RESULTS
γ-GT e γ-GTrel can be distinguished on the basis of their substrate specificity: the first is active on both G-p-Na and GSH, the latter only on GSH. Therefore, the assay of both activities during purification steps can be used to verify the enrichment of one of the two activities with respect to the other one. In particular an enrichment of γ-GTrel should result in an increase of the ratio between the activity measured with GSH and the activity measured with G-p-Na.
1. Preliminary study on the identification and purification of γ-GT rel from bovine kidney.
At the beginning of the first year an attempt was made to purify the γ-GT rel enzyme following the protocol reported in literature for the purification of γ- GT (Huseby N.E.,1980).
18.75 g of bovine kidney were homogenated in TRIS / HCl 50 mM pH 8 following the procedure described in "Materials and Methods"; after preparation of crude extract, ultracentrifugation and precipitation with ammonium sulphate, the sample was subjected to gel filtration. 3.5 ml of crude extract (0.2 mg of protein) were loaded on a Sephacryl-S300 column (see Materials and Methods). The elution profile of gel filtration is reported in Fig. 9.
Fig. 9 Gel chromatography on Sephacryl S-300 column. Ativities measured using G-pNA (diamonds) or GSH (squares) are reported. The flow rate was 18 ml/h and 2 mL fractions were collected.
Since the ratio between the activity measured with GSH and the activity measured with G-pNA was higher in the fractions that eluted in the lower molecular weight range, fractions 80 to 93 were pooled. The specific activities of the pooled fractions resulted higher than the specific activity of the pool obtained after hydrophobic interaction chromatography.
Table. Comparison of the specific activities in the pool pool obtained after hydrophobic interaction chromatography and in the pool 80-93 obtained after gel filtration.
0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4 0,45 0,5
45 55 65 75 85 95 105
Fractions
U/ml
G-p-Na (U/mg) GSH (U/mg)
Ratio
GSH/G-p-Na
HIC pool 7,27 3,9 0,53
GF pool 0,4 2,2 5,5
3 mL of the pooled fractions were loaded on a Concanavalin A affinity column (Fig. 10).
When the activity measured with G-pNA was less than 0.004 UA/min (fraction 27) a 0-100 mM gradient of α-methyl-D-mannopyranoside was applied. No evident increase in the ratio between the activity measured with GSH and the activity measured with G-pNA was observed.
Fig. 10 Affinity chromatography on ConA affinity column. The elution profile shows the absorbance at 280 nm (triangles) and the activities measured using G-pNA (diamond) and GSH (squares). The flow rate was 10 ml/h and 1.5-mL fractions were collected. The arrow indicates when the 0- 100 mM gradient of α-methyl-D-mannopyranoside was applied.
On the basis of the results obtained in the preliminary attempt to purify γ-GT rel above described, we decided to modify the purification protocol introducing a hydrophobic interaction chromatography step before gel filtration.
18.67 g of bovine kidney were homogenated in TRIS / HCl 50 mM pH8, following the procedure described in "Materials and Methods." After preparation of crude extract, ultracentrifugation and precipitation with ammonium sulphate, the sample was subjected to hydrophobic interaction chromatography (Fig. 11). 3.5 ml of crude extract (0.19 mg of protein) were
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04
0 5 10 15 20 25 30 35
Fractions
U/ml
0 0,05 0,1 0,15 0,2 0,25 0,3 0,35
Abs 280nm
loaded on a Buthyl-Sepharose Fast Flow column equilibrated with TRIS / HCl 50 mM pH 8 supplemented with 1.5 M ammononium sulphate.
When the absorbance at 280 nm returned baseline values (fraction 14) 1.5 M SA buffer was replaced with 50 mM Tris HCl pH 8 devoid of ammonium sulfate. When the activity was less than 0,085 UA/min the buffer was replaced with 2% Triton X100 in milliQ water.
The elution profile of HIC is shown in Fig.11
Fig. 11 Hydrophobic interaction chromatography on Buthyl-Sepharose Fast Flow column. Absorbance at 280 nm (triangles) and activities measured using G-pNA (diamonds) or GSH (squares) are reported. The flow rate was 10 ml/h and 1,5-mL fractions were collected.
The absorbance at 280nm was not measured in the presence of Triton X100.
0 0,5 1 1,5 2 2,5 3
0 20 40 60 80 100
Fraction
U/ml Abs 280
2% TritonX in milliQ H2O Tris HCl
γ-Glutamyltransferase activities strongly bind to Buthyl Sepharose resin, as evidenced by the fact that only after the addition of 2% Triton X-100 the activities are eluted. Active fractions (60 -80) were pooled and 3.5 ml of pooled fractions were loaded on a Sephacryl S-300 column (Fig. 12).
Fig. 12 Gel chromatography on Sephacryl S-300 column after hydrophobi interaction step. Activities measured using G-pNA (diamonds) or GSH (squares) are shown. The absorbance at 280nm was not measured due to the presence of Triton X-100. The flow rate was 18 ml/h and 2-mL fractions were collected. The ratio between the activity measured with GSH and the activity measured with G-p-Na was calculated for the fractions assayed and reported on the elution profile (triangles).
0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16
20 30 40 50 60 70 80
Fractions
U/ml
0 1 2 3 4 5 6 7 8 9
Ratio
The increase of the ratio between the activity measured with GSH and the activity measured with G-p-Na that is evident for the lowest molecular weight fractions suggest the presence of two distinct activities, one of which is more active towards GSH and is eluted in the lower molecular weight range. On the basis of this observation the active fractions with the highest GSH/G-p-Na ratio were pooled and 3 ml of pooled fractions were loaded on a ConA affinity chromatografy column (Fig. 13).
When the activity was less than 0.004 UA/min a 0 - 100 mM gradient of the α-methyl-D-mannopyranoside was applied. (Fig.13).
0 0,002 0,004 0,006 0,008 0,01 0,012 0,014
0 2 4 6 8 10 12 14 16 18 20
Fractions
U/ml
Fig.13 ConA affinity chromatography. The elution profile shows the activities measured using G-pNA (diamonds) or GSH (squares). The flow rate was 10 ml/h and 1.5-mL fractions were collected.
The elution profile obtained showed a single peak of activity towards GSH, while the activity towards G-p-NA resulted almost undetectable. After ConA affinity chromatography, the attempt to obtain a stable active pool to be further processed was unfruitful. The inactivation of the pooled fraction obtained after ConA affinity chromatography probably depended on a rapid time dependent inactivation of the activity acting prevalently on GSH: in fact, when a sample of gel filtration pooled fractions were analyzed for activities towards GSH and GpNA at different times from GF chromatography (Fig.14), a rapid loss of the activity towards GSH was observed, with a complete inactivation after 6 days.
0 0,01 0,02 0,03 0,04 0,05
0 1 2 3 4 5 6
Days (4°C)
U/ml
G-p-Na GSH
Fig. 14 Time dependent inactivation of γGT activity assayed with GSH as substrate in a sample from S-300 pool.
The time dependent inactivation of the activity towards GSH is a further evidence of the presence of two distinct activities with difference substrate specificity, one of which (possibly γGT-rel enzyme) is less stable.
An attempt was made to stabilize the enzyme activity using various thiol- reagents (Fig. 15); after sample incubation for 24 hours with DTT no effect on the enzyme activity were evident. On the contrary the incubation with both GSH and GSSG seemed to preserve and, in the case of GSSG, activate the enzyme activity towards GSH.
Fig. 15 Effect of incubation in the presence of thiol reagents on the activity measured with GSH. Aliquots of S-300 pooled fractions were incubated for 24 time at 28 °C in the presence of 5 mM DTT (purple), 0,25 mM GSH (blu), 0,25 mM GSSG (orange). After incubation the activity with GSH was assayed.
0 0,0005 0,001 0,0015 0,002 0,0025 0,003 0,0035 0,004 0,0045 0,005
0
U/ml
2. Study on the identification and purification of γGT-rel from bovine spleen
A major problem arisen during the preliminary study on the identification and purification of γGT-rel from bovine kidney was the great abundance of γGT in this organ that made it difficult to obtain the identification and
separation of an activity acting only on GSH from the classic γGT activity acting on both GSH and G-pNA. Thus at the beginning of the second year we decided to use spleen instead of kidney as a possible source of γGT-rel, since in literature γGT-rel is reported to be highly expressed in spleen (Bing Z., 1998).
19.6 g of bovine spleen were homogenated in TRIS / HCl 50 mM pH 8 following the procedure described in "Materials and Methods"; after the preparation of crude extract, ultracentrifugation and precipitation with ammonium sulphate, the sample was subjected to hydrophobic interaction chromatography on a Buthyl-Sepharose Fast Flow column equilibrated with TRIS / HCl 50 mM pH 8 supplemented with 1.5 M ammononium sulphate.
When the absorbance at 280 nm returned baseline values (fraction 14) 1.5 M SA buffer was replaced with 50 mM Tris HCl pH 8 devoid of ammonium sulfate. When the activity was less than 0,085 UA/min the buffer was replaced with 2% Triton X100 in milliQ water.
The elution profile of HIC is shown in Fig.16.
0 0,005 0,01 0,015 0,02 0,025 0,03 0,035 0,04
14 19 24 29 34 39 44 49
fractions
U/ml
G-pNA GSH
Fig 16. Hydrophobic interaction chromatography on Buthyl-Sepharose Fast Flow column. Absorbance at 280 nm (triangles) and activities measured using G-pNA (diamonds) or GSH (squares) are reported. The flow rate was 10 ml/h and 1,5-mL fractions were collected. The absorbance at 280nm was not measured in the presence of Triton X100.
The active fractions of hydrophobic interaction chromatography were pooled and concentrated on Speed-Vac to 0.82 mg/ml protein; the concentrated sample was subjected to gel filtration on a Sepahcryl S-300 column (Fig.
17).
0 0,002 0,004 0,006 0,008 0,01 0,012 0,014 0,016 0,018
30 35 40 45 50 55 60 65 70
fractions
U/ml
Fig.17. Gel filtration on Sephacryl S-300 column. Activities measured using G-pNA (diamonds) or GSH (squares) are shown. The absorbance at 280nm was not measured due to the presence of Triton X-100. The flow rate was 18 ml/h and 2-mL fractions were collected.
After gel filtration, individual fractions were divided into two aliquots: the first aliquot was incubated in the presence of 0.25 mM GSSG for 24h at 28°C, while the second aliquot was incubated in the same conditions without GSSG. A significant increase of the enzyme activity measured with GSH was observed (Fig 18 and 19), while the activity measured with G-pNA as substrate remained virtually unchanged. This observation is a further evidence of the presence of a γ-glutamyltransferase activity distinct from the classic γGT activity: this activity (possibly γGT-rel) is active on GSH and is activated by GSSG treatment.
0 0,002 0,004 0,006 0,008 0,01 0,012 0,014 0,016 0,018
30 35 40 45 50 55 60 65 70
fractions
U/ml GGT
Fig. 18. Effect of GSSG incubation on the activity measured with G-pNA as substrate. The fractions of gel filtration chromatography were incubated for 24h at 28°C either in the presence (open diamonds) or in the absence of (closed diamonds) GSSG and then assayed using the synthetic substrate (G- pNA).
0 0,002 0,004 0,006 0,008 0,01 0,012
30 35 40 45 50 55 60 65 70
fractions
U/ml
Fig. 19. Effect of GSSG incubation on the activity measured with GSH as substrate. The fractions of gel filtration chromatography were incubated for 24h at 28°C either in the presence (open squares) or in the absence (closed squares) of GSSG and then assayed using GSH as substrate.
To further characterize the GSSG activation process, we pooled the low molecular weight range activated fractions (fraction 58-68) and verified the reversibility of the activation process. After treatment with DTT, a significant reduction in the activity measured with GSH was observed.
This reduction was not observed in a pool obtained with non activated frations (Fig 20).
0 0,001 0,002 0,003 0,004 0,005 0,006 0,007
CNT GSSG
no DTT DTT
Fig.20. Effect of DTT incubation on the activity measured with GSH in the pooled fractions from Sepahcryl-S300 chromatography. After incubation in the presence or in the absence (control) of GSSG (for conditions see Fig 19 and 20), low molecular weight range fractions (fraction 58-68) from Sephacryl S300 chromatography were pooled and incubated 30 min at 37°
C both in the presence (violet bars) and absence (blue bars) of 2 mM DTT.
Before and after incubation with DTT activity was assyed with GSH as substrate.
Kinetics parameters were also determined (Fig.21) for the pool obtained with activated fractions and for the pool obtained with non activated frations; we observed that the GSSG dependent activation determined a significant decrease in the apparent Km towards GSH with apparently no change in the apparent Vmax.
0 1 2 3 4 5 6 7 8 9 10
-1,5 -0,5 0,5 1,5 2,5 3,5 4,5 5,5 6,5 7,5 8,5
1/[S]
1/V
Fig 21. Effect of the GSSG dependent activation on the kinetic parameters towards GSH. The fractions from the gel filtration chromatography, preincubated for 24h at 28 ° C both in the presence (orange line) or in the absence (green line) of GSSG (for details see Fig. 19 and Fig. 20), were assayed with different concentration of GSH (ranging from 0,125 mM to 1,5 mM) in order to evaluate kinetic parameters.
3. Development of a colorimetric method for the determination of glutathione
In the attempt to purify γGTrel, we obtained large amount of partially purified γGT from bovine kidney. By using this enzyme as ancillary enzyme we developed an end point coupled enzymatic assay for the determination of reduced and oxidized glutathione.
The determination of GSH was based on the measurement of cysteine produced from cysteinylglycine by leucyl aminopeptidase (LAP), after the transpeptidation reaction catalyzed by γ-GT.
The incubation mixture contained, in a final volume of 250 µL, 8 mM MgCl2, 0.2 mM MnCl2, 40 mM Gly-Gly, 50 mU/mL γ-GT, and 50 mU/mL LAP in 32 mM Tris–HCl pH 8.5.
To measure the concentration of total glutathione the reducing agent dithiothreitol was added to the standard assay mixture at a concentration of 2 mM; to measure the concentration of GSSG, free GSH was masked by preincubating the sample in the presence of 1 mM iodoacetamide at 45°C for 60 min. After the incubation the sample was added to the standard incubation mixture supplemented with 2 mM DTT for the reduction of GSSG.
Relying on standard curves of Fig. 22 and Fig.23 we measured the content of glutathione in acid extracts of bovine lens and followed the NADPH dependent reduction of GSSG by glutathione reductase.
Fig. 22 Calibration curves for GSH using standard GSH solutions (range, 20–
200 µM)
Fig. 23 Calibration curves for GSSG using standard GSSG solutions (range, 20–160 µM)
Three different acid extracts were analyzed and the GSH concentrations observed were 7.7, 7.5, 7.8 µmol/g wet weight, respectively, while GSSG was not detectable.
After addition of 0.500 mM GSH to three different lens acid extracts, the mean of GSH concentrations determined was 0.502 + 0.02. Similarly, after addition of 0.100 mM GSSG the mean of GSSG concentrations determined was 0.106 + 0.002 (Table).
Table. Recovery of GSH and GSSG from spiked bovine lens acid extracts
When the NADPH dependent reduction of GSSG to GSH catalyzed by the enzyme glutathione reductase was followed by using the proposed method, an excellent correlation between the production of GSH and the consumption of GSSG was observed; the reduction of GSSG was consistent with the the oxidation of NADPH followed through the decrease in absorbance at 340 nm (Fig. 24).
Fig. 24 Time course of the NADPH dependent reduction of GSSG catalyzed by glutathione reductase. GSH and GSSG (indicated by circles and squares) concentrations were determined from the calibration curves; NADPH concentration (triangles) was determined spectrometrically from the absorbance at 340 nm.
