ARCHIVES OF BIOCHEMISTRY AND ELIOPHYSICS Vol. 289, No. 1, August 15, pp. 161-166, 1991
Inhibition of Rat Brain Prostaglandin by Inorganic Selenocompounds Fakhrul Department
Islam,
Yasuyosbi
Watanabe,l
Hiroshi
of Neuroscience, *Osaka Bioscience Institute,
Received February
Morii,
D Synthase and Osamu Hayaishi*
6-2-4 Furuedai,
Suita-shi,
Osaka 565, Japan
12, 1991, and in revised form April 30, 1991
Various inorganic selenocompounds dose-dependently inhibited the rat brain prostaglandin (PG) D synthase, both in the purified enzyme preparation and in the crude brain supernatant. All of the quadrivalent selenium compounds tested had a very limited range of I&, values in the purified enzyme (11-12 wM) and in the brain supernatant (9-15 MM). A divalent selenium compound was also inhibitory, but a hexavalent selenium compound was ineffective. In contrast, organic selenocompounds such as selenomethionine and selenourea had no effect on the PGD synthase activity. Furthermore, sodium sulfate and sodium sulfite up to 10 rnW did not inhibit the activity. The inhibition by selenium required the preincubation of the metal with sulfhydryl compounds such as dithiothreitol (DTT), indicating that the formation of selenotrisulfide or some other adduct is essential for the inhibition. Furthermore, the inhibition was reversed by an excess amount of dithiothreitol, suggesting that the selenotrisulfide derivative o:f DTT binds to the SH group of the PGD synthase. The kinetic analysis revealed the inhibition by selenite to be noncompetitive with a Ki value of 10.1 PM. On the other hand, glutathione-dependent PGD synthase from rat spleen was much less inhibited, and PGF synthase and PGD2 11-ketoreductase activities were not inhibited by the selenium compound. o 1991 Academic Press, Inc.
Prostaglandin (PG)’ D synthase (prostaglandin-H, Disomerase, EC 5.3.99.2) is ubiquitously distributed in the central nervous system (CNS) (1, 2) as well as in the peripheral organs (3, 4) and catalyzes the conversion of PGHz to PGD:!. The brain-type PGD synthase, which exists not only in the CNS but also in the cochlea and epidydimis, is glutathione independent but requires a 1 To whom correspondence should be addressed. Fax: (06) 872-4818 ‘Abbreviations used: CNS, central nervous system; DTT, dithiothreitol; GSH, glutathione; KP13, potassium phosphate buffer; PG, prostaglandin. 0003.9861/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
sulfhydryl (SH) compound for its activity (3,4), whereas the peripheral-type PGD synthase is contained in the spleen, small intestine, lung, and other peripheral tissues and is glutathione dependent (4, 5). PGDz is the major PG in the central nervous system and exerts a variety of central actions such as induction of sleep (6, 7), modulation of neuroendocrine function (8), and modification of senses (9, 10). Selenium is an essential trace metal at low concentrations but is toxic at higher concentrations in all organisms (11). The metal is ubiquitously found in both prokaryotic and eukaryotic cells as a highly specific component of certain enzymes such as glutathione peroxidase, formate dehydrogenase, thiolase, xanthine dehydrogenase, glycine reductase, and nicotinic acid hydroxylase (12, 13). A number of biological effects of selenium compounds were shown to be influenced by SH compounds (14-19). For example, significant inhibition of RNA and DNA polymerases was obtained by selenite treatment in the presence of SH compounds (20). Sodium selenite inhibits NaK-ATPase activity (21), and this effect was reversed by excess amounts of glutathione or dithiothreitol (DTT). Also, selenite at a very low concentration inhibits the hormone binding site in the rat liver glucocorticoid receptor, and the inhibition was reversed by DTT (22). Ebselene (PZ 51, 2-phenyl-1,2-benzisoselenazol-3organic selenocom(2H)-one), an anti-inflammatory pound, was reported to inhibit the formation of cyclooxygenase and lipoxygenase products (23-25) and has attracted our interest to evaluate the effect of inorganic and organic compounds of selenium on the activity of PGD synthase in crude and purified enzyme preparations from rat brain. MATERIALS
AND
METHODS
Materials [I-‘*C]Arachidonic acid (2.0 GBq/mmol) was purchased from DuPont New England Nuclear. [l-‘*C]PGHz was prepared according to the method of Ohki et al. (26). Sodium selenite, selenium dioxide, selenium tetrachloride, selenous acid, and selenium oxychloride were purchased from Wako Pure Chemicals (Tokyo); selenium mono161
162
ISLAM
004
I
.
I
4.5 DTT
3.5
2.5
(-IogM)
FIG. 1. Amount of DTT required for the enzyme activity: Purified enzyme (0) or brain supernatant (0) was preincubated in the standard assay mixture with varying concentrations of DTT as described under Materials and Methods. The percentage formation of [l-“C]PGDP during a 1-min incubation with [l-i4C]PGH2 was subtracted by the value obtained in the absence of the enzyme.
chloride came from Rare Metallic Co., Ltd. (Tokyo); and Seleno-DLmethionine, selenourea, and dithiothreitol were from Sigma. Their purities were better than 97%. Male Wistar rats (200-250 Preparation of crude brain supernatunt. g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and sacrificed by perfusion with 250 ml of cold saline via the left ventricle of the heart. The brain was quickly removed and a 20% homogenate (w/v) was prepared in 10 mM potassium phosphate buffer (KPB), pH 8.0, by use of an Ultra-Turrax T-25 homogenizer (Janko & Funkel, West Germany) operated at top speed 10 times for 5 s each. The homogenate was centrifuged at 100,OOOgfor 60 min at 4”C, and the resulting supernatant was assayed for PGD synthase activity. PGD synthase was purified to apPurification of PGD synthase. parent homogeneity from 200 g of rat brains as described by Urade et al. (4). In the last column chromatography (CM-52 cellulose) step of the enzyme purification, no DTT was added in the buffer, so that the purified enzyme would be free of DTT. The purified enzyme was concentrated with a Centricon 10. The specific activity of the final preparation was 7085 nmol/mg protein/min, a value corresponding to that of an earlier preparation described by Urade et al. (4). Assay of enzymes. The standard assay for PGD synthase was performed in the reaction mixture containing 0.1 M KPB (pH 8.0), 0.25 mM DTT, and 0.5 pg of the purified enzyme or rat brain supernatant (70 gg protein) in a total volume of 50 ~1 and preincubated for 10 min at 25’C, unless otherwise stated. The reaction was started by the addition of 1 ~1 of 0.17 mM [l-“C]PGHz (the final concentration and specific _ radioactivity were 3.4 PM and 332 Bd0.17 nmol, respectively), and incubation was carried out for 1 min at 25°C. Termination of the reaction and extraction and quantification of the products were performed as described by Urade et al. (27). The activities of PGDz 11-ketoreductase and PGF synthase were assayed according to Watanabe et al. (28). Protein concentration was determined by the method of Lowry et al. (29) with bovine serum albumin as standard.
RESULTS
Requirement of DTT for PGD synthase activity. Shimizu et al. (30) originally used 0.5 mM DTT, and recently Urade et al. (4) used 1.0 mM DTT for their standard assay of PGD synthase from rat brain. At first we reinvestigated the requirement of DTT for PGD synthase
ET AL.
activity in the crude brain supernatant as well as in the preparation of purified enzyme, as shown in Fig. 1. In both cases DTT accelerated the enzyme activity. The maximal enhancement in the activity was about 7 times in the purified enzyme and about 3 times in the crude brain supernatant as compared with the activity in the absence of DTT. From these data, we decided to employ 0.25 mM DTT for the standard PGD synthase assay. In the Inhibition by selenite on PGD synthase actiuity. course of the study on the regulation of brain-type PGD synthase, we found inhibition by several metal ions of the enzyme activity in a crude supernatant of rat brain. The potency of the inhibition was different among the metal ions, the most potent inhibition having been observed with the quadrivalent selenium ion. The substrate of PGD synthase, [l-14C]PGHZ, is an unstable substance, and a small amount of it is converted nonenzymatically to PGFz,, PGEz, and PGDz when incubated for 1 min in the assay mixture without the enzyme. When the substrate was incubated with the enzyme for 1 min, PGD2 was the main enzymatic product, and the nonenzymatitally derived PGFz, and PGEz were also detected (Fig. 2A). In the presence of 0.1 mM selenite, 1 mM DTT, and
PGFza
PGEz
PGD2
II
PGH2
I
6-
A $ 4 -
; z 5 s 5 g E L
2 _
‘u
4-
z a
F I
o 6 -
2 -
0
’
0
5
10 Distance
15 (cm)
FIG. 2. Radiochromatograms of the products formed by the incubation of [l-i4C]PGHz. (A) [l-i4C]PGHz was enzymatically converted to PGD, when incubated with the purified enzyme and 1 mM DTT at 25°C for 1 min in the standard reaction mixture. The reaction was started by the addition of 1 aI of [l-i4C]PGHz and terminated after 1 min by the addition of ether: MeOH: 0.5 M citric acid (90: 12: 1.5, v/v/v). The organic phase (100 ~1) was spotted onto a TLC plate and developed in ether: MeOH: acetic acid (90: 2: 0.1, v/v/v) at -20°C. (B) Selenite (0.1 mM), DTT (1 mM), and the purified enzyme were preincubated in the standard reaction mixture at 25’C for 10 min, and then assayed by the addition of [l-i4C]PGHz. The positions of PGF,,, PGEz, and PGDx on TLC were identified by comparison with those of their authentic samples. Radioactivity on chromatographic plates was monitored by a Packard imaging scanner system 200. 0, origin; F, solvent front.
SELENIUM
INHIBITION
OF PROSTAGLANDIN
D SYNTHASE
163
TA.BLE I Effect of Various Orders of Preincubation with Selenite on the Activity of PGD Synthase 1st preincubation Time (rn; E + Selenite E+DTT Selenite + DTT Selenite + DTT Selenite + DTT
+ E + E
10 10 10 10 0
2nd preincubation (1 min) DTT Selenite E -
Inhibition (%) 0 0 95 95 0
: E 2 I=
50 -
25 -
,I. 0
Time
Note. E of selenite was carried terials and
= 0.5 ag of the purified PGD synthase. The concentrations and DTT were 15 and 250 pM, respectively, and the assay out in the standard assay mixture as described under MaMethods.
the purified enzyme (Fig. 2B), only a small quantity of [l-14C]PGHz was converted, being nonenzymatically changed to PGF*, , PGEz, and PGDz as in the case without the enzyme (data not shown), indicating that the inhibition of PGDz formation by the selenite was due to the inhibition of the enzyme activity and not due to the significant conversion of [l-“‘CIPGH, to other PGs or degradation products by selenite. We then studied the inhibition of PGD synthase by varying the combination and order of addition of selenite, DTT, and the enzyme in the preincubation, as shown in Table I. No inhibition was observed when the enzyme was preincubated for 10 min with selenite or DTT. Inhibition was observed when selenite and DTT were preincubated for 10 min followed by preincubation with the enzyme or simultaneous incubation of selenite, DTT, and the enzyme (Table I). Routinely, we performed preincubation of the selenium compound, DTT, and the enzyme in the buffer at 2,5”C for 10 min, and then the enzyme reaction was started by addition of [ l-14C]PGH2. However, no preincubation (only warming up of each solution) resulted in no inhibition of the enzyme reaction by selenite. When sodium selenite (10 PM), DTT (250 PM), and the purified PGD synthase were preincubated for various time intervals, a time-dependent inhibition of the synthase activity up to 10 min was observed; and over a lo-min preincubation the inhibition by selenite plateaued (Fig. 3). PGD Reversibility of selenite inhibition by excess DTT. synthase of the brain requires a SH compound as a cofactor and is inhibited by the selenium compounds. The inhibition pattern of the enzyme was very much dependent on the content of DTT used in the assay mixture. Increasing amounts of DTT reversed the selenium inhibition (Fig. 4), and a decreasing content of DTT increased the inhibitory effect by se.lenite (Fig. 4). Kinetic analysis. When different concentrations of selenite were preincubatecl with 250 pM DTT at 25°C for
20
10
30
(min)
FIG. 3. Time course of preincubation with selenite. Selenite (10 PM), DTT, and the purified enzyme were preincubated for 1, 3, 5, 10, 15, or 30 min in the standard reaction mixture as described under Materials and Methods.
10 min followed by a 1-min incubation with varying concentrations of [1-14C]PGHz, the PGD synthase activity was inhibited noncompetitively (Fig. 5), and the Ki value was 10.1 PM. Inhibition by selenium compounds. Next we examined the selenium-induced inhibition of PGD synthase activity of rat brain in terms of the valency state of selenium compounds. Inorganic compounds of selenium such as sodium selenite [Na#e(IV)OJ, selenous acid [H&Se(IV)O,], selenium tetrachloride [Se(IV)Cl,], selenium dioxide [Se(IV)O,], and selenium monochloride [Se(I)&l,] dose-dependently inhibited the activity of the purified enzyme and had similar I&, values (11-12 ELM, Fig. 6). When rat brain supernatant was preincubated at 25°C for 10 min in the presence of 250 PM DTT, all of the selenocompounds were found to cause 50% inhibition
FIG. 4. Inhibition of PGD synthase activity by selenite at various concentrations of DTT. Purified enzyme was preincubated with varying concentrations of sodium selenite as well as DTT at 25°C for 10 min and then incubated with substrate for 1 min as described under Materials and Methods. The symbols represent the amounts of DTT in the standard enzyme assay: 0.1 (01, 0.25 (01, 0.5 (A), and 1.0 mM (A).
164
ISLAM
of the PGD synthase activity over a concentration range of 9 to 15 PM (Fig. 7). Among inorganic compounds of selenium, sodium selenate [Na$e(IV)OJ, a hexavalent compound, had no effect on the synthase activity (Fig. 6B). Organic selenocompounds such as selenomethionine and selenourea did not inhibit the enzyme activity either in the purified enzyme preparation or in the crude brain supernatant (Figs. 6A and 7A). Thus, we observed that Reaction of selenite with DTT. preincubation of selenite with DTT was essential for the inhibition of the enzyme activity. It has been reported that selenite reacts with many SH compounds (19, 20, 31). To assess whether DTT reacts with selenite, we added a solution of selenite dropwise to one of DTT to give a final concentration of 25 PM selenite and 250 PM DTT, in a total volume of 2 ml and incubated the mixture at room temperature for 10, 15, or 30 min. A light yellow color developed due to the formation of the selenotrisulfide derivative of DTT as reported by Ganther (31), and the mixture had a broad maximum absorbance in the range of 240-350 nm. When 10 PM selenite was incubated with 250 PM DTT for 1, 3, 5, 10, 15, or 30 min at room temperature, -480 nm increased gradually up to 10 min. The time course of increase in Azso nm was similar to that required for enzyme inhibition by selenite and increased gradually with time, reached its maximum at around 10 min, and thereafter no change occurred in the absorption from 10 to 30 min after the start of incubation of selenite with DTT. This indicates that 10 min is sufficient for the formation of the selenotrisulfide derivative of DTT. On the other hand, no selenotrisulfide formation was observed when sodium selenate (25 PM) and DTT (250 PM) were incubated similarly to selenite and DTT. indicating that selenate did not react with DTT.
O.‘O r---
T
-1
E.z 0.08 ea F 0.06 .c E
0.04
-0.4
-0.2
0.0
0.2
0.4
[PGli2j:
0.6
0.8
1.0
phi’
plot of inhibition of PGD synthase activity FIG. 5. Lineweaver-Burk by selenite. The purified enzyme, DTT, and various concentrations of sodium selenite, (0) 15, (0) 13.5, (A) 12, (a) 10, or (ml 0 pM, were preincubated at 25’C for 10 min in the standard assay mixture. The reaction was started by addition of [1-‘4C]PGH2 at the indicated concentrations and incubated at 25°C for 1 min as described under Materials and Methods.
ET AL.
25 _
Concentration
(PM)
FIG. 6. Effects of selenium compounds of different valency on the activity of the purified PGD synthase. (A) Purified enzyme from rat brain was preincubated with various concentrations of SeCl, (A), SeO, (O), selenomethionine (01, or selenourea (A) in the standard assay mixture as described under Materials and Methods. (B) The purified enzyme was preincubated with H$eO, (O), NazSeOl (A), or Se& (A) at the indicated concentrations in the standard assay mixture. The various concentrations of Se&I, were dissolved in benzene and dried in the assay tubes under N, gas. The assay mixture was added to the tubes and PGD synthase activity was determined after a 1-min incubation at 25°C as described under Materials and Methods.
Effect of sulfur compounds. Selenium and sulfur are in the same group of the periodic table and have some similar chemical properties. Also, several enzymatic processes do not distinguish selenium from sulfur. However, sodium sulfite and sodium sulfate up to 10 mM concentration had no effect on the enzyme activity in either purified enzyme preparation or crude brain supernatant (data not shown). Effectof other metals. We also studied the effect of diand trivalent cations such as ferric chloride, zinc chloride, and cobalt chloride on the PGD synthase activity. These compounds inhibited the synthase activity dose-dependently in crude brain supernatant and showed I(&, values of 0.5 to 1 mM (data not shown). On the other hand, chlorides of chromium, thallium, and vanadium up to 2.5 mM concentration had no effect on the enzyme activity in the crude brain supernatant. These findings indicate that selenium is a very specific inhibitor of PGD synthase activity as compared with other metals.
SELENIUM
INHIBITION
OF PROSTAGLANDIN
1
100
25
25 Concentration
50
75
D SYNTHASE
165
enzymes. The mechanism of the inhibition was therefore speculated to be due to the selenotrisulfide derivative of DTT formation in the preincubation mixture between divalent or quadrivalent selenocompounds and SH compound(s). Ganther (31) has demonstrated that SH compounds react with selenium compounds in a 4:l molar ratio to form one molecule each of selenotrisulfide (RSSeSR) and disulfide (RSSR), via the reaction first proposed by Painter (32):
too0
(@l)
FIG. 7. Effects of selenium compounds of different valency on the PGD synthase activity in the rat brain supernatant. (A) Brain supernatant was preincubated with varying amounts of SeCl, (O), H2Se03 (A), SeOCl, (O), or selenomethionine (A) in the standard reaction mixture as described under Materials and Methods. The various amounts of SeOCl, dissolved in benzene were dried in the assay tubes under N2 gas. The assay mixture was added to the tubes, and then PGD synthase (B) activity was determined in the presence of 3.4 PM [l-“CIPGH,. Brain supernatant was preincubated with various amounts of Na,SeO, (0) or SeOP (0) in the standard reaction mixture as described under Materials and Methods.
Effect of SeC& on other PG-related enzymes. The inhibitory effect of SeCl, was studied on PGD2 ll-ketoreductase and PGF synthase purified to apparent homogeneity from bovine lung. Neither activity was inhibited, even by as high as 100 ~LM[SeCl, (data not shown). To know whether the selenotr:isulfide derivative of DTT inhibits the PGF synthase activity with [l-14C]PGHz used as a substrate, we added 100 PM SeCl, and 500 PM DTT to the assay mixture of PG.F synthase and preincubated the mixture for 10 min as in the case of PGD synthase; however, no inhibition was observed (data not shown). The inhibition of glutath:ione (GSH)-dependent PGD synthase activity in rat spleen supernatant (lOO,OOOg,for 60 min) was also studied. A 10% inhibition of this activity by 100 PM SeCl, in the presence of 1 mM GSH was observed. To know whether (GSH protects against the selenium inhibition, we added 1 mM GSH to the rat brain supernatant in place of DTT and preincubated the mixture with 100 PM SeCl, for 10 min at 25°C. As a result, about 85% of the enzyme activity was inhibited, whereas 1 mM DTT and 100 pM SeC14 inhibited 100% of the enzyme activity in rat brain supernatant under the standard assay conditions. DISCUSSION In the present study, we demonstrated the inhibition of PGD synthase by divale:nt and quadrivalent inorganic selenocompounds. The specificity of the inorganic selenocompounds was shown in terms of metal compounds, organic and inorganic selenocompounds, and PG-related
4RSH + H2Se03 + RSSeSR + RSSR + 3Hz0. It has been reported that selenotrisulfide is a very toxic substance and is responsible for the inhibition of amino acid transport (33), protein synthesis (16), and gene expression (17). A possible mechanism of inhibition of ribonuclease activity by selenite was reported by Ganther and Corcoran (34). When selenite was added to the ribonuclease, the activity was inhibited due to the formation of selenotrisulfide in which the selenium bridges with two SH groups of the enzyme, i.e.,
/E\ S \Se/S Frenkel et al. (20) have reported that SH groups of RNA and DNA polymerases were unable to react with selenite itself, but were able to be substituted by the added selenotrisulfide derivative, thereby forming the above type of adduct. On the basis of these findings, it is likely that the SH groups of the PGD synthase are unable to react with quadrivalent selenocompounds, but are able to be substituted by the selenotrisulfide formed by the action of selenium compounds and DTT in the preincubation mixture, thereby forming the above type of product. Such an exchange reaction may easily occur as reported by Nakagawa et al. (35, 36). The reversibility of selenium inhibition by DTT has been reported by Tashima et al. (22) and Bergad and Rathbun (21). Inhibition of glucocorticoid receptor binding was observed with 125 PM selenite and 2.5 mM DTT restored the binding (22). Furthermore, 1 mM selenite inhibited ATPase activity by about 45%, but the addition of 10 mM DTT restored the activity up to 84%. These findings are in agreement with ours, that an excess amount of DTT reversed the selenium inhibition. This also supports the possible involvement of selenotrisulfide in the selenium inhibition of PGD synthase. As far as we know, the selenium content in the mammalian brain has not yet been reported, but selenium toxicity in a few areas of China has been reported. In affected
166
ISLAM
individuals its typical level in the blood was approximately 40 PM (37), whereas the concentration of selenium in normal human blood is 2.5 PM (38). In the presence of a low concentration (0.1 mM) of DTT, the IC& value of selenite for PGD synthase was 5.0 PM, indicating that the selenium concentration might regulate PGDz biosynthesis under such pathological conditions. One of the candidates of the endogenous source of sulfhydryl compounds to produce adduct with inorganic selenium for inhibition of the PGD synthase might be glutathione, since preincubation of low concentration of glutathione (0.1 mM) and sodium selenite caused an inhibition of the brain-type PGD synthase activity, although it is difficult to demonstrate an accurate balance between protective and inhibitory (adduct-producing) effects of glutathione on PGD synthase activity. The specificity of selenium inhibition of PGD synthase might also be a tool for elucidation of the physiological role of PGDz in the central nervous system as well as in the peripheral tissues. ACKNOWLEDGMENTS We are grateful to Dr. K. Watanabe, Dept. of Enzymes and Metabolism of our institute, for a generous gift of PGD, 11-ketoreductase and PGF synthase and useful discussions. We also thank Dr. L. D. Frye for critical reading of the manuscript. This work was supported in part by the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency, Japan, and by grants from the Naito Foundation, Sankyo Foundation for Life Science, and Sankyo Co., Ltd. F.I. is a recipient of an STA Fellowship.
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Inhibition of Rat Brain Prostaglandin by Inorganic Selenocompounds Fakhrul Department
Islam,
Yasuyosbi
Watanabe,l
Hiroshi
of Neuroscience, *Osaka Bioscience Institute,
Received February
Morii,
D Synthase and Osamu Hayaishi*
6-2-4 Furuedai,
Suita-shi,
Osaka 565, Japan
12, 1991, and in revised form April 30, 1991
Various inorganic selenocompounds dose-dependently inhibited the rat brain prostaglandin (PG) D synthase, both in the purified enzyme preparation and in the crude brain supernatant. All of the quadrivalent selenium compounds tested had a very limited range of I&, values in the purified enzyme (11-12 wM) and in the brain supernatant (9-15 MM). A divalent selenium compound was also inhibitory, but a hexavalent selenium compound was ineffective. In contrast, organic selenocompounds such as selenomethionine and selenourea had no effect on the PGD synthase activity. Furthermore, sodium sulfate and sodium sulfite up to 10 rnW did not inhibit the activity. The inhibition by selenium required the preincubation of the metal with sulfhydryl compounds such as dithiothreitol (DTT), indicating that the formation of selenotrisulfide or some other adduct is essential for the inhibition. Furthermore, the inhibition was reversed by an excess amount of dithiothreitol, suggesting that the selenotrisulfide derivative o:f DTT binds to the SH group of the PGD synthase. The kinetic analysis revealed the inhibition by selenite to be noncompetitive with a Ki value of 10.1 PM. On the other hand, glutathione-dependent PGD synthase from rat spleen was much less inhibited, and PGF synthase and PGD2 11-ketoreductase activities were not inhibited by the selenium compound. o 1991 Academic Press, Inc.
Prostaglandin (PG)’ D synthase (prostaglandin-H, Disomerase, EC 5.3.99.2) is ubiquitously distributed in the central nervous system (CNS) (1, 2) as well as in the peripheral organs (3, 4) and catalyzes the conversion of PGHz to PGD:!. The brain-type PGD synthase, which exists not only in the CNS but also in the cochlea and epidydimis, is glutathione independent but requires a 1 To whom correspondence should be addressed. Fax: (06) 872-4818 ‘Abbreviations used: CNS, central nervous system; DTT, dithiothreitol; GSH, glutathione; KP13, potassium phosphate buffer; PG, prostaglandin. 0003.9861/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
sulfhydryl (SH) compound for its activity (3,4), whereas the peripheral-type PGD synthase is contained in the spleen, small intestine, lung, and other peripheral tissues and is glutathione dependent (4, 5). PGDz is the major PG in the central nervous system and exerts a variety of central actions such as induction of sleep (6, 7), modulation of neuroendocrine function (8), and modification of senses (9, 10). Selenium is an essential trace metal at low concentrations but is toxic at higher concentrations in all organisms (11). The metal is ubiquitously found in both prokaryotic and eukaryotic cells as a highly specific component of certain enzymes such as glutathione peroxidase, formate dehydrogenase, thiolase, xanthine dehydrogenase, glycine reductase, and nicotinic acid hydroxylase (12, 13). A number of biological effects of selenium compounds were shown to be influenced by SH compounds (14-19). For example, significant inhibition of RNA and DNA polymerases was obtained by selenite treatment in the presence of SH compounds (20). Sodium selenite inhibits NaK-ATPase activity (21), and this effect was reversed by excess amounts of glutathione or dithiothreitol (DTT). Also, selenite at a very low concentration inhibits the hormone binding site in the rat liver glucocorticoid receptor, and the inhibition was reversed by DTT (22). Ebselene (PZ 51, 2-phenyl-1,2-benzisoselenazol-3organic selenocom(2H)-one), an anti-inflammatory pound, was reported to inhibit the formation of cyclooxygenase and lipoxygenase products (23-25) and has attracted our interest to evaluate the effect of inorganic and organic compounds of selenium on the activity of PGD synthase in crude and purified enzyme preparations from rat brain. MATERIALS
AND
METHODS
Materials [I-‘*C]Arachidonic acid (2.0 GBq/mmol) was purchased from DuPont New England Nuclear. [l-‘*C]PGHz was prepared according to the method of Ohki et al. (26). Sodium selenite, selenium dioxide, selenium tetrachloride, selenous acid, and selenium oxychloride were purchased from Wako Pure Chemicals (Tokyo); selenium mono161
162
ISLAM
004
I
.
I
4.5 DTT
3.5
2.5
(-IogM)
FIG. 1. Amount of DTT required for the enzyme activity: Purified enzyme (0) or brain supernatant (0) was preincubated in the standard assay mixture with varying concentrations of DTT as described under Materials and Methods. The percentage formation of [l-“C]PGDP during a 1-min incubation with [l-i4C]PGH2 was subtracted by the value obtained in the absence of the enzyme.
chloride came from Rare Metallic Co., Ltd. (Tokyo); and Seleno-DLmethionine, selenourea, and dithiothreitol were from Sigma. Their purities were better than 97%. Male Wistar rats (200-250 Preparation of crude brain supernatunt. g) were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and sacrificed by perfusion with 250 ml of cold saline via the left ventricle of the heart. The brain was quickly removed and a 20% homogenate (w/v) was prepared in 10 mM potassium phosphate buffer (KPB), pH 8.0, by use of an Ultra-Turrax T-25 homogenizer (Janko & Funkel, West Germany) operated at top speed 10 times for 5 s each. The homogenate was centrifuged at 100,OOOgfor 60 min at 4”C, and the resulting supernatant was assayed for PGD synthase activity. PGD synthase was purified to apPurification of PGD synthase. parent homogeneity from 200 g of rat brains as described by Urade et al. (4). In the last column chromatography (CM-52 cellulose) step of the enzyme purification, no DTT was added in the buffer, so that the purified enzyme would be free of DTT. The purified enzyme was concentrated with a Centricon 10. The specific activity of the final preparation was 7085 nmol/mg protein/min, a value corresponding to that of an earlier preparation described by Urade et al. (4). Assay of enzymes. The standard assay for PGD synthase was performed in the reaction mixture containing 0.1 M KPB (pH 8.0), 0.25 mM DTT, and 0.5 pg of the purified enzyme or rat brain supernatant (70 gg protein) in a total volume of 50 ~1 and preincubated for 10 min at 25’C, unless otherwise stated. The reaction was started by the addition of 1 ~1 of 0.17 mM [l-“C]PGHz (the final concentration and specific _ radioactivity were 3.4 PM and 332 Bd0.17 nmol, respectively), and incubation was carried out for 1 min at 25°C. Termination of the reaction and extraction and quantification of the products were performed as described by Urade et al. (27). The activities of PGDz 11-ketoreductase and PGF synthase were assayed according to Watanabe et al. (28). Protein concentration was determined by the method of Lowry et al. (29) with bovine serum albumin as standard.
RESULTS
Requirement of DTT for PGD synthase activity. Shimizu et al. (30) originally used 0.5 mM DTT, and recently Urade et al. (4) used 1.0 mM DTT for their standard assay of PGD synthase from rat brain. At first we reinvestigated the requirement of DTT for PGD synthase
ET AL.
activity in the crude brain supernatant as well as in the preparation of purified enzyme, as shown in Fig. 1. In both cases DTT accelerated the enzyme activity. The maximal enhancement in the activity was about 7 times in the purified enzyme and about 3 times in the crude brain supernatant as compared with the activity in the absence of DTT. From these data, we decided to employ 0.25 mM DTT for the standard PGD synthase assay. In the Inhibition by selenite on PGD synthase actiuity. course of the study on the regulation of brain-type PGD synthase, we found inhibition by several metal ions of the enzyme activity in a crude supernatant of rat brain. The potency of the inhibition was different among the metal ions, the most potent inhibition having been observed with the quadrivalent selenium ion. The substrate of PGD synthase, [l-14C]PGHZ, is an unstable substance, and a small amount of it is converted nonenzymatically to PGFz,, PGEz, and PGDz when incubated for 1 min in the assay mixture without the enzyme. When the substrate was incubated with the enzyme for 1 min, PGD2 was the main enzymatic product, and the nonenzymatitally derived PGFz, and PGEz were also detected (Fig. 2A). In the presence of 0.1 mM selenite, 1 mM DTT, and
PGFza
PGEz
PGD2
II
PGH2
I
6-
A $ 4 -
; z 5 s 5 g E L
2 _
‘u
4-
z a
F I
o 6 -
2 -
0
’
0
5
10 Distance
15 (cm)
FIG. 2. Radiochromatograms of the products formed by the incubation of [l-i4C]PGHz. (A) [l-i4C]PGHz was enzymatically converted to PGD, when incubated with the purified enzyme and 1 mM DTT at 25°C for 1 min in the standard reaction mixture. The reaction was started by the addition of 1 aI of [l-i4C]PGHz and terminated after 1 min by the addition of ether: MeOH: 0.5 M citric acid (90: 12: 1.5, v/v/v). The organic phase (100 ~1) was spotted onto a TLC plate and developed in ether: MeOH: acetic acid (90: 2: 0.1, v/v/v) at -20°C. (B) Selenite (0.1 mM), DTT (1 mM), and the purified enzyme were preincubated in the standard reaction mixture at 25’C for 10 min, and then assayed by the addition of [l-i4C]PGHz. The positions of PGF,,, PGEz, and PGDx on TLC were identified by comparison with those of their authentic samples. Radioactivity on chromatographic plates was monitored by a Packard imaging scanner system 200. 0, origin; F, solvent front.
SELENIUM
INHIBITION
OF PROSTAGLANDIN
D SYNTHASE
163
TA.BLE I Effect of Various Orders of Preincubation with Selenite on the Activity of PGD Synthase 1st preincubation Time (rn; E + Selenite E+DTT Selenite + DTT Selenite + DTT Selenite + DTT
+ E + E
10 10 10 10 0
2nd preincubation (1 min) DTT Selenite E -
Inhibition (%) 0 0 95 95 0
: E 2 I=
50 -
25 -
,I. 0
Time
Note. E of selenite was carried terials and
= 0.5 ag of the purified PGD synthase. The concentrations and DTT were 15 and 250 pM, respectively, and the assay out in the standard assay mixture as described under MaMethods.
the purified enzyme (Fig. 2B), only a small quantity of [l-14C]PGHz was converted, being nonenzymatically changed to PGF*, , PGEz, and PGDz as in the case without the enzyme (data not shown), indicating that the inhibition of PGDz formation by the selenite was due to the inhibition of the enzyme activity and not due to the significant conversion of [l-“‘CIPGH, to other PGs or degradation products by selenite. We then studied the inhibition of PGD synthase by varying the combination and order of addition of selenite, DTT, and the enzyme in the preincubation, as shown in Table I. No inhibition was observed when the enzyme was preincubated for 10 min with selenite or DTT. Inhibition was observed when selenite and DTT were preincubated for 10 min followed by preincubation with the enzyme or simultaneous incubation of selenite, DTT, and the enzyme (Table I). Routinely, we performed preincubation of the selenium compound, DTT, and the enzyme in the buffer at 2,5”C for 10 min, and then the enzyme reaction was started by addition of [ l-14C]PGH2. However, no preincubation (only warming up of each solution) resulted in no inhibition of the enzyme reaction by selenite. When sodium selenite (10 PM), DTT (250 PM), and the purified PGD synthase were preincubated for various time intervals, a time-dependent inhibition of the synthase activity up to 10 min was observed; and over a lo-min preincubation the inhibition by selenite plateaued (Fig. 3). PGD Reversibility of selenite inhibition by excess DTT. synthase of the brain requires a SH compound as a cofactor and is inhibited by the selenium compounds. The inhibition pattern of the enzyme was very much dependent on the content of DTT used in the assay mixture. Increasing amounts of DTT reversed the selenium inhibition (Fig. 4), and a decreasing content of DTT increased the inhibitory effect by se.lenite (Fig. 4). Kinetic analysis. When different concentrations of selenite were preincubatecl with 250 pM DTT at 25°C for
20
10
30
(min)
FIG. 3. Time course of preincubation with selenite. Selenite (10 PM), DTT, and the purified enzyme were preincubated for 1, 3, 5, 10, 15, or 30 min in the standard reaction mixture as described under Materials and Methods.
10 min followed by a 1-min incubation with varying concentrations of [1-14C]PGHz, the PGD synthase activity was inhibited noncompetitively (Fig. 5), and the Ki value was 10.1 PM. Inhibition by selenium compounds. Next we examined the selenium-induced inhibition of PGD synthase activity of rat brain in terms of the valency state of selenium compounds. Inorganic compounds of selenium such as sodium selenite [Na#e(IV)OJ, selenous acid [H&Se(IV)O,], selenium tetrachloride [Se(IV)Cl,], selenium dioxide [Se(IV)O,], and selenium monochloride [Se(I)&l,] dose-dependently inhibited the activity of the purified enzyme and had similar I&, values (11-12 ELM, Fig. 6). When rat brain supernatant was preincubated at 25°C for 10 min in the presence of 250 PM DTT, all of the selenocompounds were found to cause 50% inhibition
FIG. 4. Inhibition of PGD synthase activity by selenite at various concentrations of DTT. Purified enzyme was preincubated with varying concentrations of sodium selenite as well as DTT at 25°C for 10 min and then incubated with substrate for 1 min as described under Materials and Methods. The symbols represent the amounts of DTT in the standard enzyme assay: 0.1 (01, 0.25 (01, 0.5 (A), and 1.0 mM (A).
164
ISLAM
of the PGD synthase activity over a concentration range of 9 to 15 PM (Fig. 7). Among inorganic compounds of selenium, sodium selenate [Na$e(IV)OJ, a hexavalent compound, had no effect on the synthase activity (Fig. 6B). Organic selenocompounds such as selenomethionine and selenourea did not inhibit the enzyme activity either in the purified enzyme preparation or in the crude brain supernatant (Figs. 6A and 7A). Thus, we observed that Reaction of selenite with DTT. preincubation of selenite with DTT was essential for the inhibition of the enzyme activity. It has been reported that selenite reacts with many SH compounds (19, 20, 31). To assess whether DTT reacts with selenite, we added a solution of selenite dropwise to one of DTT to give a final concentration of 25 PM selenite and 250 PM DTT, in a total volume of 2 ml and incubated the mixture at room temperature for 10, 15, or 30 min. A light yellow color developed due to the formation of the selenotrisulfide derivative of DTT as reported by Ganther (31), and the mixture had a broad maximum absorbance in the range of 240-350 nm. When 10 PM selenite was incubated with 250 PM DTT for 1, 3, 5, 10, 15, or 30 min at room temperature, -480 nm increased gradually up to 10 min. The time course of increase in Azso nm was similar to that required for enzyme inhibition by selenite and increased gradually with time, reached its maximum at around 10 min, and thereafter no change occurred in the absorption from 10 to 30 min after the start of incubation of selenite with DTT. This indicates that 10 min is sufficient for the formation of the selenotrisulfide derivative of DTT. On the other hand, no selenotrisulfide formation was observed when sodium selenate (25 PM) and DTT (250 PM) were incubated similarly to selenite and DTT. indicating that selenate did not react with DTT.
O.‘O r---
T
-1
E.z 0.08 ea F 0.06 .c E
0.04
-0.4
-0.2
0.0
0.2
0.4
[PGli2j:
0.6
0.8
1.0
phi’
plot of inhibition of PGD synthase activity FIG. 5. Lineweaver-Burk by selenite. The purified enzyme, DTT, and various concentrations of sodium selenite, (0) 15, (0) 13.5, (A) 12, (a) 10, or (ml 0 pM, were preincubated at 25’C for 10 min in the standard assay mixture. The reaction was started by addition of [1-‘4C]PGH2 at the indicated concentrations and incubated at 25°C for 1 min as described under Materials and Methods.
ET AL.
25 _
Concentration
(PM)
FIG. 6. Effects of selenium compounds of different valency on the activity of the purified PGD synthase. (A) Purified enzyme from rat brain was preincubated with various concentrations of SeCl, (A), SeO, (O), selenomethionine (01, or selenourea (A) in the standard assay mixture as described under Materials and Methods. (B) The purified enzyme was preincubated with H$eO, (O), NazSeOl (A), or Se& (A) at the indicated concentrations in the standard assay mixture. The various concentrations of Se&I, were dissolved in benzene and dried in the assay tubes under N, gas. The assay mixture was added to the tubes and PGD synthase activity was determined after a 1-min incubation at 25°C as described under Materials and Methods.
Effect of sulfur compounds. Selenium and sulfur are in the same group of the periodic table and have some similar chemical properties. Also, several enzymatic processes do not distinguish selenium from sulfur. However, sodium sulfite and sodium sulfate up to 10 mM concentration had no effect on the enzyme activity in either purified enzyme preparation or crude brain supernatant (data not shown). Effectof other metals. We also studied the effect of diand trivalent cations such as ferric chloride, zinc chloride, and cobalt chloride on the PGD synthase activity. These compounds inhibited the synthase activity dose-dependently in crude brain supernatant and showed I(&, values of 0.5 to 1 mM (data not shown). On the other hand, chlorides of chromium, thallium, and vanadium up to 2.5 mM concentration had no effect on the enzyme activity in the crude brain supernatant. These findings indicate that selenium is a very specific inhibitor of PGD synthase activity as compared with other metals.
SELENIUM
INHIBITION
OF PROSTAGLANDIN
1
100
25
25 Concentration
50
75
D SYNTHASE
165
enzymes. The mechanism of the inhibition was therefore speculated to be due to the selenotrisulfide derivative of DTT formation in the preincubation mixture between divalent or quadrivalent selenocompounds and SH compound(s). Ganther (31) has demonstrated that SH compounds react with selenium compounds in a 4:l molar ratio to form one molecule each of selenotrisulfide (RSSeSR) and disulfide (RSSR), via the reaction first proposed by Painter (32):
too0
(@l)
FIG. 7. Effects of selenium compounds of different valency on the PGD synthase activity in the rat brain supernatant. (A) Brain supernatant was preincubated with varying amounts of SeCl, (O), H2Se03 (A), SeOCl, (O), or selenomethionine (A) in the standard reaction mixture as described under Materials and Methods. The various amounts of SeOCl, dissolved in benzene were dried in the assay tubes under N2 gas. The assay mixture was added to the tubes, and then PGD synthase (B) activity was determined in the presence of 3.4 PM [l-“CIPGH,. Brain supernatant was preincubated with various amounts of Na,SeO, (0) or SeOP (0) in the standard reaction mixture as described under Materials and Methods.
Effect of SeC& on other PG-related enzymes. The inhibitory effect of SeCl, was studied on PGD2 ll-ketoreductase and PGF synthase purified to apparent homogeneity from bovine lung. Neither activity was inhibited, even by as high as 100 ~LM[SeCl, (data not shown). To know whether the selenotr:isulfide derivative of DTT inhibits the PGF synthase activity with [l-14C]PGHz used as a substrate, we added 100 PM SeCl, and 500 PM DTT to the assay mixture of PG.F synthase and preincubated the mixture for 10 min as in the case of PGD synthase; however, no inhibition was observed (data not shown). The inhibition of glutath:ione (GSH)-dependent PGD synthase activity in rat spleen supernatant (lOO,OOOg,for 60 min) was also studied. A 10% inhibition of this activity by 100 PM SeCl, in the presence of 1 mM GSH was observed. To know whether (GSH protects against the selenium inhibition, we added 1 mM GSH to the rat brain supernatant in place of DTT and preincubated the mixture with 100 PM SeCl, for 10 min at 25°C. As a result, about 85% of the enzyme activity was inhibited, whereas 1 mM DTT and 100 pM SeC14 inhibited 100% of the enzyme activity in rat brain supernatant under the standard assay conditions. DISCUSSION In the present study, we demonstrated the inhibition of PGD synthase by divale:nt and quadrivalent inorganic selenocompounds. The specificity of the inorganic selenocompounds was shown in terms of metal compounds, organic and inorganic selenocompounds, and PG-related
4RSH + H2Se03 + RSSeSR + RSSR + 3Hz0. It has been reported that selenotrisulfide is a very toxic substance and is responsible for the inhibition of amino acid transport (33), protein synthesis (16), and gene expression (17). A possible mechanism of inhibition of ribonuclease activity by selenite was reported by Ganther and Corcoran (34). When selenite was added to the ribonuclease, the activity was inhibited due to the formation of selenotrisulfide in which the selenium bridges with two SH groups of the enzyme, i.e.,
/E\ S \Se/S Frenkel et al. (20) have reported that SH groups of RNA and DNA polymerases were unable to react with selenite itself, but were able to be substituted by the added selenotrisulfide derivative, thereby forming the above type of adduct. On the basis of these findings, it is likely that the SH groups of the PGD synthase are unable to react with quadrivalent selenocompounds, but are able to be substituted by the selenotrisulfide formed by the action of selenium compounds and DTT in the preincubation mixture, thereby forming the above type of product. Such an exchange reaction may easily occur as reported by Nakagawa et al. (35, 36). The reversibility of selenium inhibition by DTT has been reported by Tashima et al. (22) and Bergad and Rathbun (21). Inhibition of glucocorticoid receptor binding was observed with 125 PM selenite and 2.5 mM DTT restored the binding (22). Furthermore, 1 mM selenite inhibited ATPase activity by about 45%, but the addition of 10 mM DTT restored the activity up to 84%. These findings are in agreement with ours, that an excess amount of DTT reversed the selenium inhibition. This also supports the possible involvement of selenotrisulfide in the selenium inhibition of PGD synthase. As far as we know, the selenium content in the mammalian brain has not yet been reported, but selenium toxicity in a few areas of China has been reported. In affected
166
ISLAM
individuals its typical level in the blood was approximately 40 PM (37), whereas the concentration of selenium in normal human blood is 2.5 PM (38). In the presence of a low concentration (0.1 mM) of DTT, the IC& value of selenite for PGD synthase was 5.0 PM, indicating that the selenium concentration might regulate PGDz biosynthesis under such pathological conditions. One of the candidates of the endogenous source of sulfhydryl compounds to produce adduct with inorganic selenium for inhibition of the PGD synthase might be glutathione, since preincubation of low concentration of glutathione (0.1 mM) and sodium selenite caused an inhibition of the brain-type PGD synthase activity, although it is difficult to demonstrate an accurate balance between protective and inhibitory (adduct-producing) effects of glutathione on PGD synthase activity. The specificity of selenium inhibition of PGD synthase might also be a tool for elucidation of the physiological role of PGDz in the central nervous system as well as in the peripheral tissues. ACKNOWLEDGMENTS We are grateful to Dr. K. Watanabe, Dept. of Enzymes and Metabolism of our institute, for a generous gift of PGD, 11-ketoreductase and PGF synthase and useful discussions. We also thank Dr. L. D. Frye for critical reading of the manuscript. This work was supported in part by the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency, Japan, and by grants from the Naito Foundation, Sankyo Foundation for Life Science, and Sankyo Co., Ltd. F.I. is a recipient of an STA Fellowship.
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