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Catalytic effects by thioltransferase on the transfer of methylmercury and p-mercuribenzoate from macromolecules to low molecular weight thiol compounds
Toxicology, 10 (1978) 115--122
© Elsevier/North-Holland Scientific Publishers Ltd.
CATALYTIC EFFECTS BY THIOLTRANSFERASE ON THE T R A N S F E R OF METHYLMERCURY AND p-MERCURIBENZOATE FROM MACROMOLECULES TO LOW MOLECULAR WEIGHT THIOL COMPOUNDS
STELLAN ERIKSSON and ANDERS SVENSON* Department o f Biochemistry, Arrhenius Laboratory, Fack, S-106 91 Stockholm and *Swedish Water and Air Pollution Research Institute, POB 210 60 S-100 31 Stockholm (Sweden)
(Received September 6th, 1977) (Revision received February 2nd, 1978) (Accepted February 9th, 1978)
SUMMARY
Thiol agarose and glyceraldehyde-3-phosphate dehydrogenase were blocked with methylmercury or p-mercuribenzoate. The exchange of mercurials between the thiol-containing polymers and glutathione or dithioerythritol was investigated. The activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited by blocking thiol-groups with the mercury compounds. Inhibition was reversible when a short period of inactivation was used. Inactivation for longer periods resulted in reduced regain of enzyme activity. The activity was in part regained when either of the 2 thiol compounds was added. Thioltransferase, known to catalyze thiol-disulfide exchange reactions, increased the regain of glyceraldehyde-3-phosphate dehydrogenase activity to nearly the original value. Here, thioltransferase is proposed to catalyze the transfer of organomercurial from one thiol complex to another. Some consequences of the observations in vivo are discussed.
INTRODUCTION
Monosubstituted organomercurials readily form complexes with thiol compounds. The complexes are usually very strong with equilibrium constants of 10~°--10~° M -1 , cf. [1]. Although strong, the complexes may undergo displacement with other thiol compounds at measurable rates. Abbreviations: DTE, dithioerythritol; EDTA, ethylenediamino tetraacetic acid disodium salt; G3PD, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; GSSG, glutathione disulfide; MeHg, methylmercury; PMB, p-mercuribenzoate ; TT, thioltransferase.
115
Metabolism of mercury in nature was shown to occur from organic mercury compounds to inorganic mercury [2,3] as well as in the reverse direction [4], catalyzed by microorganisms. There is a continuous intake of mercury with food and the strongly absorbed methylmercury (MeHg) is partially accumulated and/or eliminated [2]. The fate of intracellular mercury is little investigated, but the main part is found in the cytosol [5]. Noteworthy, the organomercury mercaptides of cysteine were found in urine [6] and bile [7]. The purpose nf this investigation was to elucidate the metabolic events leading from high molecular weight mercaptides of alkyl/aryl mercurials to more easily eliminated products. The role of thioltransferase, earlier known to catalyze thiol-disulfide exchange [8], was investigated in such a detoxification reaction. Thioltransferase is the enzyme of many animal tissues responsible for the reduction of disulfide groups via a thiol transfer. In the cell the process is coupled to the reduction of glutathione disulfide (GSSG) catalyzed by another enzyme, glutathione reductase. In vitro thioltransferase has been assigned activity with a number of other disulfides and thiol compounds as substrates (EC 1.8.4.1, 1.8.4.3 and 1.8.4.4) [9,10]. MATERIALS AND METHODS Thiol agarose was prepared from Activated Thiol-Sepharose 4B, obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. The commercial product was swollen and washed according to the manufacturer's instructions. Swollen gel (10 g wet, packed wt.) was suspended in 0.1 M Na-borate, pH 8.5, containing 20 mM dithioerythritol (DTE) (Sigma Chem. Co., St Louis, USA). The suspension was gently agitated and after 20 min at 25°C the gel was collected by filtration and washed with buffer. Freshly prepared thiol gels were used in the experiments. The thiol content of the gel was determined with 2,2'
116
After 15 min of mixing, reduced glutathione (GSH) was added to give 50, 100 and 500 t~M concentrations in 3 parallel tests. The suspensions were gently agitated and after 10, 20 and 30 min the gel particles were separated from their surrounding m e d i u m b y centrifugation. From this medium samples of 0.10 ml were withdrawn for radioactivity analysis. In another series of experiments a mixture of GSSG and GSH was used. The concentrations at the start o f the experiments were 21 and 8 pM respectively. In parallel tests thioltransferase was added to 0.033 and 0.067 pM. Glyceraldehyde-3-phosphate dehydrogenase (G3PD, EC 1.2.1.12) was used as a naturally occurring high molecular weight polythiol c o m p o u n d and substrate in the assay system. A 10 mg/ml suspension o f the enzyme from rabbit muscle was obtained from Boehringer, Mannheim, Germany. E n z y m e activity was inhibited in a mixture of G3PD (7 pM) and 0.5 mM p-mercuribenzoate (PMB, obtained from Sigma Chem. Co.). The volume of the mixture was 0.10 ml and the composition of the buffer was 0.2 M Tris--HC1, pH 7.5. The mixture was incubated at 20°C for 5 min. Inactive G3PD was reactivated b y dilution of 10 t~l of the incubation mixture in 0.1 mM DTE or GSH dissolved in buffer to a final vol. of 0.10 ml. The activity of reactivated G3PD was determined spectrophotometrically according to a standard procedure [ 1 4 ] , after 5 min of reactivation. Reactivation with and w i t h o u t the presence of thioltransferase was performed. Thioltransferase was prepared from bovine liver using a modification of the procedure described by Eriksson and Guthenberg [ 1 0 ] . The specific activity obtained with GSH and cystine was 250 p m o l / m i n • mg. At the start o f the reactivation experiments 20 pl o f a 4.6 pM solution o f thioltransferase were added to the medium, and the activity of G3PD was determined as described above. R E S U L T S AND DISCUSSION
The analysis o f radioactive m e t h y l m e r c u r y in the supernatant of the suspension of methylmercury-thiol agarose showed that a constant level was reached after 10 min of reaction, when the first sample was taken. Even when the concentration of GSH was lower (50 uM) the exchange was t o o rapid to be followed with any accuracy b y this technique. Addition of thioltransferase had, therefore, no measurable effect on the exchange. The comparatively low equilibrium constants with m e t h y l m e r c u r y and the gel m a y result from the polythiol function of the gel. Constants of l 0 s --106 M -1 were estimated as compared with those o f m e t h y l m e r c u r y and cysteine or glutathione (1016 M -1 [1]). The low equilibrium constants may thus explain w h y the non-enzymatic exchange was achieved within 10 min. The release of m e t h y l m e r c u r y with the mixture of 8 pM GSH and 21 pM GSSG was considerably slow as shown in Fig. 1. Upon addition of thioltransferase the initial rate was markedly increased, cf. Fig. 1. In this experiment thioltransferase is supposed to act in a two-steps reaction (Fig. 2). In Eqn. 1 the reduction o f GSSG was catalyzed with thiols on the agarose
117
200
i
i
i
100
b E
Z
0
i
50
0
13me (min}
, 100
, 1200
Fig. 1. Effect of thioltransferase on the exchange of methylmercury between thiol agarose and glutathione. The concentration of radioactive methylmercury in the supernatant of the suspension of methylmercury-thiol agarose mercaptide plotted as a function of time. The rate of exchange was determined in a mixture of GSSG and GSH alone (--~--), and with thioltransferase added (--o--). Experimental details in the text.
gel as the second substrate. In the second step (Eqn. 2) MeHg was transferred from the agarose to GSH, eventually catalyzed by thioltransferase. This possible effect was further investigated in a series of experiments. The synthetic thiol agarose might reflect the properties of a protein with polythiol function. G3PD was chosen as an example of naturally occurring polythiol compounds. If inhibited b y mercurials, the degree of its reactivation could easily be measured. Furthermore this enzyme holds a central position in cell metabolism and it is localized in the cytosol fraction, and is thus easily attacked by mercurials appearing intracellularly. G3PD from rabbit muscle contains 14 SH groups, 11 of which are reactive and 4 are essential for activity [ 1 5 ] . Although there seems to be a similar n u m b e r of SH equivalents in the 2 assay systems those o f G3PD are thus exMeHg
M,Hg S
SH
SH
~ll/l
I Il l
* GSSG
GSI
Ill/l/l/
MeHg S }
1L
Eq. 1
S-S I
+ GSH
"ILl' MeHg
GS
I
S
/
GS
I
SH S // // /
/
J"
( ond / or A )
I
GsH
- ,
SH SH S /.r , , , ' ' / , ' ,
• MeHg-SG
Eq. 2
Fig. 2. Thioltransferase mediating the reduction of GSSG by thiol agarose gel (Eqn.1). The transfer of methylmercury from the thiol agarose gel to GSH (Eqn. 2). GSH, glutathione. GSSG, glutathione disulfide; MeHg, methylmercury; TT, thioltransferase.
118
TABLE
I
INACTIVATION DEHYDROGENASE
Components
AND a
REACTIVATION
of the different
Inactivation
PMB
incubations
OF
GLYCERALDEHYDE-3-PHOSPHATE
b
Activity %
Reactivation
MeHg
G3PD
PMB
MeHg
G3PD
DTE
GSH
TT
0
--
7
0
--
0.7
100
--
1
500
--
7
50
--
0.7
0
--
0
2
500
--
7
50
--
0.7
100
--
0
68
500
--
7
50
--
0.7
100
--
1
97
50
--
7
5
--
0.7
100
--
0
65
50 500 500
----
7 7 7
5 50 50
----
0.7 0.7 0.7
100 ---
-100 100
1 0 1
97 52 58
500
--
7
50
--
0.7
--
100
2.5
86
500
--
7
50
--
0.7
--
10
0
33
500 ---
-500 500
7 7 7
50 ---
-50 50
0.7 0.7 0.7
--
1
0 100
10 ---
0
37 8 53
--
--
500 500
7 7
---
50 50
0.7 0.7
100 100
---
0.5 1
66 89
--
500
7
--
50
0.7
100
--
2.5
94
Abbreviations:
PMB,
aThe
assays were run in duplicate.
ball
concentrations
are
given
in
uM.
methylmercuric chloride; G3PD, glyceraldehyde-3-phosphate thioerythritol; GSH, glutathione; TT, thioltransferase. CThe activity system.
of
G3PD
was
60
nmol
of NADH
oxidized
1
100 c
p-mercuribenzoate; dehydrogenase;
MeHg, DTE,
per rain in the standard
di-
assay
pected to have a more variable availability and reactivity to mercurials. The inactivation of G3PD with mercurials as well as the regain of activity with DTE and GSH and the regain catalyzed by thioltransferase are presented in Table I. G3PD was almost completely inactivated by 500 pM PMB. Regain o f activity measured 5 min after the addition o f thiol c o m p o u n d was variable, and was d e p e n d e n t on the species o f thiol c o m p o u n d and its concentration. Thus DTE was f o u n d to be more effective than GSH as a displacer o f PMB from the active site thiols o f G3PD. Increasing the concentration o f GSH leads t o a higher rate o f exchange. Addition o f thioltransferase (1 pM) to reactivation medium containing DTE resulted in almost complete reactivation of G3PD after only 5 min. The reaction scheme is presented in Fig. 3. The c o n c e n t r a t i o n o f thioltransferase in bovine liver as well as in the assay system is 1 pM. A control experiment with G3PD and thioltransferase without substrate resulted in a small degree of reactivation or protection (Table I).
119
-S-Hg-R G3PD
÷ HS-R'
spontaneous
TT
,
1 -SH ÷ R-Hg-S-R' G3PD
reactivated
Fig. 3. Thioltransferase catalyzing the transfer of alkyl/aryl mercury from glyceraldehyde3-phosphate dehydrogenase to low molecular weight compounds. Abbreviations are listed in Fig. 2 and Table 1.
In vivo a still faster reactivation could be expected t h a n t h a t shown in the table. The complex test system measures the activity of thioltransferase indirectly and the natural liver c o n t e n t of 5--10 mM GSH in the assay system would give too rapid reaction velocity to be accurately followed. Estimating the K m value o f GSH to be in the mM range, the reaction velocity would increase proportionally with GSH. Thioltransferase contains one thiol, group probably constituting a part of the active site, because the enzyme activity is abolished by thiol-blocking reagents. There is thus a minor contribution of thiol groups from thioltransferase that might act non-enzymatically. In liver cells the ratio of GSH: thioltransferase is estimated to be 10,000 : 1, respectively. With the introduction of heavy metals into proteins there is a concomitant risk for secondary changes of the protein. Exposure o f the e n z y m e to 500 ~M PMB for more than 5 min gave a lower degree of reactivation. The incubation periods used for inactivation were 5, 7.5 and 45 min, and this t r e a t m e n t resulted in 97, 65 and 3% o f activity of G3PD after reactivation with DTE and thioltransferase. This may be explained by an irreversible change in c o n f o r m a t i o n o f the e n z y m e when highly blocked with PMB [1,16,17]. Therefore a preincubation period of 5 min was chosen during which time reactivation was n o t disturbed by secondary changes. In addition thioltransferase would catalyze the detoxification o f a protein-mercury mercaptide in vivo by means o f GSH, as soon as the heavy metal mercaptide appears, i.e., before a conformational change has taken place, leaving the protein in a euphoristic state [18]. The same degree of reactivation was obtained when the PMB concentration was 50 pM as compared with 500 pM in the standard procedure. Methylmercury also inhibited the activity of G3PD. In general removal o f MeHg from G3PD was more difficult t h a n removal of PMB both when treated with DTE alone or in combination with thioltransferase. The degree o f reactivation of G3PD increased when the a m o u n t of thioltransferase was increased. The effect o f thioltransferase on the exchange o f organomercuric ions from G3PD to GSH and DTE shows t h a t the e n z y m e can accept mercury mercaptides as a substrate. To our knowledge this has n o t been demonstrated earlier. The general picture t h a t emerges from these experiments is t h a t the thioltransferase acts as a catalyst for the transfer of
120
an organomercurial from one thiol complex to another. The mercury moiety thus replaces a sulfenyl part of a disulfide substrate. A survey of the distribution o f organomercury c o m p o u n d s at the subcellular level showed that part of the mercury is b o u n d to membranes and to subcellular particles, but the main portion in liver was found in the cytosol [ 2 , 5 ] . This latter fraction is probably bound to proteins containing thiol groups, cf. [2]. GSH is the most abundant cellular non-protein thiol [19]. The occurrence of the cysteine organomercury mercaptide as an excretion product [6,7] suggests that GSH is involved in detoxification of the mercaptide between proteins and mercury. The complex between GSH and the mercury derivative would thereafter be degraded to the cysteine moiety alone, in parallel to the metabolic degradation of xenobiotics leading to mercapturic acids
[20]. The observation that thioltransferase enhances the exchange of mercurials from one mercaptide complex to another might be clinically useful with respect to detoxification of mercurials. ACKNOWLEDGEMENTS
This work was in part supported by grants (to S.E.) from the foundations of Harald Jeansson and Hahns (Royal Academy of Science).
REFERENCES 1 J.L. Webb, Enzyme and Metabolic Inhibitors, Vol. 2, Academic Press, London, 1966, p. 729. 2 F. Berglund, M. Berlin, G. Birke, R. CederlSf, U. von Euler, L. Friberg, B. Holmstedt, E. Jonsson, K.G. Lfining, C. Ramel, S. Skerfving, ~. Swensson and S. Tejning, Nord. Hyg. Tidskr., Suppl., 52 (1971) 4. 3 K. Furukawa, T. Suzuki and K. Tonomura, Agric. Biol. Chem., 33 (1969) 128. 4 S. Jensen and A. JernelSv, Nature, 223 (1967) 753. 5 T. Norseth, Biochem. Pharmacol., 16 (1967) 1645. 6 I.M. Weiner, R.I. bevy and G.H. Mudge, J. Pharmacol. Exp. Ther., 138 (1962) 96. 7 T. Norseth and T.W. Clarkson, Arch. Environ. Health, 21 (1970) 717. 8 P. AskelSf, K. Axelsson, S. Eriksson and B. Mannervik, FEBS Lett., 38 (1974) 263. 9 S. Eriksson and B. Mannervik, Biochim. Biophys. Acta, 212 (1970) 518. 10 S. Eriksson and C. Guthenberg, Biochem. Pharmacol., 24 (1975) 241. 11 D.R. Grassetti and J.F. Murray, Jr., Arch. Biochem. Biophys., 82 (1967) 41. 12 K. Brocklehurst, J. Carlsson, M.P.J. Kierstan and E.M. Crook, Biochem. J., 133 (1973) 573. 13 T. Stuchbury, M. Shipton, R. Norris, J.P.G. Malthouse, K. Brocklehurst, J.A.L. Herbert and H. Suschitzky, Biochem. J., 151 (1975) 417. 14 H.U. Bergmeyer, (Ed.) Methoden der Enzymatischen Analyse, Verlag Chemie, Weinheim, 1974, p. 495. 15 R.E. Amelunxen, D.O. Cart and S. Grisolia, Biochim. Biophys. Acta, 113 (1966) 184. 16 K. Kirschner and G. Johannin, Hoppe-Seylers Z. Physiol. Chem., 348 (1967) 1235. 17 L. Kastenschmidt, J. Kastenschmidt and E. Helmreich, Biochemistry, 7 (1968) 3590.
121
18 19 20
122
E. Racker, Glutathione as a coenzyme in intermediary metabolism, in S. Colowick (Ed.), Glutathione: a Symposium, Academic Press, London, 1954, p. 166. P.C. Jocelyn, Biochemistry of the SH-Group, Academic Press, London, 1972, p. 10. E. Boyland and L.F. Chasseaud, The role of glutathione and glutathione S-transferase in mercapturic acid biosynthesis, in A.H. Meister (Ed.), Adv. Enzymol., Vol. 32, Academic Press, London, 1969, p. 173.
© Elsevier/North-Holland Scientific Publishers Ltd.
CATALYTIC EFFECTS BY THIOLTRANSFERASE ON THE T R A N S F E R OF METHYLMERCURY AND p-MERCURIBENZOATE FROM MACROMOLECULES TO LOW MOLECULAR WEIGHT THIOL COMPOUNDS
STELLAN ERIKSSON and ANDERS SVENSON* Department o f Biochemistry, Arrhenius Laboratory, Fack, S-106 91 Stockholm and *Swedish Water and Air Pollution Research Institute, POB 210 60 S-100 31 Stockholm (Sweden)
(Received September 6th, 1977) (Revision received February 2nd, 1978) (Accepted February 9th, 1978)
SUMMARY
Thiol agarose and glyceraldehyde-3-phosphate dehydrogenase were blocked with methylmercury or p-mercuribenzoate. The exchange of mercurials between the thiol-containing polymers and glutathione or dithioerythritol was investigated. The activity of glyceraldehyde-3-phosphate dehydrogenase was inhibited by blocking thiol-groups with the mercury compounds. Inhibition was reversible when a short period of inactivation was used. Inactivation for longer periods resulted in reduced regain of enzyme activity. The activity was in part regained when either of the 2 thiol compounds was added. Thioltransferase, known to catalyze thiol-disulfide exchange reactions, increased the regain of glyceraldehyde-3-phosphate dehydrogenase activity to nearly the original value. Here, thioltransferase is proposed to catalyze the transfer of organomercurial from one thiol complex to another. Some consequences of the observations in vivo are discussed.
INTRODUCTION
Monosubstituted organomercurials readily form complexes with thiol compounds. The complexes are usually very strong with equilibrium constants of 10~°--10~° M -1 , cf. [1]. Although strong, the complexes may undergo displacement with other thiol compounds at measurable rates. Abbreviations: DTE, dithioerythritol; EDTA, ethylenediamino tetraacetic acid disodium salt; G3PD, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathione; GSSG, glutathione disulfide; MeHg, methylmercury; PMB, p-mercuribenzoate ; TT, thioltransferase.
115
Metabolism of mercury in nature was shown to occur from organic mercury compounds to inorganic mercury [2,3] as well as in the reverse direction [4], catalyzed by microorganisms. There is a continuous intake of mercury with food and the strongly absorbed methylmercury (MeHg) is partially accumulated and/or eliminated [2]. The fate of intracellular mercury is little investigated, but the main part is found in the cytosol [5]. Noteworthy, the organomercury mercaptides of cysteine were found in urine [6] and bile [7]. The purpose nf this investigation was to elucidate the metabolic events leading from high molecular weight mercaptides of alkyl/aryl mercurials to more easily eliminated products. The role of thioltransferase, earlier known to catalyze thiol-disulfide exchange [8], was investigated in such a detoxification reaction. Thioltransferase is the enzyme of many animal tissues responsible for the reduction of disulfide groups via a thiol transfer. In the cell the process is coupled to the reduction of glutathione disulfide (GSSG) catalyzed by another enzyme, glutathione reductase. In vitro thioltransferase has been assigned activity with a number of other disulfides and thiol compounds as substrates (EC 1.8.4.1, 1.8.4.3 and 1.8.4.4) [9,10]. MATERIALS AND METHODS Thiol agarose was prepared from Activated Thiol-Sepharose 4B, obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. The commercial product was swollen and washed according to the manufacturer's instructions. Swollen gel (10 g wet, packed wt.) was suspended in 0.1 M Na-borate, pH 8.5, containing 20 mM dithioerythritol (DTE) (Sigma Chem. Co., St Louis, USA). The suspension was gently agitated and after 20 min at 25°C the gel was collected by filtration and washed with buffer. Freshly prepared thiol gels were used in the experiments. The thiol content of the gel was determined with 2,2'
116
After 15 min of mixing, reduced glutathione (GSH) was added to give 50, 100 and 500 t~M concentrations in 3 parallel tests. The suspensions were gently agitated and after 10, 20 and 30 min the gel particles were separated from their surrounding m e d i u m b y centrifugation. From this medium samples of 0.10 ml were withdrawn for radioactivity analysis. In another series of experiments a mixture of GSSG and GSH was used. The concentrations at the start o f the experiments were 21 and 8 pM respectively. In parallel tests thioltransferase was added to 0.033 and 0.067 pM. Glyceraldehyde-3-phosphate dehydrogenase (G3PD, EC 1.2.1.12) was used as a naturally occurring high molecular weight polythiol c o m p o u n d and substrate in the assay system. A 10 mg/ml suspension o f the enzyme from rabbit muscle was obtained from Boehringer, Mannheim, Germany. E n z y m e activity was inhibited in a mixture of G3PD (7 pM) and 0.5 mM p-mercuribenzoate (PMB, obtained from Sigma Chem. Co.). The volume of the mixture was 0.10 ml and the composition of the buffer was 0.2 M Tris--HC1, pH 7.5. The mixture was incubated at 20°C for 5 min. Inactive G3PD was reactivated b y dilution of 10 t~l of the incubation mixture in 0.1 mM DTE or GSH dissolved in buffer to a final vol. of 0.10 ml. The activity of reactivated G3PD was determined spectrophotometrically according to a standard procedure [ 1 4 ] , after 5 min of reactivation. Reactivation with and w i t h o u t the presence of thioltransferase was performed. Thioltransferase was prepared from bovine liver using a modification of the procedure described by Eriksson and Guthenberg [ 1 0 ] . The specific activity obtained with GSH and cystine was 250 p m o l / m i n • mg. At the start o f the reactivation experiments 20 pl o f a 4.6 pM solution o f thioltransferase were added to the medium, and the activity of G3PD was determined as described above. R E S U L T S AND DISCUSSION
The analysis o f radioactive m e t h y l m e r c u r y in the supernatant of the suspension of methylmercury-thiol agarose showed that a constant level was reached after 10 min of reaction, when the first sample was taken. Even when the concentration of GSH was lower (50 uM) the exchange was t o o rapid to be followed with any accuracy b y this technique. Addition of thioltransferase had, therefore, no measurable effect on the exchange. The comparatively low equilibrium constants with m e t h y l m e r c u r y and the gel m a y result from the polythiol function of the gel. Constants of l 0 s --106 M -1 were estimated as compared with those o f m e t h y l m e r c u r y and cysteine or glutathione (1016 M -1 [1]). The low equilibrium constants may thus explain w h y the non-enzymatic exchange was achieved within 10 min. The release of m e t h y l m e r c u r y with the mixture of 8 pM GSH and 21 pM GSSG was considerably slow as shown in Fig. 1. Upon addition of thioltransferase the initial rate was markedly increased, cf. Fig. 1. In this experiment thioltransferase is supposed to act in a two-steps reaction (Fig. 2). In Eqn. 1 the reduction o f GSSG was catalyzed with thiols on the agarose
117
200
i
i
i
100
b E
Z
0
i
50
0
13me (min}
, 100
, 1200
Fig. 1. Effect of thioltransferase on the exchange of methylmercury between thiol agarose and glutathione. The concentration of radioactive methylmercury in the supernatant of the suspension of methylmercury-thiol agarose mercaptide plotted as a function of time. The rate of exchange was determined in a mixture of GSSG and GSH alone (--~--), and with thioltransferase added (--o--). Experimental details in the text.
gel as the second substrate. In the second step (Eqn. 2) MeHg was transferred from the agarose to GSH, eventually catalyzed by thioltransferase. This possible effect was further investigated in a series of experiments. The synthetic thiol agarose might reflect the properties of a protein with polythiol function. G3PD was chosen as an example of naturally occurring polythiol compounds. If inhibited b y mercurials, the degree of its reactivation could easily be measured. Furthermore this enzyme holds a central position in cell metabolism and it is localized in the cytosol fraction, and is thus easily attacked by mercurials appearing intracellularly. G3PD from rabbit muscle contains 14 SH groups, 11 of which are reactive and 4 are essential for activity [ 1 5 ] . Although there seems to be a similar n u m b e r of SH equivalents in the 2 assay systems those o f G3PD are thus exMeHg
M,Hg S
SH
SH
~ll/l
I Il l
* GSSG
GSI
Ill/l/l/
MeHg S }
1L
Eq. 1
S-S I
+ GSH
"ILl' MeHg
GS
I
S
/
GS
I
SH S // // /
/
J"
( ond / or A )
I
GsH
- ,
SH SH S /.r , , , ' ' / , ' ,
• MeHg-SG
Eq. 2
Fig. 2. Thioltransferase mediating the reduction of GSSG by thiol agarose gel (Eqn.1). The transfer of methylmercury from the thiol agarose gel to GSH (Eqn. 2). GSH, glutathione. GSSG, glutathione disulfide; MeHg, methylmercury; TT, thioltransferase.
118
TABLE
I
INACTIVATION DEHYDROGENASE
Components
AND a
REACTIVATION
of the different
Inactivation
PMB
incubations
OF
GLYCERALDEHYDE-3-PHOSPHATE
b
Activity %
Reactivation
MeHg
G3PD
PMB
MeHg
G3PD
DTE
GSH
TT
0
--
7
0
--
0.7
100
--
1
500
--
7
50
--
0.7
0
--
0
2
500
--
7
50
--
0.7
100
--
0
68
500
--
7
50
--
0.7
100
--
1
97
50
--
7
5
--
0.7
100
--
0
65
50 500 500
----
7 7 7
5 50 50
----
0.7 0.7 0.7
100 ---
-100 100
1 0 1
97 52 58
500
--
7
50
--
0.7
--
100
2.5
86
500
--
7
50
--
0.7
--
10
0
33
500 ---
-500 500
7 7 7
50 ---
-50 50
0.7 0.7 0.7
--
1
0 100
10 ---
0
37 8 53
--
--
500 500
7 7
---
50 50
0.7 0.7
100 100
---
0.5 1
66 89
--
500
7
--
50
0.7
100
--
2.5
94
Abbreviations:
PMB,
aThe
assays were run in duplicate.
ball
concentrations
are
given
in
uM.
methylmercuric chloride; G3PD, glyceraldehyde-3-phosphate thioerythritol; GSH, glutathione; TT, thioltransferase. CThe activity system.
of
G3PD
was
60
nmol
of NADH
oxidized
1
100 c
p-mercuribenzoate; dehydrogenase;
MeHg, DTE,
per rain in the standard
di-
assay
pected to have a more variable availability and reactivity to mercurials. The inactivation of G3PD with mercurials as well as the regain of activity with DTE and GSH and the regain catalyzed by thioltransferase are presented in Table I. G3PD was almost completely inactivated by 500 pM PMB. Regain o f activity measured 5 min after the addition o f thiol c o m p o u n d was variable, and was d e p e n d e n t on the species o f thiol c o m p o u n d and its concentration. Thus DTE was f o u n d to be more effective than GSH as a displacer o f PMB from the active site thiols o f G3PD. Increasing the concentration o f GSH leads t o a higher rate o f exchange. Addition o f thioltransferase (1 pM) to reactivation medium containing DTE resulted in almost complete reactivation of G3PD after only 5 min. The reaction scheme is presented in Fig. 3. The c o n c e n t r a t i o n o f thioltransferase in bovine liver as well as in the assay system is 1 pM. A control experiment with G3PD and thioltransferase without substrate resulted in a small degree of reactivation or protection (Table I).
119
-S-Hg-R G3PD
÷ HS-R'
spontaneous
TT
,
1 -SH ÷ R-Hg-S-R' G3PD
reactivated
Fig. 3. Thioltransferase catalyzing the transfer of alkyl/aryl mercury from glyceraldehyde3-phosphate dehydrogenase to low molecular weight compounds. Abbreviations are listed in Fig. 2 and Table 1.
In vivo a still faster reactivation could be expected t h a n t h a t shown in the table. The complex test system measures the activity of thioltransferase indirectly and the natural liver c o n t e n t of 5--10 mM GSH in the assay system would give too rapid reaction velocity to be accurately followed. Estimating the K m value o f GSH to be in the mM range, the reaction velocity would increase proportionally with GSH. Thioltransferase contains one thiol, group probably constituting a part of the active site, because the enzyme activity is abolished by thiol-blocking reagents. There is thus a minor contribution of thiol groups from thioltransferase that might act non-enzymatically. In liver cells the ratio of GSH: thioltransferase is estimated to be 10,000 : 1, respectively. With the introduction of heavy metals into proteins there is a concomitant risk for secondary changes of the protein. Exposure o f the e n z y m e to 500 ~M PMB for more than 5 min gave a lower degree of reactivation. The incubation periods used for inactivation were 5, 7.5 and 45 min, and this t r e a t m e n t resulted in 97, 65 and 3% o f activity of G3PD after reactivation with DTE and thioltransferase. This may be explained by an irreversible change in c o n f o r m a t i o n o f the e n z y m e when highly blocked with PMB [1,16,17]. Therefore a preincubation period of 5 min was chosen during which time reactivation was n o t disturbed by secondary changes. In addition thioltransferase would catalyze the detoxification o f a protein-mercury mercaptide in vivo by means o f GSH, as soon as the heavy metal mercaptide appears, i.e., before a conformational change has taken place, leaving the protein in a euphoristic state [18]. The same degree of reactivation was obtained when the PMB concentration was 50 pM as compared with 500 pM in the standard procedure. Methylmercury also inhibited the activity of G3PD. In general removal o f MeHg from G3PD was more difficult t h a n removal of PMB both when treated with DTE alone or in combination with thioltransferase. The degree o f reactivation of G3PD increased when the a m o u n t of thioltransferase was increased. The effect o f thioltransferase on the exchange o f organomercuric ions from G3PD to GSH and DTE shows t h a t the e n z y m e can accept mercury mercaptides as a substrate. To our knowledge this has n o t been demonstrated earlier. The general picture t h a t emerges from these experiments is t h a t the thioltransferase acts as a catalyst for the transfer of
120
an organomercurial from one thiol complex to another. The mercury moiety thus replaces a sulfenyl part of a disulfide substrate. A survey of the distribution o f organomercury c o m p o u n d s at the subcellular level showed that part of the mercury is b o u n d to membranes and to subcellular particles, but the main portion in liver was found in the cytosol [ 2 , 5 ] . This latter fraction is probably bound to proteins containing thiol groups, cf. [2]. GSH is the most abundant cellular non-protein thiol [19]. The occurrence of the cysteine organomercury mercaptide as an excretion product [6,7] suggests that GSH is involved in detoxification of the mercaptide between proteins and mercury. The complex between GSH and the mercury derivative would thereafter be degraded to the cysteine moiety alone, in parallel to the metabolic degradation of xenobiotics leading to mercapturic acids
[20]. The observation that thioltransferase enhances the exchange of mercurials from one mercaptide complex to another might be clinically useful with respect to detoxification of mercurials. ACKNOWLEDGEMENTS
This work was in part supported by grants (to S.E.) from the foundations of Harald Jeansson and Hahns (Royal Academy of Science).
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18 19 20
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E. Racker, Glutathione as a coenzyme in intermediary metabolism, in S. Colowick (Ed.), Glutathione: a Symposium, Academic Press, London, 1954, p. 166. P.C. Jocelyn, Biochemistry of the SH-Group, Academic Press, London, 1972, p. 10. E. Boyland and L.F. Chasseaud, The role of glutathione and glutathione S-transferase in mercapturic acid biosynthesis, in A.H. Meister (Ed.), Adv. Enzymol., Vol. 32, Academic Press, London, 1969, p. 173.