M. Morohoshi N. FEBS Lett. 1993; 323: 135-140Crossref PubMed Scopus (95) Google Scholar), ion channels (13Dulhunty A. Gage P. Curtis S. Chelvanayagam G. Board P. J. Biol. Chem. 2001; 276: 3319-3323Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), and the eukaryotic translation elongation factor 1Bγ (eEF1Bγ) (14Jeppesen M.G. Ortiz P. Shepard W. Kinzy T.G. Nyborg J. Andersen G.R. J. Biol. Chem. 2003; 278: 47190-47198Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15Koonin E.V. Mushegian A.R. Tatusov R.L. Altschul S.F. Bryant S.H. Bork P. Valencia A. Protein Sci. 1994; 3: 2045-2054Crossref PubMed Scopus (131) Google Scholar). The functions of the GST domain in these proteins remain poorly understood, although the eEF1Bγ from rice has recently been shown to possess a GST activity (16Kobayashi S. Kidou S. Ejiri S. Biochem. Biophys. Res. Commun. 2001; 288: 509-514Crossref PubMed Scopus (30) Google Scholar). This result was surprising, because eEF1B was previously thought only to be involved in protein synthesis, and no enzymatic activity had been proposed for the eEF1Bγ subunit. Previously, the other two subunits (eEF1Bα and β) of eEF1B were shown to function to recycle elongation factor 1A (eEF1A) complexed with GDP back to the active eEF1A·GTP form (17van Damme H.T. Amons R. Karssies R. Timmers C.J. Janssen G.M. Moller W. Biochim. Biophys. Acta. 1990; 1050: 241-247Crossref PubMed Scopus (92) Google Scholar, 18Janssen G.M. Moller W. Eur. J. Biochem. 1988; 171: 119-129Crossref PubMed Scopus (107) Google Scholar). This eEF1A·GTP complex is then able bind to an aminoacyl tRNA, forming a ternary complex that is able to enter the A-site of the ribosome, with the hydrolysis of GTP. In contrast to the wealth of information on GSTs and related proteins in other organisms, little is known about thiol-xenobiotic conjugation in trypanosomes. Although there has been one report of low levels of GST activity in T. cruzi (19Yawetz A. Agosin M. Comp. Biochem. Physiol. 1981; 68B: 237-243Google Scholar), this has been disputed (20Moutiez M. Meziani-Cherif D. Aumercier M. Sergheraert C. Tartar A. Chem. Pharm. Bull. (Tokyo). 1994; 42: 2641-2644Crossref Scopus (45) Google Scholar). In addition, no GST activities have been reported in the related Leishmania sp. or T. brucei. However, because the major low molecular mass thiol in these organisms is trypanothione, it has been proposed that these trypanosomatids instead possess a trypanothione S-transferase or TST (5Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (691) Google Scholar). This activity may also be involved in the resistance of Leishmania sp. to the metalloid antimony (21Legare D. Richard D. Mukhopadhyay R. Stierhof Y.D. Rosen B.P. Haimeur A. Papadopoulou B. Ouellette M. J. Biol. Chem. 2001; 276: 26301-26307Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), which is currently the front-line drug for the treatment of leishmaniasis. It has been found that the acquisition of high level antimony resistance in L. tarentolae requires the overproduction of trypanothione, the overexpression of metal-thiol conjugate transporters and a third unidentified trypanothione-dependent factor (22Grondin K. Haimeur A. Mukhopadhyay R. Rosen B.P. Ouellette M. EMBO J. 1997; 16: 3057-3065Crossref PubMed Scopus (167) Google Scholar). This factor was postulated to be a TST activity involved in the formation of an antimony-trypanothione conjugate. Indeed, in an analogous system, the mechanism of resistance of mammalian cells to arsenite involves the efflux of the metalloid, which is thought to be facilitated by the overexpression of a GST-pi (23Liu J. Chen H. Miller D.S. Saavedra J.E. Keefer L.K. Johnson D.R. Klaassen C.D. Waalkes M.P. Mol. Pharmacol. 2001; 60: 302-309Crossref PubMed Scopus (206) Google Scholar, 24Wang H.F. Lee T.C. Biochem. Biophys. Res. Commun. 1993; 192: 1093-1099Crossref PubMed Scopus (65) Google Scholar). Here, we report the detection of TST activities in trypanosomatids and the purification and characterization of the Crithidia fasciculata TST. This is identified as the ribosomal elongation factor eEF1B complex and the subunit containing the TST active site defined as eEF1Bγ. Materials—All reagents were standard commercial products and of the highest available purity. Trypanothione and glutathionylspermidine were obtained from Bachem. Glutathione affinity agaroses were obtained from Sigma, all other chromatographic resins and columns were from Amersham Biosciences. Rat liver GST was purchased as a mixture of isoforms from Sigma. Cell Culture—C. fasciculata choanomastigotes; T. cruzi epimastigotes; T. brucei bloodstream form; and L. major, L. infantum, and L. tarentolae promastigotes were grown as described previously (25Ariyanayagam M.R. Fairlamb A.H. Mol. Biochem. Parasitol. 2001; 115: 189-198Crossref PubMed Scopus (173) Google Scholar, 26Haimeur A. Brochu C. Genest P.A. Papadopoulou B. Ouellette M. Mol. Biochem. Parasitol. 2000; 108: 131-135Crossref PubMed Scopus (121) Google Scholar). Enzyme Assays—GST was assayed at 25 °C in 100 mm (Na+) phosphate, pH 6.5, with 1 mm 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mm GSH as substrates. The rate of formation of the glutathione S-dinitrobenzene conjugate was followed at 340 nm, using the published extinction coefficient of 9.6 mm cm-1 (27Habig W.H. Jakoby W.B. Methods Enzymol. 1981; 77: 398-405Crossref PubMed Scopus (2098) Google Scholar). TST activity was measured under identical conditions with T[SH]2 and CDNB being added to 400 μm. Assay reactions were initiated with thiol, which was produced immediately before addition to assays by mixing trypanothione disulfide (T[S]2) with a 2-fold excess of tris(2-carboxyethyl)phosphine and a 5-fold excess of NaOH. The absorbance coefficient for the T[SDNB]2 conjugate was measured by allowing assays to proceed to completion and found to be 9.2 mm-1 cm-1 per millimolar of sulfhydryl group. One unit of TST activity corresponds to 1 μmol of sulfhydryl group conjugated per minute. Where necessary, in TST assays with alternative electrophilic substrates, the pH and substrate concentrations given for the corresponding GST assay (27Habig W.H. Jakoby W.B. Methods Enzymol. 1981; 77: 398-405Crossref PubMed Scopus (2098) Google Scholar) were altered to minimize the rate of the spontaneous reaction. The activities with these substrates were calculated using the absorbance coefficients for the corresponding GSH conjugates. In the case of the peroxidase, dehydroascorbate reductase and thioltransferase assays, tris(2-carboxyethyl)phosphine was omitted and T[S]2 was reduced by 10 μg ml-1 trypanothione reductase and 540 μm NADPH, before addition of the second substrate. For enzyme purification, aliquots of fractions were screened for TST activity using a Molecular Devices Thermomax plate reader. In this assay, samples were diluted in the plate with water to give a final volume of 50 μl, and the reactions were initiated by the addition of 200 μl of a mixture containing buffer, dithiothreitol (DTT), and substrates. The final assay mixture contained 800 μm CDNB, 1 mm DTT, 50 μm T[S]2, and 100 mm (Na+) phosphate, pH 6.5. The average rate of two blank assays was subtracted from these rates. Protein concentrations were measured using the procedure of Bradford (28Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217547) Google Scholar), using BSA as the analytical standard. All trypanosomatid extracts were produced as described below for C. fasciculata, and mouse liver was extracted by disruption in a Dounce homogenizer and then processed as for C. fasciculata cells. Purification of the C. fasciculata TST—Unless otherwise stated, all the steps in the following purification were performed at 4 °C. Frozen C. fasciculata cell pellets were thawed on ice and resuspended in an equal volume of ice-cold lysis buffer, giving in the final mixture 75 mm (Na+) phosphate, pH 7.2, 2 mm DTT, 1 mm EDTA, 1 mm benzamidine, 1 mm phenanthroline, 3 μg ml-1 leupeptin, 250 μm 4-(2-aminoethyl-)benzenesulfonyl fluoride, and 1 μm pepstatin A. The cells were lysed by sonication (4 × 30 s pulses at 20-μm amplitude from a 19-mm enddiameter probe) in a Sanyo Soniprep 150 sonicator with cooling to <4 °C in an ice-salt bath between pulses. After centrifugation for 80 min at 40,000 × g, the resulting supernatant was brought to a final concentration of 4% (w/v) polyethylene glycol 6000 (PEG) over 5 min, by the dropwise addition of PEG from a 50% (w/v) stock, stirred for 30 min, and centrifuged for 30 min at 30,000 × g. The supernatant was adjusted to 9% (w/v) PEG and centrifuged as before. The resulting pellet was resuspended in 150 ml of buffer A (25 mm (Na+) Bis-tris, 1 mm EDTA, 1 mm DTT, pH 6.5) and then centrifuged to remove insoluble material. The clarified supernatant was applied at 2 ml min-1 to a 100-ml (19 × 2.6 cm) Amersham Biosciences Q-Sepharose anion exchange column equilibrated in buffer A. After washing with 2 column volumes of buffer A, bound proteins were eluted at 1 ml min-1 with a linear gradient of 200–500 mm NaCl in the same buffer, and active fractions were pooled. Following overnight dialysis against 2 liters of 550 mm (NH4)2SO4 in buffer B (25 mm (Na+) HEPES, 1 mm EDTA, 1 mm DTT, pH 7), the sample was applied at 2 ml min-1 to an 85-ml (16 × 2.6 cm) Amersham Biosciences phenyl-Sepharose (low substitution) column equilibrated with 550 mm (NH4)2SO4 in buffer B. The column was washed with 5 column volumes of the same buffer, and TST activity eluted with 250 ml of buffer B. Active fractions were pooled and concentrated to 1 ml by vacuum ultrafiltration in a Sartorius collodion bag. The concentrated TST was applied to a 319-ml (2.6 × 60 cm) Superdex 200 26/60 size-exclusion column equilibrated with buffer C (50 mm (Na+) HEPES, 300 mm NaCl, 0.01% (w/v) sodium azide, pH 7.5) and eluted at a flow rate of 2 ml min-1. Fractions with TST activity were pooled and concentrated as before. Analytical Chromatography—Analytical size-exclusion chromatography was carried out using a 24-ml (1 × 30 cm) Superdex 200 HR 10/30 size-exclusion column equilibrated with buffer C, with separations performed at a flow rate of 0.5 ml min-1. This procedure was also used to calculate the relative molecular weight (Mr) of proteins, using carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa) as analytical standards. Analytical anion-exchange chromatography was carried out using a 6-ml (1.6 × 3 cm) Amersham Biosciences Resource Q column under the same buffer conditions as the Q-Sepharose step in the TST purification scheme. The sample was applied at 1 ml min-1, and the column was washed with 2 column volumes of buffer. The bound proteins were then eluted with a complex gradient that went from 0–200 mm NaCl in 6 ml, 200–500 mm NaCl in 60 ml, and then 500–1000 mm NaCl in 6 ml, all in buffer A. Analysis of Products of TST Reaction—Assays for mass spectrometric analysis were performed in 200 mm ammonium acetate, pH 6.5, with the other conditions as before. The reactions were followed at 340 nm and after 30 min 2-hydroxyethyl disulfide was added to 10 mm, oxidizing the T[SH]2 by disulfide exchange and thus quenching the trypanothione reaction. The products were then lyophilized in glass vials, re-dissolved in water and diluted 1:500 with 1:1 acetonitrile and water. Samples were analyzed on a Micromass Ultima electrospray mass spectrometer in positive mode. Several scans were combined, background-subtracted, and smoothed to produce the final spectra. Cross-linking Analysis—All samples and controls were dialyzed overnight against 55 mm (Na+) phosphate, pH 7.5, reaction buffer, protein samples were then diluted to 1 mg ml-1 in reaction buffer. The amine-reactive homobifunctional reagent bis(sulfosuccinimidyl)suberate (BS3) was added, from a freshly prepared 10 mm stock in 5 mm sodium succinate, pH 5, to the required final concentration. The reactions were incubated at room temperature for 40 min and then quenched by the addition of ethanolamine to a final concentration of 5 mm. Aldolase was used as a positive control and BSA and carbonic anhydrase as negative controls. Samples from the reactions were then analyzed by using either 10% SDS-PAGE gels (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar), to examine complexes less than 150 kDa or 6% Weber SDS-PAGE (30Davies G.E. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 651-656Crossref PubMed Scopus (719) Google Scholar) to examine complexes greater than 150 kDa. Dissociation of Complex—For thiocyanate disassociation, samples of TST in buffer C were incubated on ice with 2 mm DTT for 3 h in the presence or absence of 1.5 m NaSCN. The proteins were then applied to a Superdex 200 HR 10/30 column equilibrated with either buffer C or this buffer plus 500 mm NaSCN, and eluted at 0.5 ml min-1. Because NaSCN inhibits the TST assay, 200-μl samples from each column fraction were dialyzed against TST assay buffer (100 mm (Na+) phosphate, pH 6.5) before analysis by enzyme assay and SDS-PAGE. Inhibition and Chemical Modification of TST—Modification with N-ethylmaleimide (NEM) was carried out in an 80-μl volume at room temperature in buffer C. Samples of TST (16 μg and 61 milliunits) were incubated with 100 μm DTT for 5 min, and then NEM was added to a final concentration of 400 μm. After a 15-min incubation, the reactions were quenched by the addition of DTT to 625 μm. Inhibition of TST by Cibacron blue F3G-A was determined under standard assay conditions, in the presence or absence of 0.5% (w/v) BSA. Routinely, TST was preincubated with the inhibitor, but no decrease in inhibition was observed when assays were initiated with enzyme. Dye concentrations were determined spectrophotometrically, using the absorbance coefficients of ϵm = 11.6 (622 nm) for Cibacron blue (31Ashton A.R. Gideon G.M. Biochem. J. 1978; 175: 501-506Crossref PubMed Scopus (87) Google Scholar) and ϵm = 4.2 (610 nm) for reactive blue 4 (32Small D.A.P. Lowe C.R. Atkinson T. Bruton C.J. Eur. J. Biochem. 1982; 128: 119-123Crossref PubMed Scopus (37) Google Scholar). The rate of time-dependent inhibition of TST by chlorotriazine dyes was measured in reaction mixtures of 100 μl containing 70 milliunits (18 μg) samples of TST and varying concentrations of dye in 20 mm (Na+) phosphate, pH 7.5. The reactions were allowed to proceed at room temperature, with 5-μl samples being removed at intervals to determine residual TST activity. These samples were assayed by 100-fold dilution into assay mixtures containing 0.5% (w/v) BSA, to prevent inhibition by any unreacted dye. Incubations with CDNB were performed under identical conditions, and samples were assayed in standard assay conditions. T[SH]2 and GSH were generated in incubations by the addition of 600 μm NADPH, 1 milliunit of trypanothione reductase or glutathione reductase, and 500 μm T[S]2 or GSSG. The resulting data were fitted by non-linear regression analysis to the single exponential decay function and expressed as a percentage of the activity at zero time. Samples of proteins for mass spectrometry were desalted by dialysis against one liter of water for 3 h at 4 °C, before analysis by MALDI-TOF mass spectrometry on a PerSeptive Biosystems Voyager-DE STR mass spectrometer. Cloning of eEF1BG1 and eEF1BG2 from C. fasciculata—A partial clone of the C. fasciculata eEF1BG1 was obtained by RT-PCR from cDNA prepared using the Cells-to-cDNA kit (Ambion). The sense primer (5′-CGCTATATAAGTATCAGTTTCTGTA-3′) contained 25 nucleotides of the mini-exon sequence that is trans-spliced onto the 5′-end of all trypanosomatid mRNA transcripts (33Perry K.L. Watkins K.P. Agabian N. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8190-8194Crossref PubMed Scopus (131) Google Scholar, 34Gabriel A. Sisodia S.S. Cleveland D.W. J. Biol. Chem. 1987; 262: 16192-16199Abstract Full Text PDF PubMed Google Scholar). The degenerate antisense primer (5′-CCYTCCCANGCNAGGTAKTC-3′) was designed to the tryptic peptide ITDYLA(F/W)EGPTIPLPV. PCR was performed in 50-μl volume reactions containing template cDNA, 20 ng ml-1 sense and antisense primers, 250 μm dNTPs, 5 units of Taq polymerase (Promega), and Taq buffer plus 1.5 mm MgCl2. The reactions were placed in a thermocycler that had been preheated to 95 °C and subjected to the following: 95 °C for 5 min, 30 cycles of 95 °C for 1 min, 55 °C for 1 min, then 72 °C for 2 min, and finally the reactions were heated to 72 °C for 10 min. PCR products were then cloned into the pCR-Blunt II-TOPO plasmid using the Zero Blunt TOPO PCR cloning kit (Invitrogen). Southern blotting was carried out by the standard capillary transfer method (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1989Google Scholar), using the RT-PCR clone of eEF1BG1 as a probe. The probe was labeled using the Gene Images labeling kit and hybridization detected with the Gene Images dioxetane detection module (Amersham Biosciences). PCR amplification of C. fasciculata eEF1BG intergenic regions was carried out using sense (5′-GGAGCTGTTTGACTGGGAGGAGAT-3′) and antisense primers (5′-GAAGTCCGCCGTCTCGTTGTC-3′) as described above, with the substitution of Pfu polymerase and Pfu buffer (Promega) for Taq polymerase. Four intergenic regions were amplified, cloned, and sequenced. Multiple primers were then designed to their 5′ and 3′ regions. One pair of these primers (sense 5′-GCACCGGCGTACCTGATGACTT-3′ and antisense 5′-TTAGTGGCCACTGATGCGACAGC-3′) produced a product that was cloned, sequenced, and identified as an eEF1Bγ gene (eEF1BG2). This gene was then cloned into the expression vector pET15b (Novagen), and recombinant protein was expressed and purified according to the manufacturer's instructions. Nomenclature—The terminology of eukaryotic elongation factors is somewhat confusing. In this report we have used the IUBMB nomenclature recommendations (available www.chem.qmul.ac.uk/iubmb/misc/trans.html), with the elongation factor 1B complex, previously named eEF-1βγδ being referred to as eEF1B. The complex's subunits are, in order of increasing size, elongation factor 1B α (previously eEF-1β), now referred to as eEF1Bα; elongation factor 1B β (previously eEF-1δ) referred to as eEF1Bβ and elongation factor 1B γ (previously eEF-1γ) referred to as eEF1Bγ. The SWISS-PROT identifier is used when referring to previously characterized elongation factors. Detection and Initial Characterization of Trypanothione S-Transferase Activities in Trypanosomatids—Clarified extracts of various organisms and cell types were assayed for S-transferase activities (Table I). L. tarentolae (a lizard parasite) was included, because this organism has been frequently used in studies on Leishmania antimony resistance. Mouse liver extract was assayed as a positive control for the GST assay and trypanothione reductase activity was used to confirm adequate extraction of the parasites. No GST activity was detected in the trypanosomatids. Instead a trypanothione S-transferase (TST) activity was detected in C. fasciculata, all the Leishmania sp. and T. brucei. The TST activity in mouse liver extracts is probably due to the ability of GSTs to use T[SH]2 as an alternative substrate, although with a specific activity 100-fold less than that given with GSH. Because TST activity was highest in C. fasciculata and L. major, these activities were further characterized. Activity in these extracts was proportional to the amount of protein added, heat labile, and, when analyzed by size-exclusion chromatography the activity eluted close to the void volume of the column, showing Mr values greater than 400,000 (data not shown). This is in contrast to the GST activity in the mouse liver extract, which eluted with an Mr of 45,000 (this value being identical to the reported Mr of the three major mouse glutathione S-transferases) (36Warholm M. Jensson H. Tahir M.K. Mannervik B. Biochemistry. 1986; 25: 4119-4125Crossref PubMed Scopus (56) Google Scholar).Table ISpecific activities of TST, GST, and trypanothione reductase in soluble extracts of trypanosomatids All enzyme activities were assayed as described in the relevant section under "Experimental Procedures" and corrected for non-enzymatic background rates.OrganismCell typeTSTGSTTrypanothione reductasemilliunits mg-1C. fasciculataChoanomastigotes16.7<0.1aLess than the limit of detection920L. majorPromastigotes6.4<0.1580L. infantumPromastigotes2.9<0.180L. tarentolaePromastigotes0.4<0.1430T. cruziEpimastigotes<0.1aLess than the limit of detection<0.1510T. bruceiBloodstream form2.8<0.130M. musculusLiver231970NDbND, not determineda Less than the limit of detectionb ND, not determined Open table in a new tab A pilot study showed that C. fasciculata TST activity was not retained on S-hexylglutathione, glutathione disulfide, or glutathione coupled to epoxy-activated agarose; in contrast to the mouse liver GSTs, which were completely retained on S-hexylglutathione-agarose (data not shown). Purification of C. fascic
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