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• W2045915921 abstract "Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculataby aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 μmol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting K m values for Mg2+-ATP, GSH, and spermidine of 0.25 ± 0.02, 2.51 ± 0.33, and 0.47 ± 0.09 mm, respectively, and a k cat of 415 ± 78 min−1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (K i = 0.08 mm) and trypanothione exerted a feedback inhibition competitive with GSH (K i = 0.48 mm). Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculataby aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 μmol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting K m values for Mg2+-ATP, GSH, and spermidine of 0.25 ± 0.02, 2.51 ± 0.33, and 0.47 ± 0.09 mm, respectively, and a k cat of 415 ± 78 min−1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (K i = 0.08 mm) and trypanothione exerted a feedback inhibition competitive with GSH (K i = 0.48 mm). Glutathionylspermidine synthetase (GspS) 1The abbreviations used are: GspS, glutathionylspermidine synthetase; Gsp, glutathionylspermidine; TSH, trypanothione; TS, trypanothione synthetase; DTT, dithiothreitol; bis-Tris propane, 1,3-bis[tris(hydroxymethy)methylamino]propane; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography. 1The abbreviations used are: GspS, glutathionylspermidine synthetase; Gsp, glutathionylspermidine; TSH, trypanothione; TS, trypanothione synthetase; DTT, dithiothreitol; bis-Tris propane, 1,3-bis[tris(hydroxymethy)methylamino]propane; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography. catalyzes the first of two steps of trypanothione biosynthesis, the synthesis of glutathionylspermidine (Gsp) from GSH and spermidine with the consumption of ATP (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar). Trypanothione (N 1,N 8-bis(glutathionyl)spermidine; TSH) is a metabolite unique to trypanosomatids such asTrypanosoma sp., Leishmania sp., andCrithidia fasciculata (2Shim H. Fairlamb A.H. J. Gen. Microbiol. 1988; 134: 807-817Google Scholar). These parasites comprise pathogens causing widespread and difficult to treat tropical diseases such as African sleeping sickness (Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense), Chagas disease (Trypanosoma cruzi), kala azar (Leishmania donovani), oriental sore (Leishmania tropica), and mucocutaneous leishmaniasis (Leishmania braziliensis). Others (e.g. Trypanosoma congolense) affect domestic animals, whereas C. fasciculata is pathogenic to insects only. Since the discovery of TSH in 1985 (3Fairlamb A.H. Blackburn P. Ulrich P. Chait B.T. Cerami A. Science. 1985; 227: 1485-1487Google Scholar, 4Fairlamb A.H. Cerami A. Mol. Biochem. Parasitol. 1985; 14: 187-198Google Scholar), the pathways for its synthesis and utilization have attracted considerable interest as potential targets for selective therapeutic intervention (5Schirmer R.H. Müller J.G. Krauth-Siegel R.L. Ang. Chemie (Int. Ed.). 1995; 34: 141-154Google Scholar, 6Fairlamb A. H Biochemist. 1996: 11-16Google Scholar). In all trypanosomatids, TSH substitutes for GSH in the defense against hydroperoxides and derived reactive oxygen species because of its ability to reduce peroxides either enzymatically (7Penketh P.G. Klein R.A. Mol. Biochem. Parasitol. 1986; 20: 111-121Google Scholar, 8Penketh P.G. Kennedy W.P.K. Patton C.L. Sartorelli A.C. FEBS Lett. 1987; 2: 427-431Google Scholar, 9Henderson G.B. Fairlamb A.H. Cerami A. Mol. Biochem. Parasitol. 1987; 24: 39-45Google Scholar) or spontaneously (10Carnieri E.G.S. Moreno S.N.J. Docampo R. Mol. Biochem. Parasitol. 1993; 61: 79-86Google Scholar). It thereby protects the parasitic trypanosomatids, which apparently are deficient in catalase and glutathione peroxidases (11Boveris A. Sies H. Martino E.E. Docampo R. Turrens J.F. Stoppani A.O.M. Biochem. J. 1980; 188: 643-648Google Scholar), against oxidative stress for instance during host-defense reactions (9Henderson G.B. Fairlamb A.H. Cerami A. Mol. Biochem. Parasitol. 1987; 24: 39-45Google Scholar, 12Babior B.M. Kipnes R.S. Curnutte J.T. J. Clin. Invest. 1973; 52: 741-744Google Scholar, 13Klebanoff S.J. Rosen H. Oxygen Free Radicals and Tissue Damage. Ciba Foundation Symposia 65 Exepta Medica, Amsterdam1979: 263-284Google Scholar). Trypanothione disulfide thus formed is reduced by the NADPH-dependent trypanothione reductase (14Krauth-Siegel R.L. Schöneck R. FASEB J. 1995; 9: 1138-1146Google Scholar, 15Bailey S. Smith K. Fairlamb A.H. Hunter W.N. Eur. J. Biochem. 1993; 213: 67-75Google Scholar), a flavoprotein homologous to glutathione reductase that, together with glutathione peroxidases (16Flohé L. Dolphin D. Poulson R. Avramovic A. Glutathione: Chemical, Biochemical, and Medical Aspects. John Wiley & Sons, Inc., New York1989: 643-731Google Scholar, 17Ursini F. Maiorino M. Brigelius-Flohé R. Aumann K.D. Roveri A. Schomburg D. Flohé L. Methods Enzymol. 1995; 252: 38-53Google Scholar), constitutes a major part of the defense system of the host (18Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Google Scholar, 19Flohé L. Giertz H. Beckmann R. Bonta L. Bray M.A. Parnham M.J. Handbook of Inflammation. Elsevier Science Publishers B.V., Amsterdam1985: 255-281Google Scholar). The precursor of TSH, Gsp, may have a distinct biological role. It was first identified inEscherichia coli (20Tabor H. Tabor C.W. J. Biol. Chem. 1975; 250: 2648-2654Google Scholar), where it remains unprocessed to TSH due to the apparent lack of TSH synthetase. In E. coli, GspS, and consequently Gsp, is prominent in the stationary phase (20Tabor H. Tabor C.W. J. Biol. Chem. 1975; 250: 2648-2654Google Scholar,21Smith K. Borges A. Ariyanayagam M.R. Fairlamb A.H. Biochem. J. 1995; 312: 465-469Google Scholar). Similarly, in C. fasciculata, Gsp increases substantially during the transition from growth phase to stationary phase, while TSH simultaneously drops (22Henderson G.B. Yamaguchi M. Novoa L. Fairlamb A.H. Cerami A. Biochemistry. 1990; 29: 3924-3929Google Scholar). These fluctuations of GSH conjugates or the associated variations in cellular spermidine levels have tentatively been implicated in growth regulation (2Shim H. Fairlamb A.H. J. Gen. Microbiol. 1988; 134: 807-817Google Scholar,20Tabor H. Tabor C.W. J. Biol. Chem. 1975; 250: 2648-2654Google Scholar, 21Smith K. Borges A. Ariyanayagam M.R. Fairlamb A.H. Biochem. J. 1995; 312: 465-469Google Scholar). The first enzyme of TSH synthesis, GspS, has been isolated once in trace amounts from C. fasciculata (0.5 mg from 500 g, wet cell mass) and preliminarily characterized in terms of the apparentM r, kinetic parameters, and substrate specificity (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar). An enzyme catalyzing the analogous reaction inE. coli has recently been cloned. Surprisingly, this GspS also exhibits a substantial amidase activity with Gsp as substrate. The simultaneous catalysis of Gsp synthesis and breakdown results in an apparently futile ATP consumption, the biological role of which remains speculative (23Bollinger J.M. Kwon D.S. Huisman G.W. Kolter R. Walsh C.T. J. Biol. Chem. 1995; 270: 14031-14041Google Scholar, 24Kwon D.S. Lin C.-H. Chen S. Coward J.K. Walsh C.T. Bollinger J.M. J. Biol. Chem. 1997; 272: 2429-2436Google Scholar). Since E. coli does not produce TSH, its GspS obviously has to be seen in a biological context distinct from trypanosomal TSH metabolism, and also the structural and phylogenetic relationship of bacterial and trypanosomal GspS remains to be investigated. Here we report a convenient isolation procedure for GspS from C. fasciculata that allows an in depth analysis of this enzyme. Various physicochemical parameters, preliminary amino acid sequence data, and the kinetic mechanism of the enzyme are presented. Protein concentrations were determined by the method of Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). Bovine serum albumin was used as a standard. The assays were carried out at 25.0 °C in a volume of 0.9 ml containing 50 mm bis-Tris-propane, 50 mm Tris, pH 7.5, 5 mm MgSO4, 1 mm EDTA, 5 mm DTT, 5 mm ATP, 10 mm GSH, and 10 mm spermidine (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar). The assay for trypanothione synthetase (TS) was carried out as described by Smith et al. (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar). Aliquots were taken after 20 min. For thiol analysis, a precolumn derivatization with the fluorescent thiol-specific reagent, monobromobimane (Calbiochem), was used as described previously (2Shim H. Fairlamb A.H. J. Gen. Microbiol. 1988; 134: 807-817Google Scholar). All samples for HPLC analysis were diluted 4-fold with water. Separation and analytical conditions were as described previously (26Fairlamb A.H. Henderson G.B. Bacchi C.J. Cerami A. Mol. Biochem. Parasitol. 1987; 24: 185-191Google Scholar). HPLC analysis was performed with a Jasco-HPLC-system consisting of an autosampler (851-AS), a pump (PU-980), a ternary gradient unit (LG-980–02), and a highly sensitive fluorescence detector (FP-920), which enabled a precise analysis of the small product peak within numerous other and larger ones. An external standard (0.04 mm Gsp) was used for integration calibration of the samples. The malachite green colorimetric assay for liberation of inorganic phosphate (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar) was used for fast detection of GspS activity during purification after column chromatography and for GspS localization on gels. C. fasciculatawas grown in a medium previously described (27Le Trant N. Meshnick S.R. Kitchener K. Eaton J.W. Cerami A. J. Biol. Chem. 1983; 258: 125-130Google Scholar) in a 100-liter fermenter at 27 °C with continuous stirring (200 rpm) and aeration (0.1 volume/volume/min). Organisms were harvested in the late logarithmic growth phase by continuous flow centrifugation. The pellet was resuspended with 100 mm HEPES buffer (pH 7.5) containing 1 mm DTT and 1 mm MgSO4. After centrifugation, the cells were stored at −20 °C. 250 g of cells were suspended in 250 ml of 20 mm bis-Tris-propane buffer, pH 7.5, disrupted by freezing in liquid nitrogen and thawing. The crude homogenate was subjected to an aqueous two-phase extraction at room temperature. All other operations were performed at 4 °C. For extraction of GspS aqueous two-phase systems (total mass, 900 g) were prepared by weighing in concentrated solutions of the phase components and finally the crude extract (Fig. 1). A poly(ethylene glycol) (PEG)/phosphate system containing 7.5% (w/w) PEG6000, 13% (w/w) sodium-potassium phosphate, pH 7.0, and 40% crude homogenate (or water in the blank systems) was used. The mixture was gently shaken for 10 min at room temperature and separated by centrifugation at 5000 × g. The top phase was sucked off and applied to a bottom phase of a blank system. After mixing, centrifugation, and separation of the phases, the PEG-rich top phase of the second phase extraction was mixed with a blank bottom phase, adjusted to pH 6.0 with HCl. This third system was mixed again, centrifuged, and separated. Now the GspS was found in the phosphate-rich bottom phase. The phosphate-rich third bottom phase and other pooled enzyme fractions were diafiltrated with a membrane with a cut-off of 30 kDa (Filtron Minisette) using a Pro Flux M12 diafiltrator (Amicon) at 0.2 megapascals and a 500-fold volume of 2 mmbis-Tris-propane buffer, pH 8.0. A BioLogic-System (Bio-Rad) was used at 4 °C for all chromatographies. The diafiltrated protein mixture was applied onto a Resource Q column (6 ml) (Pharmacia Biotech Inc.) equilibrated with 2 mm bis-Tris-propane buffer, pH 8.0. After washing with 10 column volumes of equilibration buffer, the bound proteins were eluted at a flow rate of 1 ml/min with a gradient of 0.0–0.4 m KCl (100% B) as follows: t = 0 min, B = 0%; t = 20 min,B = 15%; t = 40 min, B= 15%; t = 60 min, B = 30%;t = 120 min, B = 30%;t = 150 min, B = 100%. The GspS eluted at 0.27 m KCl, and the pooled active fractions were diafiltrated with 2 mm bis-Tris-propane buffer, pH 6.0. The diafiltrated proteins were applied onto Poros 20 Pi (0.46 × 10 cm, 1.7 ml) (Perseptive Biosystems) equilibrated with 2 mmbis-Tris-propane buffer, pH 6.0. After washing with 10 column volumes of equilibration buffer, bound proteins were eluted at a flow rate of 4 ml/min with a gradient of 0–1 m NaCl (100% B) as follows:t = 0 min, B = 0%; t = 8 min, B = 35%; t = 16 min,B = 35%; t = 17 min, B= 37%; t = 21 min, B = 37%;t = 25 min, B = 100%. GspS eluted at 0.7 m NaCl. Pooled active fractions were adjusted to 1 m ammonium sulfate and applied onto a hydrophobic interaction chromatography column Poros 20 PE (0.46 × 10 cm, 1.7 ml) (Perseptive Biosystems) equilibrated with 20 mm bis-Tris-propane buffer, pH 8.0, containing 1m ammonium sulfate, washed with 10 column volumes of equilibration buffer, and eluted with a linear gradient of 1–0m ammonium sulfate and a flow rate of 4 ml/min over 7.5 min. GspS eluted at 0.75 m ammonium sulfate. Pooled active fractions were diafiltrated with 10 mm bis-Tris-propane buffer, pH 6.8. The diafiltrated fraction was applied onto a Mono P HR 5/20 column (4 ml) (Pharmacia) for anion exchange chromatography. The column was equilibrated with 10 mmbis-Tris-propane buffer, pH 6.8. After washing with 10 column volumes of equilibration buffer, bound proteins were eluted with a gradient of 0–1 m NaCl (100% B) as follows: t = 0 min, B = 0%; t = 20 min,B = 25%; t = 40 min, B= 25%; t = 60 min, B = 50%;t = 80 min, B = 50%; t= 100 min, B = 100%. The flow rate was 1 ml/min. GspS eluted at 0.45 m NaCl. Proteins were applied onto a gel permeation chromatography column, Superose 12 (HR 10/30) (Pharmacia), equilibrated with 20 mmbis-Tris-propane buffer, pH 7.5, containing 0.15 m NaCl, and eluted with a flow rate of 0.3 ml/min. Blue dextran (2,000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), and carbonic anhydrase (30 kDa) were used as standards. The subunit molecular weight was determined by SDS-PAGE (28Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) using a PhastGel Gradient 8-25 (Pharmacia) with the following molecular mass standards: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). The native molecular weight was determined by native PAGE using a PhastGel Gradient 8–25 (Pharmacia) with the same molecular mass standards and additionally thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and lactate dehydrogenase (140 kDa). The isoelectric point was determined by isoelectric focusing using a PhastGel IEF 3–9 (Pharmacia) with a broad pI calibration kit and by titration curve analysis with PhastGel IEF 3–9. The latter technique is a two-dimensional electrophoresis. In the first dimension, a pH gradient is generated. The gel is then rotated clockwise 90°, and the sample is applied perpendicular to the pH gradient across the middle of the gel (29Rosengreen A. Bjellquist B. Gasparic V. Radola B.J. Graesslin D. Electrofocusing and Isotachophoresis. W. de Gruyter, Berlin1977: 165-171Google Scholar). For detection of GspS activity after native PAGE or isoelectric focusing, the gels were cut into two pieces; one was silver-stained for protein detection, and the second was incubated for 15 min at room temperature in a solution of 100 mm HEPES, pH 7.0, 5 mm MgSO4, 1 mm EDTA, 5 mm DTT, 10 mm GSH, 10 mmspermidine, and 2 mm ATP. After 15 min, 2.5 ml of a staining solution containing malachite green, ammonium molybdate, and Tween 20 (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar) was added. Lanes containing active GspS showed a dark green color after few minutes. The GspS content in partially purified samples was determined using SDS capillary electrophoresis (Bio-Rad) on the basis of the absorption at 280 nm and assuming an identical absorption coefficient for all proteins in the sample. SDS-PAGE of purified GspS was performed at a constant current of 20 mA in a separating gel (7.5% T). For blotting, the proteins were transferred for 1.5 h onto a polyvinylidene difluoride membrane at 40 V/70 mA in a buffer containing 25 mm Tris base, 192 mm glycine, and 10% (v/v) methanol. The blot was stained with Coomassie Blue. For peptide sequencing, the band corresponding to a molecular mass of 78 kDa was cut out. This material was washed and digested with endoproteinase Lys-C as described before (30Maiorino M. Roche C. Kieß M. Koenig K. Gawlik D. Matthes M. Naldini E. Pierce R. Flohé L. Eur. J. Biochem. 1996; 238: 838-844Google Scholar) and separated by reversed-phase HPLC (30Maiorino M. Roche C. Kieß M. Koenig K. Gawlik D. Matthes M. Naldini E. Pierce R. Flohé L. Eur. J. Biochem. 1996; 238: 838-844Google Scholar). Peptide peaks were detected at 214 nm and collected manually. Aliquots of 15–30 μl were applied directly to biobrene-coated, precycled glass fiber filters of a sequencer (Applied Biosystems 470A) with standard gas phase programs of the manufacturer. All kinetic experiments were carried out at 25.0 °C in a volume of 0.9 ml containing 50 mm bis-Tris-propane, 50 mm Tris, pH 7.5, 1 mm EDTA, 5 mmDTT, and variable concentrations of ATP (0.10, 0.13, 0.18, 0.28, and 0.66 mm), GSH (0.36, 0.47, 0.66, 1.11, and 3.57 mm), and spermidine (0.36, 0.47, 0.66, 1.11, and 3.57 mm), respectively. The enzymatic tests for kinetic studies except the ADP inhibition studies were performed in the presence of phosphoenolpyruvate (10 mm) and pyruvate kinase (0.5 units). A fixed magnesium concentration of 5 mm and a GspS content of 0.072 mg (0.923 μm) was used. Aliquots were taken at 15 and 30 min. GspS activity was analyzed by product determination as described above. 31P NMR spectra were recorded on a Bruker ARX 400 NMR spectrometer (at 162 MHz and locked to the deuterium resonance of D2O) to detect potential partial reactions. The experiments were carried out at 25.0 °C in a volume of 0.6 ml containing 50 mm bis-Tris-propane, 50 mm Tris, pH 7.5, 5 mm MgSO4, 1 mm EDTA, 5 mm DTT, in the presence of 20% D2O. Spectra were recorded at the beginning of the experiment and after the addition of the substrates (5 mm ATP, 10 mm GSH, and 10 mm spermidine). The purification strategy outlined under “Experimental Procedures” resulted in a GspS preparation with a specific activity of 37.6 units/mg at an overall yield of about 20%. The purification factor achieved was 12,500. As is seen from TableI, the phase distribution system applied proved to be highly efficient in enriching GspS.Table IPurification of glutathionylspermidine synthetaseVolumeProteinSpecific activityPurification factorYieldmlmgunits/mg%Crude extract3807600.00.0031100First extraction into top phase170221.00.09231107Second extraction into top phase170119.00.1294381Extraction into bottom phase50550.60.1996653Diafiltration120048.00.258363Resource Q64.21.137025Poros 20 Pi80.85.8194324Poros 20 PE50.212.2406713Mono P50.137.61253319 Open table in a new tab The optimized procedure was based on a factorial design of phase compositions (31Menge U. Todd P. Sikdar S.K. Bier M. Frontiers in Bioprocessing II. ACS Books, Washington, D. C.1992: 331-339Google Scholar), i.e. PEG6000/phosphate (7.5/13% (w/w)), PEG4000/phosphate (8/14% (w/w)), PEG1550/phosphate (9/18% (w/w)), each tested at pH 4.0, 5.5, and 7.0 and containing 40% cell lysate. By centrifugation, the cell debris was concentrated in a gum-like interphase if the pH of the system was ≥5.5. A graphical evaluation of the experimental data (not shown) clearly demonstrated a significant increase in the partition coefficient of GspS with increasing pH and a decrease in the partition coefficient of the total protein with increasing molecular weight of PEG. The best system, containing 7.5% (w/w) PEG6000, 13% (w/w) phosphate, pH 7.0, yielded an extraction of GspS into the top phase (Fig. 1) with a purification factor of 30 in one step. Some residual turbidity left in the top phase of the initial extraction could be eliminated by a second extraction step, mixing the primary top phase with a bottom phase of an identical blank system. By these systems, a proteolytic activity, as observed with casein yellow, and an ATPase activity were quantitatively removed by extraction into the bottom phases. Simultaneously, GspS was completely separated from TS activity. While GspS was recovered completely in the top phase, TS activity was extracted into the bottom phase (Fig. 1), but it proved to be unstable and was not purified further. This confirms, in contrast to previous assumptions (22Henderson G.B. Yamaguchi M. Novoa L. Fairlamb A.H. Cerami A. Biochemistry. 1990; 29: 3924-3929Google Scholar), the existence of two distinct enzymes involved in trypanothione biosynthesis (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar). After two extractions into top phases, GspS was essentially free of interfering enzymatic activities and could be precisely quantitated. The final chromatographic purification of GspS, however, was impaired by the high phosphate concentration and viscosity of the top phase in which the enzyme was dissolved. GspS was therefore extracted from the second top phase into the bottom phase of a third system by lowering its pH to 6.0 without loss of activity. The GspS in the phosphate-rich bottom phase was diafiltrated and then could be loaded onto a Resource Q column. The specific activity of GspS obtained after additional chromatographic steps was about 6-fold higher than achieved before (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar), and also the yields compared favorably with previous experience. In fact, GspS thus purified appeared homogeneous by SDS-PAGE (Fig. 2) and by titration curve analysis (Fig. 3).Figure 3Titration curve analysis of the homogeneous glutathionylspermidine synthetase. 1. dimension, isoelectric focusing, run without protein sample; 2. dimension, native PAGE.View Large Image Figure ViewerDownload (PPT) A subunit molecular mass of GspS of 78 kDa was estimated by SDS-PAGE, and an identical value was obtained by gradient gel electrophoresis of the native enzyme. In the latter case, the identity of the 78-kDa band with GspS was confirmed by activity staining, i.e. phosphate liberation upon incubation with Mg2+-ATP, GSH, and spermidine (not shown). Also, gel permeation chromatography on Superose 12 indicated a comparable molecular mass (79 kDa). A small activity peak eluted at about 170 kDa, suggesting a slight tendency of the enzyme to dimerize. In essence, however, GspS of C. fasciculata was present as a monomeric enzyme of 78 kDa. Its isoelectric point deduced from isoelectric focusing was at pH 4.6. Functional characterization of GspS of C. fasciculata was performed with a 400-fold purified preparation, i.e. with the fraction obtained after step 6 in Table I, since the pure enzyme, even when stored at 4 °C and −20 °C, almost completely lost its activity within 1 day. According to purity analysis by capillary electrophoresis, the partially purified preparation contained 33% GspS. At this stage of purification, GspS activity was stable in 20 mm bis-Tris-propane, pH 8.0, and in the presence of 8 mm DTT for more than 2 months at −20 °C. However, it lost its activity within 1 day in the presence of 1 mphosphate and 1 m ammonium sulfate. At room temperature, the enzyme could be stored for more than 24 h without loss of activity, which guaranteed reliable functional studies at the temperature optimum between 25 and 30 °C. Biosynthetic activity required the presence of magnesium ions. In partially purified GspS, we did not observe any TS activity, at a detection limit of ≥1% of the corresponding GspS rate. We could also exclude an amidase activity of C. fasciculata GspS, which had been described for the corresponding E. coli enzyme (23Bollinger J.M. Kwon D.S. Huisman G.W. Kolter R. Walsh C.T. J. Biol. Chem. 1995; 270: 14031-14041Google Scholar,24Kwon D.S. Lin C.-H. Chen S. Coward J.K. Walsh C.T. Bollinger J.M. J. Biol. Chem. 1997; 272: 2429-2436Google Scholar), since Gsp was not hydrolyzed by C. fasciculata GspS under experimental conditions that would have detected a hydrolytic activity at a 1% level of the synthetase activity. TSH hydrolysis was also not detected (not shown). These findings contrast markedly with a relative amidase activity of 18% (pH 7.5) or 35% (pH 8.5) of the synthetase activity reported for E. coli GspS. Also, the pH optimum of the C. fasciculata enzyme (Fig. 4) is higher by nearly 1 pH unit (pH 7.6–7.8) than that of E. coli GspS (pH 6.8). As already observed by Smith et al. (1Smith K. Nadeau K. Bradley M. Walsh C. Fairlamb A.H. Protein Sci. 1992; 1: 874-883Google Scholar), N-terminal amino acid sequencing proved unsuccessful, obviously due to an N-terminal blocking group. After proteolytic cleavage with endoproteinase Lys-C, however, a total of 11 peptides could be recovered from HPLC in a quality to allow sequencing. Of these peptides, seven could unambiguously be aligned to the deduced GspS sequence of E. coli recently published by Bollinger et al. (23Bollinger J.M. Kwon D.S. Huisman G.W. Kolter R. Walsh C.T. J. Biol. Chem. 1995; 270: 14031-14041Google Scholar) (Table II). GspS of E. coli and of C. fasciculata thus appeared to be phylogenetically related. However, based on the limited sequence information, the sequence similarity between these enzymes, with only 40% identity, appears rather low.Table IIPeptides of glutathionylspermidine synthetase from Crithidia fasciculata(10)VPFGEVQGYAPGHIPAYSNK(29)+*** + ***** + +**133SIITGLDSPFAAI(145)*** * + *(191)TYEPTE(196)* **(202)NEIPRPLTHK(211)** * +(227)LDLNDPAE(234)** **++(500)ILPIIYHNHPDHPAILRAE(518)****++ * * +* +(535)IVGRVGRNVTITDG(548)*+** * *+ +The amino acid numbering corresponds to the E. coli sequence (23Bollinger J.M. Kwon D.S. Huisman G.W. Kolter R. Walsh C.T. J. Biol. Chem. 1995; 270: 14031-14041Google Scholar). *, identical amino acids; +, similar amino acids found in GspS from E. coli. Open table in a new tab The amino acid numbering corresponds to the E. coli sequence (23Bollinger J.M. Kwon D.S. Huisman G.W. Kolter R. Walsh C.T. J. Biol. Chem. 1995; 270: 14031-14041Google Scholar). *, identical amino acids; +, similar amino acids found in GspS from E. coli. The analysis of the kinetic mechanism by steady-state kinetics were performed by means of direct product (Gsp) detection at fixed time points. The time points were set to yield less than 15% consumption of the limiting substrate but more than 0.002 mm Gsp for a reliable quantification. In the beginning, nonlinear Lineweaver-Burk plots were obtained that could be attributed to product (ADP) inhibition (see below) and, at an ATP concentration above 1 mm, also to substrate inhibition (not shown). When ADP accumulation was avoided by coincubation with phosphoenolpyruvate/pyruvate kinase and the concentrations of ATP were kept constant at levels below 1 mm, linear double-reciprocal plots were observed. Fig. 5,A–C, illustrates enzyme-normalized Lineweaver-Burk plots, each" @default.
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• W2045915921 title "Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase from Crithidia fasciculata" @default.
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