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An electron spin resonance assay of glutathione S-conjugate transport
JOURNALOF
biochemical and biophysical methods J. B&hem.
Biophys. Methods 33 (1996) 65-71
An electron spin resonance assay of glutathione S-conjugate transport Lukasz Pulaski *, Grzegorz Bartosz Depariment
of Molecular
Received
Biophysics,
Uniwrsi&
of kid5
13 March 1995: revised 7 August
Bnnacha
I2 / 16, 90-237 trid5
1995: accepted 7 August
Poland
1995
Abstract A method for studying export of glutathione S-conjugates from cells is proposed based on the use of the spin label tempo-maleimide. This compound is conjugated intracellularly with glutathione and the concentration of the exported conjugate is measured in cell supernatants in an spin resonance spectrometer after reoxidation with ferricyanide. This method allows for measurements of micromolar concentrations of the conjugate and requires low amounts of cells.
electron
Keywords:
Erythrocyte;
Transport;
Electron spin resonance;
Glutathione
S-conjugate:
Tempo-maleimide
1. Introduction One of the main functions of the ubiquitous tripeptide glutathione is the detoxication of reactive electrophiles of exo- and endogenous origin. Such compounds may be modified (detoxication phase l), conjugated to glutathione by glutathione S-transferases (detoxication phase 2) and then exported out of animal cells or deposited into vacuoles of plant cells (detoxication phase 3) [l-3]. In recent years a growing interest has been observed in studies of the phase 3 of glutathione-linked detoxication, i.e., the active transport of glutathione S-conjugates out of the cytoplasm [1,3-71. This process may be of medical and toxicological importance since cisplatin and other chemotherapeutics
CDNB, I-chloro-2,4-dinitrobenzene: DNP-SG, 2,4-diAbbreviations: B-SG, bimane-S-glutathione; nitrophenyWglutathione; ESR, electron spin resonance: mBCI, monochlorobimane; tempo-maleimide. (2,2,6,6-tetramethyl-l-piperidynyloxy)maleimide; TLC. thin-layer chromatography; TM-SC, tempo-maleimide-S-glutathione. * Corresponding author. Tel.: (48-42) 354-476; Fax: (48-42) 354-473. 0165-022X/96/$15.00
SSDl 0165-022X(95)00036-4
Copyright
0 1996 Elsevier Science B.V. All rights reserved.
[4,5] as well as metabolites of some pesticides [I J] may be exported from cells via the ‘glutathione S-conjugate pump’. The classic whole-cell method of studies of transport of glutathione S-conjugates consists of loading the cells with CDNB and calorimetric monitoring of the appearance of the formed conjugate (DNP-SC) in extracellular medium 17-l I]. A serious disadvantage of this method is its low sensitivity unless a radiolabeled CDNB analog (fluorodinitrobenzene) is used and efflux of radioactivity is measured [ 12,131. However, apart from the radioactive hazard. this technique is much more expensive. In this paper, we propose a method for studies of glutathione S-conjugate export from cells employing a commonly used spin label tempo-maleimide which is sensitive. requires low amounts of cells and may be convenient for users of ESR spectrometers. In a separate paper, we proposed another sensitive method based on measurement of fluorescence of a bimane conjugate of glutathione [ 141.
2. Materials
and methods
Blood was obtained from healthy donors (young males) by venipuncture after informed consent. Erythrocytes were collected after centrifugation at 3000 X ,q for 5 min. washed 3 times with PBS t IS0 mM NaCI. IO mM phosphate buffer, pH 7.4) and used within 24 h. Tempo-maleimide and all the other reagents were from Sigma (Deisenhofen. Germany) and were of analytical grade. All values are averaged from at least 3 experiments (mean * SD).
Erythrocytes were suspended to a hematocrit 50% in buffer Z (138 mM NaCI, 5 mM KCI. I mM MgCl?. IO mM glucose. 8 mM phosphate buffer, pH 7.4). Tempo-maleimide was added from a stock ethanol solution (IO mM). After 5-min incubation at room temperature the erythrocytes were washed three times at 4°C with buffer Z and suspended in this buffer to hematocrit 50%. After specified times of incubation at 37°C aliquots were withdrawn. the erythrocytes were sedimented (1 min, I5 000 X g, 2°C) and the supematant was collected. When hemolysis was checked, it was always found to be below 17~. The ESR spectra of the supernatants were measured in an ESP-300E spectrometer (Bruker) after reoxidation of the label with 1 mM potassium ferricyanide. Concentration of TM-SG in the supernatants was estimated either by double integration of ESR spectra or by measurement of the height of the middle peak of the spectrum (‘peak picking procedure’). In the double integration procedure, tempo solutions of different concentrations were used as standards. The height of the middle peak in the spectrum (Fig. 1) was found to be proportional to the label concentration for the whole concentration range of TM-SG employed. Its use can be recommended because the measurement is simple and can be done even on spectra taken in old-fashioned, non-computerized spectrometers. For measurements of intracellular conjugate concentration, erythrocytes. after preincubation with tempo-maleimide and washing off the excess label. were centrifuged to a
t. Pulaski. G. Bartm:
/ J. Brocherrr. Bioph,w. Methods 33 ( IYYfil
Fig. 1. ESR spectrum of TM-Xi
65- 71
67
in supernaranrs of erythrocytesuspensions.
hematocrit of 90% and lysed with an equal volume of water. Hemoglobin, which interfered with the measurements by quenching the ESR signal, was removed by centrifugation through Centricon C-30 filters (Amicon) and ESR spectra of so prepared samples were measured. TLC demonstrated that all the spin label was present as TM-W in the hemolysates. For competitive inhibition experiments cells were first preincubated for 5 min with 100 FM tempo-maleimide and afterwards (without washing) with appropriate concentrations of CDNB (for 15 min) or mBC1 (for 5 min) to rule out competition at the conjugation level. Since TM-SG transport rate remained constant in these conditions for 60 min, the subsequent preincubations did not interfere with the results.
3. Results 3.1. Identification
of TM-SG
Upon incubation of human erythrocytes with tempo-maleimide a compound giving an ESR signal was excreted to the medium in a time-dependent manner. This compound was identified as tempo-maleimide glutathione S-conjugate (tempo-maleimide-s-glutathione, TM-SG) by means of TLC on Silicagel 60 (Merck, Darmstadt). After developing the chromatogram in n-propanol/acetic acid/H20 (10: 1:5) the TLC plate was divided into 15 zones covering the distance between application site and solvent front. The silicagel from each zone was scraped off, suspended in 30% ethanol and the ESR spectra were measured. Fig. 2 shows the distribution of ESR signal amplitude in chromatograms of erythrocyte suspension supematant after incubation with tempo-maleimide, for a solution of TM-SG obtained by non-enzymatic conjugation of tempo-maleimide to excess glutathione at pH 7.0, and for the tempo-maleimide. The R, of TM-SG in this solvent system was found to be 0.47.
68
L. Putmki,
G. Bartos: / J. Biochem. Biophys. Mrih0d.v 33
(1 Y96)
65- 71
25
2
15
1
*
R:,=,
z
=
I
r-E 05
+A
*L 0 0
02
06
04
08
1
Rf Fig. 2. Distribution of ESR signal amplitude in thin-layer chromatograms (Silicagel 60. n-propanol/acetic acid/water, IO: I :5): E, TM-Xi exported from erythrocytes: A, TM-SC obtained by non-enzymatic conjugation of tempo-maleimide with glutathione: L. free tempo-maleimide.
3.2. Kinetics of TM-SG export .from the erythroqtes
The time dependence of extracellular concentration of TM-SG, reflecting the conjugate export from the erythrocytes, was found to be linear (correlation coefficient r = 0.98) for incubation times up to 60 min after preincubation with 100 FM tempomaleimide (Fig. 3). Lineweaver-Burk plot of the concentration dependence of TM-SG export (Fig. 4) could be fitted to a single line corresponding to a K, of 939 + 15 FM and a V,,, of 420 i 48 nmol/(ml X h).
0
0
IO
20
30
40
50
60
Time [min]
Fig. 3. Time course of TM-SC export from erythrocytes mide.
after 5 min incubation
with 100 FM tempo-malei-
t. Pulaski.
G. Burtosz / J. Biochem.
-0 02
0
002
3.3. Inhibition
Methods
004 l/S
Fig. 4. Lineweaver-Burk
Biophp.
006
33
(19%)65-
008
71
69
01
[l/pm]
plot of transport rate dependence
on intracellular
TM-SG concentration
of TM-SG export from the etythrocytes
TM-SG export was strongly inhibited by high concentrations of sodium ortho-vanadate and sodium fluoride, known potent inhibitors of erythrocyte glutathione conjugate pump [ 151. Intracellular formation of other substrates for the glutathione S-conjugate pump, DNP-SG (by treatment of erythrocytes with CDNB) [8,9,15] and B-SG (by treatment with mBC1) [ 141 also decreased the export rate of TM-SG (Table 1).
4. Discussion
Tempo-maleimide penetrating into the erythrocytes reacts mainly (in approx. 70-80%) with intracellular glutathione (the remainder is bound to hemoglobin and other proteins, including membrane proteins). The conjugate formed (TM-SG) is exported out of the cells via the glutathione S-conjugate pump. The latter conclusion can be drawn from the inhibition of the TM-SG export by o-vanadate and fluoride which block the export of DNP-SG [ 151, the inhibition of TM-SG export by other intracellularly formed glutathione conjugates, B-SG and DNP-SG (Table 1) and from the competitive inhibition of
Table I Inhibition
of TMSG transport
in human erythrocytes Transport
Control 250 PM o-vanadate 20 mM fluoride 50 IJ.M CDNB 250 )LM CDNB 1OOOmMCDNB 50 PM mBC1
84.8 8.3 11.7 44.6 14.6 5.5 69.8
rate (nmol/(ml
X h))
Relative transport rate (SC) 100 9.8 13.8 52.6 17.2 6.5 82.3
70
i.. Prtfmki.
G. Bortos: / J. Biociturrr. Biopiys.
Methods 33
f IYY61
65-7f
the active transport of [‘HIDNP-SC into inside-out erythrocyte membrane vesicles by TM-SG (Bartosz. Sies and Akerboom. unpublished). Therefore, measurement of the TM-SG export is a valid means of studying the glutathione S-conjugate pump. Advantages of this technique include low concentrations of the conjugate which can be measured (starting from several FM) and low volumes of samples to be analysed (tens of p_ll. We studied the transport in erythrocytes but this method may be even more useful for cells of other types, available in much lower quantities. Thus. the method proposed is simple and cheap in many respects. provided an ESR spectrometer is available. Nitroxide spin labels are reduced in cells, mainly to non-paramagnetic hydroxylamine derivatives which can be reoxidized by ferricyanide. Also the tempo-maleimide and TM-SG are partly reduced inside erythrocytes and TM-SG is transported partly in the reduced form. Therefore. reoxidation of the conjugate is needed before the measurement. One should be careful. however, to use low concentrations of ferricyanide because higher concentrations would quench the signal. Under our conditions, the intensity of the TM-SG signal in erythrocyte supernatants increased by about 30% after addition of ferricyanide. If the spin labels were completely reduced inside the cells (which may be the case with other cell types). one could think of a transport assay in whole cell suspensions, adding ferricyanide to extracellular medium and monitoring the increase in signal intensity in cell suspension. Careful studies of the concentration dependence of DNP-SG export from erythrocytes [I I] or its import into inside-out vesicles [ lS,l6] showed the existence of two kinetic components of the transport, one of high affinity (low K,,) and low capacity, and another of low affinity (high K,, 1 and high capacity. The Lineweaver-Burk plot of the concentration dependence of TM-SG transport shows no clear-cut biphasicity (Fig. 4) with apparent K,,, value corresponding apparently to the low affinity component of the ‘glutathione S-conjugate pump’ (for DNP-SG: 897 pM [16]). The heterogeneity of the transport substrate (TM-SG with the nitroxide group intact or reduced) also did not result in any apparent heterogeneity of the Lineweaver-Burk plot which suggests that the reduction of the nitroxide group does not have any significant effect on the affinity of the conjugate for the transporter.
5. Simplified
description
of the method and its advantages
The method proposed consists in an electron spin resonance (ESR) assay of a spin-labeled glutathione S-conjugate exported from erythrocytes. Upon incubation of the erythrocytes with a maleimide spin label (tempo-maleimide), tempo-maleimide-.Yglutathione (TM-SG) is formed inside the cells. The time course of TM-SG export from the cells is monitored by ESR measurement of its concentration in cell suspension supernatants after reoxidation of the label with I mM potassium ferricyanide. The method consumes little time and material. allows an easy determination of glutathione S-conjugate transport kinetics in intact erythrocytes and other cells, and avoids the use of radioactivity or noxious chemicals.
15 Pubski,
G. Bartosz/J.
Biochem.
Biophys. Methods
33 (1996165-71
71
Acknowledgements This study was supported by the grant No. 6 P203 001 04 of the Committee for Scientific Research (Poland). We are indebted to the Department of Biophysics, Medical University of t6di, for the supply of human erythrocytes.
References glutathione S-conjugate export pump, Trends Biochem. Sci., 17 ( 1992) 453-468 [21 Sandermann Jr., H., Higher plant metabolism of xenobiotics: the ‘green liver’ concept, Pharmacogenetics, 4 (1994) 225-241 glutathione [31 Martinoia, E., Grill, E., Tommasini, R., Kreuz, K. and Amrhein, N., ATP-dependent conjugate ‘export’ pump in the vacuolar membrane of plants, Nature, 364 (1993) 247-249 [41 Zimniak, P. and Awasthi, Y.C., ATP-dependent transport systems for organic anions, Hepatology, 17 (1993) 330-339 [51 Ishikawa, T., Wright, CD. and Ishizuka, H., GS-X pump is functionally overexpressed in cis-diamminodichloroplatinum (ID-resistant human leukemia HL-60 cells and down-regulated by cell differentiation, J. Biol. Chem., 269 (1994) 29085-29093 cis- diamminedichloroplatinum(lI) metabolism 161Ishikawa, T. and Ali-Osman, F., Glutathione-associated and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance, J. Biol. Chem., 268 (1993) 20116- 20125 [7] Olive, C. and Board, P., Glutathione S-conjugate transport by cultured human cells, Biochim. Biophys. Acta, 1224 (1994) 264-268 [8] Board. P.G., Transport of glutathione S-conjugate from human erythrocytes, 124 (1981) 163- 165 [9] Awasthi, Y.C., Misra, Cl., Rassin, D.K. and Srivastava, S.K., Detoxification of xenobiotics by glutathione S-transferases in erythrocytes: the transport of the conjugate of glutathione and I-chloro- 2,4-dinitrobenzene, Br. J. Haematol., 55 (1983) 419-425 [lo] LaBelle, E.F., Singh, S.V.. Srivastava, S.K. and Awasthi, Y.C., Evidence for different transport systems for oxidized glutathione and S-dinitrophenyl glutathione in human erythrocytes. B&hem. Biophys. Res. Commun., 139. (1986) 538-544 [I I] Eckert, K.-G. and Eyer, P., Formation and transport of xenohiotic glutathione S-conjugates in red cells, B&hem. Pharmacol., 35 (1986) 325-329 [12] Oude Elferink, R.P.J., Bakker, C.T.M. and Jansen, P.L.M., Glutathione- conjugate transport by human colon adenocarcinoma cells (Caco-2 cells), Biochem. J., 290 (1993) 759-764 [131 Oude Elferink, R.P.J., Ottenhoff, R., Radominska, A., Hofmann, A.F., Kuipers, F. and Jansen, P.L.M., Inhibition of glutathione-conjugate secretion from isolated hepatocytes by dipolar bile acids and other organic anions, B&hem. J., 274 (1991) 281-286 in human erythrocytes, Biochim. Biophys. iI41 PuIaski, t. and Bartosz, Cl., Transport of bimane-S-glutathione Acta, 1268 (1996) 279-284. in [151 Akerboom, T.P.M., Bartosz, G. and Sies, H., Low- and high-K, transport of dinitrophenylglutathione inside-out vesicles from human erythrocytes, Biochim. Biophys. Acta, 1103 (1992) 115-l 19 [I61Bartosz, G., Sies, H. and Akerboom, T.P.M., Organic anions exhibit distinct inhibition patterns on the low-K, and high-g, transport of S-(2,4-dinitrophenyl)glutathione through the human erythrocyte membrane, Biochem. J., 292 (1993) 171-174
111Ishikawa, T., The ATP-dependent
biochemical and biophysical methods J. B&hem.
Biophys. Methods 33 (1996) 65-71
An electron spin resonance assay of glutathione S-conjugate transport Lukasz Pulaski *, Grzegorz Bartosz Depariment
of Molecular
Received
Biophysics,
Uniwrsi&
of kid5
13 March 1995: revised 7 August
Bnnacha
I2 / 16, 90-237 trid5
1995: accepted 7 August
Poland
1995
Abstract A method for studying export of glutathione S-conjugates from cells is proposed based on the use of the spin label tempo-maleimide. This compound is conjugated intracellularly with glutathione and the concentration of the exported conjugate is measured in cell supernatants in an spin resonance spectrometer after reoxidation with ferricyanide. This method allows for measurements of micromolar concentrations of the conjugate and requires low amounts of cells.
electron
Keywords:
Erythrocyte;
Transport;
Electron spin resonance;
Glutathione
S-conjugate:
Tempo-maleimide
1. Introduction One of the main functions of the ubiquitous tripeptide glutathione is the detoxication of reactive electrophiles of exo- and endogenous origin. Such compounds may be modified (detoxication phase l), conjugated to glutathione by glutathione S-transferases (detoxication phase 2) and then exported out of animal cells or deposited into vacuoles of plant cells (detoxication phase 3) [l-3]. In recent years a growing interest has been observed in studies of the phase 3 of glutathione-linked detoxication, i.e., the active transport of glutathione S-conjugates out of the cytoplasm [1,3-71. This process may be of medical and toxicological importance since cisplatin and other chemotherapeutics
CDNB, I-chloro-2,4-dinitrobenzene: DNP-SG, 2,4-diAbbreviations: B-SG, bimane-S-glutathione; nitrophenyWglutathione; ESR, electron spin resonance: mBCI, monochlorobimane; tempo-maleimide. (2,2,6,6-tetramethyl-l-piperidynyloxy)maleimide; TLC. thin-layer chromatography; TM-SC, tempo-maleimide-S-glutathione. * Corresponding author. Tel.: (48-42) 354-476; Fax: (48-42) 354-473. 0165-022X/96/$15.00
SSDl 0165-022X(95)00036-4
Copyright
0 1996 Elsevier Science B.V. All rights reserved.
[4,5] as well as metabolites of some pesticides [I J] may be exported from cells via the ‘glutathione S-conjugate pump’. The classic whole-cell method of studies of transport of glutathione S-conjugates consists of loading the cells with CDNB and calorimetric monitoring of the appearance of the formed conjugate (DNP-SC) in extracellular medium 17-l I]. A serious disadvantage of this method is its low sensitivity unless a radiolabeled CDNB analog (fluorodinitrobenzene) is used and efflux of radioactivity is measured [ 12,131. However, apart from the radioactive hazard. this technique is much more expensive. In this paper, we propose a method for studies of glutathione S-conjugate export from cells employing a commonly used spin label tempo-maleimide which is sensitive. requires low amounts of cells and may be convenient for users of ESR spectrometers. In a separate paper, we proposed another sensitive method based on measurement of fluorescence of a bimane conjugate of glutathione [ 141.
2. Materials
and methods
Blood was obtained from healthy donors (young males) by venipuncture after informed consent. Erythrocytes were collected after centrifugation at 3000 X ,q for 5 min. washed 3 times with PBS t IS0 mM NaCI. IO mM phosphate buffer, pH 7.4) and used within 24 h. Tempo-maleimide and all the other reagents were from Sigma (Deisenhofen. Germany) and were of analytical grade. All values are averaged from at least 3 experiments (mean * SD).
Erythrocytes were suspended to a hematocrit 50% in buffer Z (138 mM NaCI, 5 mM KCI. I mM MgCl?. IO mM glucose. 8 mM phosphate buffer, pH 7.4). Tempo-maleimide was added from a stock ethanol solution (IO mM). After 5-min incubation at room temperature the erythrocytes were washed three times at 4°C with buffer Z and suspended in this buffer to hematocrit 50%. After specified times of incubation at 37°C aliquots were withdrawn. the erythrocytes were sedimented (1 min, I5 000 X g, 2°C) and the supematant was collected. When hemolysis was checked, it was always found to be below 17~. The ESR spectra of the supernatants were measured in an ESP-300E spectrometer (Bruker) after reoxidation of the label with 1 mM potassium ferricyanide. Concentration of TM-SG in the supernatants was estimated either by double integration of ESR spectra or by measurement of the height of the middle peak of the spectrum (‘peak picking procedure’). In the double integration procedure, tempo solutions of different concentrations were used as standards. The height of the middle peak in the spectrum (Fig. 1) was found to be proportional to the label concentration for the whole concentration range of TM-SG employed. Its use can be recommended because the measurement is simple and can be done even on spectra taken in old-fashioned, non-computerized spectrometers. For measurements of intracellular conjugate concentration, erythrocytes. after preincubation with tempo-maleimide and washing off the excess label. were centrifuged to a
t. Pulaski. G. Bartm:
/ J. Brocherrr. Bioph,w. Methods 33 ( IYYfil
Fig. 1. ESR spectrum of TM-Xi
65- 71
67
in supernaranrs of erythrocytesuspensions.
hematocrit of 90% and lysed with an equal volume of water. Hemoglobin, which interfered with the measurements by quenching the ESR signal, was removed by centrifugation through Centricon C-30 filters (Amicon) and ESR spectra of so prepared samples were measured. TLC demonstrated that all the spin label was present as TM-W in the hemolysates. For competitive inhibition experiments cells were first preincubated for 5 min with 100 FM tempo-maleimide and afterwards (without washing) with appropriate concentrations of CDNB (for 15 min) or mBC1 (for 5 min) to rule out competition at the conjugation level. Since TM-SG transport rate remained constant in these conditions for 60 min, the subsequent preincubations did not interfere with the results.
3. Results 3.1. Identification
of TM-SG
Upon incubation of human erythrocytes with tempo-maleimide a compound giving an ESR signal was excreted to the medium in a time-dependent manner. This compound was identified as tempo-maleimide glutathione S-conjugate (tempo-maleimide-s-glutathione, TM-SG) by means of TLC on Silicagel 60 (Merck, Darmstadt). After developing the chromatogram in n-propanol/acetic acid/H20 (10: 1:5) the TLC plate was divided into 15 zones covering the distance between application site and solvent front. The silicagel from each zone was scraped off, suspended in 30% ethanol and the ESR spectra were measured. Fig. 2 shows the distribution of ESR signal amplitude in chromatograms of erythrocyte suspension supematant after incubation with tempo-maleimide, for a solution of TM-SG obtained by non-enzymatic conjugation of tempo-maleimide to excess glutathione at pH 7.0, and for the tempo-maleimide. The R, of TM-SG in this solvent system was found to be 0.47.
68
L. Putmki,
G. Bartos: / J. Biochem. Biophys. Mrih0d.v 33
(1 Y96)
65- 71
25
2
15
1
*
R:,=,
z
=
I
r-E 05
+A
*L 0 0
02
06
04
08
1
Rf Fig. 2. Distribution of ESR signal amplitude in thin-layer chromatograms (Silicagel 60. n-propanol/acetic acid/water, IO: I :5): E, TM-Xi exported from erythrocytes: A, TM-SC obtained by non-enzymatic conjugation of tempo-maleimide with glutathione: L. free tempo-maleimide.
3.2. Kinetics of TM-SG export .from the erythroqtes
The time dependence of extracellular concentration of TM-SG, reflecting the conjugate export from the erythrocytes, was found to be linear (correlation coefficient r = 0.98) for incubation times up to 60 min after preincubation with 100 FM tempomaleimide (Fig. 3). Lineweaver-Burk plot of the concentration dependence of TM-SG export (Fig. 4) could be fitted to a single line corresponding to a K, of 939 + 15 FM and a V,,, of 420 i 48 nmol/(ml X h).
0
0
IO
20
30
40
50
60
Time [min]
Fig. 3. Time course of TM-SC export from erythrocytes mide.
after 5 min incubation
with 100 FM tempo-malei-
t. Pulaski.
G. Burtosz / J. Biochem.
-0 02
0
002
3.3. Inhibition
Methods
004 l/S
Fig. 4. Lineweaver-Burk
Biophp.
006
33
(19%)65-
008
71
69
01
[l/pm]
plot of transport rate dependence
on intracellular
TM-SG concentration
of TM-SG export from the etythrocytes
TM-SG export was strongly inhibited by high concentrations of sodium ortho-vanadate and sodium fluoride, known potent inhibitors of erythrocyte glutathione conjugate pump [ 151. Intracellular formation of other substrates for the glutathione S-conjugate pump, DNP-SG (by treatment of erythrocytes with CDNB) [8,9,15] and B-SG (by treatment with mBC1) [ 141 also decreased the export rate of TM-SG (Table 1).
4. Discussion
Tempo-maleimide penetrating into the erythrocytes reacts mainly (in approx. 70-80%) with intracellular glutathione (the remainder is bound to hemoglobin and other proteins, including membrane proteins). The conjugate formed (TM-SG) is exported out of the cells via the glutathione S-conjugate pump. The latter conclusion can be drawn from the inhibition of the TM-SG export by o-vanadate and fluoride which block the export of DNP-SG [ 151, the inhibition of TM-SG export by other intracellularly formed glutathione conjugates, B-SG and DNP-SG (Table 1) and from the competitive inhibition of
Table I Inhibition
of TMSG transport
in human erythrocytes Transport
Control 250 PM o-vanadate 20 mM fluoride 50 IJ.M CDNB 250 )LM CDNB 1OOOmMCDNB 50 PM mBC1
84.8 8.3 11.7 44.6 14.6 5.5 69.8
rate (nmol/(ml
X h))
Relative transport rate (SC) 100 9.8 13.8 52.6 17.2 6.5 82.3
70
i.. Prtfmki.
G. Bortos: / J. Biociturrr. Biopiys.
Methods 33
f IYY61
65-7f
the active transport of [‘HIDNP-SC into inside-out erythrocyte membrane vesicles by TM-SG (Bartosz. Sies and Akerboom. unpublished). Therefore, measurement of the TM-SG export is a valid means of studying the glutathione S-conjugate pump. Advantages of this technique include low concentrations of the conjugate which can be measured (starting from several FM) and low volumes of samples to be analysed (tens of p_ll. We studied the transport in erythrocytes but this method may be even more useful for cells of other types, available in much lower quantities. Thus. the method proposed is simple and cheap in many respects. provided an ESR spectrometer is available. Nitroxide spin labels are reduced in cells, mainly to non-paramagnetic hydroxylamine derivatives which can be reoxidized by ferricyanide. Also the tempo-maleimide and TM-SG are partly reduced inside erythrocytes and TM-SG is transported partly in the reduced form. Therefore. reoxidation of the conjugate is needed before the measurement. One should be careful. however, to use low concentrations of ferricyanide because higher concentrations would quench the signal. Under our conditions, the intensity of the TM-SG signal in erythrocyte supernatants increased by about 30% after addition of ferricyanide. If the spin labels were completely reduced inside the cells (which may be the case with other cell types). one could think of a transport assay in whole cell suspensions, adding ferricyanide to extracellular medium and monitoring the increase in signal intensity in cell suspension. Careful studies of the concentration dependence of DNP-SG export from erythrocytes [I I] or its import into inside-out vesicles [ lS,l6] showed the existence of two kinetic components of the transport, one of high affinity (low K,,) and low capacity, and another of low affinity (high K,, 1 and high capacity. The Lineweaver-Burk plot of the concentration dependence of TM-SG transport shows no clear-cut biphasicity (Fig. 4) with apparent K,,, value corresponding apparently to the low affinity component of the ‘glutathione S-conjugate pump’ (for DNP-SG: 897 pM [16]). The heterogeneity of the transport substrate (TM-SG with the nitroxide group intact or reduced) also did not result in any apparent heterogeneity of the Lineweaver-Burk plot which suggests that the reduction of the nitroxide group does not have any significant effect on the affinity of the conjugate for the transporter.
5. Simplified
description
of the method and its advantages
The method proposed consists in an electron spin resonance (ESR) assay of a spin-labeled glutathione S-conjugate exported from erythrocytes. Upon incubation of the erythrocytes with a maleimide spin label (tempo-maleimide), tempo-maleimide-.Yglutathione (TM-SG) is formed inside the cells. The time course of TM-SG export from the cells is monitored by ESR measurement of its concentration in cell suspension supernatants after reoxidation of the label with I mM potassium ferricyanide. The method consumes little time and material. allows an easy determination of glutathione S-conjugate transport kinetics in intact erythrocytes and other cells, and avoids the use of radioactivity or noxious chemicals.
15 Pubski,
G. Bartosz/J.
Biochem.
Biophys. Methods
33 (1996165-71
71
Acknowledgements This study was supported by the grant No. 6 P203 001 04 of the Committee for Scientific Research (Poland). We are indebted to the Department of Biophysics, Medical University of t6di, for the supply of human erythrocytes.
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111Ishikawa, T., The ATP-dependent