Vol. 131, No. 3
JOURNAL OF BACTUROLOGY, Sept. 1977, p. 1013-1015 Copyright C 1977 American Society for Microbiology
Printed in U.S.A.
Inhibition of Thiamine Transport in Saccharomyces cerevisiae by Thiamine Disulfides AKIO IWASHIMA,* YASUO WAKABAYASHI, AND YOSHITSUGU NOSE Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto, Japan Received for publication 19 May 1977
Both thiamine disulfide and O-benzoyl thiamine disulfide, which are thiolform derivatives of thiamine, strongly inhibited thiamine transport in Saccharomyces cerevisiae. The inhibition appeared to be due to a high affinity of the analogs for yeast cell membranes, in which thiamine transport component(s) may be integrated. Previous studies have shown that transport of thiamine across cell membranes ofSaccharomyces cerevisiae is mediated by a specific transport system (5, 10). The transport process is pH, temperature, and energy dependent, and it displays structural specificity and saturation kinetics (apparent Km of 0.18 ,uM). Several thiamine derivatives, such as pyrithiamine, are also taken up by yeast similarly to thiamine and appear to act as competitive inhibitors (6, 7). During a further investigation into the structural specificity of the thiamine transport system in S. cerevisiae, we found that disulfide derivatives of thiamine, particularly 0-benzoylthiamine disulfide (BTDS), strongly inhibit the uptake of the vitamin by whole yeast cells. In this communication we present data suggesting that thiamine disulfides specifically inhibit yeast thiamine transport by interacting with some transport component(s), which appears to be firmly attached to the cell membrane, of the thiamine transport system in S. cerevisiae. Initial rates of thiamine uptake by S. cerevisiae, which was obtained as a clonal isolate of commercial bakers' yeast (Oriental Yeast Co., Ltd., Osaka, Japan) as previously reported (7), in the presence of thiamine disulfides at various concentrations are shown in Table 1. Both thiamine disulfide, prepared as described by Kawasaki et al. (8), and BTDS (a gift from Tanabe Seiyaku Co., Ltd., Osaka, Japan) markedly inhibited thiamine uptake by yeast cells, and the inhibition by the latter was much stronger than that by the former. The extent of inhibition of thiamine transport by BTDS was not significantly increased even if the analog was added to the cell suspensions during preincubation, and the inhibition could be reversed by washing the cells with 0.05 M potassium phosphate buffer (pH 5.0). These findings would rule out the possibility that BTDS re-
acts with some transport component(s) to yield a covalently bound thiamine derivative, as indicated in an affinity label for yeast biotin transport component(s) with biotinyl-p-nitrophenyl ester (1). The kinetics of thiamine transport shows that both thiamine disulfide and BTDS competitively inhibit thiamine uptake, with apparent K{ values of 57 nM for thiamine disulfide and 1.8 nM for BTDS (Fig. 1). Since the K{ value for pyrithiamine, which is known as one of the most potent antimetabolites of thiamine, is 0.11 ,uM, the affinity of BTDS to the yeast thiamine transport system appears to be approximately 60-fold higher than that of pyrithiamine. Suzuoki (10) showed that thiamine propyl disulfide cannot be taken up by bakers' yeast cells unless it is converted to thiamine by reduction. Subsequently, it was also demonstrated with Escherichia coli (2) that thiamine propyl disulfide is not directly taken up by the cells, but is first reduced to thiamine on the outer surface of the cell membrane (probably by a nonenzymatic process), which is then transported via the uptake system specific for thiamine. Table 2 shows intracellular thiamine levels after incubation of yeast cells with BTDS. At a concentration of 0.1 uM, BTDS inhibited thiamine uptake 91.8%, whereas incubation of the cells with BTDS at the same concentration only caused a slight elevation of the intracellular thiamine level, which corresponds to 1.1% of the amount of thiamine transported. The thiamine level still remained at 3.1% even with incubation with 1 ,uM BTDS. These results indicate that the inhibition of thiamine transport by BTDS is not due to an intracellular accumulation of thiamine and consequent regulation, but appears to be due to specific competition between thiamine and BTDS on the cell membrane before they enter into the cells. An earlier investigation has shown that pyrithiamine
1013
1014
NOTES
J. BACTERIOL.
TABLE 1. Inhibition of thiamine transport by thiamine disulfidesa Thiamine uptake Addition (,uM) (nmol/mg [dry wt] per 2 min) None ...................... 12.0 Thiamine disulfide (0.25) ..... 7.90 Thiamine disulfide (0.5) ...... 4.71 Thiamine disulfide (1.0) ...... 3.17 BTDS (0.005) ................ 6.63 BTDS (0.01) ................. 4.96 BTDS (0.02) ................. 2.93 a Five milliliters of yeast cell suspension (73 ,ug [dry weight]/mi) in 0.05 M potassium phosphate buffer (pH 5.0) containing 0.1 M glucose was preincubated for 15 min at 37°C and then exposed simultaneously to 1 ,uM ['4C]thiamine (15.5 mCi/ mmol) and various concentrations of thiamine disulfide or BTDS as indicated, followed by further incubation at 37°C for 2 min. The uptake of [14C]thiamine was measured as previously described (5).
3
-
Thiamine disulfid
2
BTDS
is taken up by the same transport system as thiamine in S. cerevisiae (7). The uptake of pyrithiamine was also strongly inhibited by BTDS (Table 3), suggesting that the inhibitory effect of BTDS is exerted on a common transport system for thiamine and pyrithiamine in
yeast.
In the yeast thiamine transport reaction, the presence of the intact pyrimidine moiety of the thiamine molecule seems to be important, at least for recognition by the transport component(s) on the cell membrane, as judged by the facts that, whereas thiamine analogs, such as oxythiamine, in which the pyrimidine moiety is modified do not appreciably affect thiamine transport, modification of the thiazole moiety inhibits transport greatly, as shown in pyrithiamine and dimethialium (5). However, the remarkable inhibitory action of BTDS, which exceeds by an order of magnitude that of pyrithiamine, cannot be ascribed only to its structural analogy to thiamine, but it is supposed that there is some additional mechanism(s) in the inhibition induced by BTDS. One of the distinct chemical properties of
both thiamine disulfide and BTDS compared with thiamine and pyrithiamine is their greater solubility in organic solvents. For instance, the partition coefficients at pH 6.6 (25°C) of thiamine, thiamine disulfide, and BTDS have been reported to be 0.02, 2.6, and 550 for n-butanol and below 0.0001, 0.004, and 28 for benzene, respectively (3). Thus, it was suggested that the inhibitory action of these TABLE 2. Intracellular thiamine leuels of yeast cells after incubation with thiamine and BTDSa Addition (,uM)
I
1 I/S (xIO7M)
2
FIG. 1. Demonstration of competitive inhibition of thiamine transport by thiamine disulfide and BTDS. After preincubation for 15 min at 37°C, yeast cell suspensions (18 pg [dry weight]/mg) were incubated for 1 min with varying concentrations of [14C]thiamine in the presence of 0.2 ,M thiamine disulfide or 4 nM BTDS. Abscissa, Reciprocal thiamine concentration; ordinate, thiamine uptake, in nmollmg (dry weight) per min.
Intracellular thiamine (nmollmg [dry wt])
None ...................... 0.12 Thiamine (1.0) ............... 7.66 Thiamine (1.0), BTDS (0.1) ... 0.74 BTDS (0.1) .................. 0.20 BTDS (1.0) .................. 0.35 a Intracellular thiamine amount was determined as previously described (7) after incubation for 2 min at 37°C. TABLE 3.
EffectofBTDSonuptakeofpyrithiaminea Addition (uM)
Pyrithiamine uptake
None ....... 100 BTDS (0.01) ... .... 32.9 BTDS (0.1) ....... 6.3 a The uptake of pyrithiamine was assayed as previously described (7). BTDS was added to yeast cell suspensions simultaneously with 1 ,tM pyrithia-
mine.
VOL. 131, 1977
thiamine disulfides probably depends on their high solubility to lipid components in the yeast cell membranes. The inhibition of thiamine disulfides, furthermore, appeared to be specific for thiamine transport in S. cerevisiae, since BTDS did not affect the uptake of pyridoxine, which can be taken up by yeast cells (9) and has a similar structure to the pyrimidine moiety of thiamine, at 10-fold concentrations of the vitamin (data not shown). On the other hand, with E. coli K-12 BTDS did not inhibit thiamine transport, even at a 10-fold concentration of thiamine, which may reflect the difference in lipid composition between the two microorganisms. The transport component(s) responsible for such inhibition by thiamine disulfides in S. cerevisiae is still obscure, but it seems to be present firmly attached to the cell membrane, since thiamine-binding activity (4) found in soluble fractions of sonic extracts of yeast cells (6) was not affected by BTDS at the concentration that is inhibitory for yeast thiamine transport. LITERATURE CITED 1. Becker, J. M., M. Wilchek, and E. Katchalski. 1971.
NOTES
2.
3.
4.
5. 6.
7. 8.
9.
10.
1015
Irreversible inhibition of biotin transport in yeast by biotinyl-p-nitrophenyl ester. Proc. Natl. Acad. Sci. U.S.A. 68:2604-2607. Fujii, A., T. Kawasaki, and Y. Nose. 1970. Uptake of thiamine propyl disulfide in Escherichia coli. J. Vitaminol. 16:39-44. Harada, K., K. Kohno, I. Daira, I. Saito, and I. Utsumi. 1965. Studies on thiamine disulfide. XIII. Blood thiamine levels by the intravenous administration of O-acylthiamine disulfide and some properties of 0butyroylthiamine disulfide. Vitamins 32:464-471. Iwashima, A., A. Matsuura, and Y. Nose. 1971. Thiamine-binding protein ofEscherichia coli. J. Bacteriol. 108:1419-1421. Iwashima, A., H. Nishino, and Y. Nose. 1973. Carriermediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta 330:222-234. Iwashima, A., and Y. Nose. 1976. Regulation of thiamine transport in Saccharomyces cerevisiae. J. Bacteriol. 128:855-857. Iwashima, A., Y. Wakabaywahi, and Y. Nose. 1975. Thiamine transport mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta 413:243-247. Kawaski, C., T. Horio, and I. Daira. 1963. Formation of thiochrome. VI. Formation of thiamine disulfide and thiochrome from thiamine by ferricyanide and its dependency to pH of the reaction media. Vitamins 26:298-302. Shane, B., and E. E. Snell. 1976. Transport and metabolism of vitamin B,, in the yeast Saccharomyces carlsbergensis 4228. J. Biol. Chem. 251:1042-1051. Suzuoki, J. 1955. Thiamine uptake by yeast cells. J. Biochem. (Tokyo) 42:27-39.
JOURNAL OF BACTUROLOGY, Sept. 1977, p. 1013-1015 Copyright C 1977 American Society for Microbiology
Printed in U.S.A.
Inhibition of Thiamine Transport in Saccharomyces cerevisiae by Thiamine Disulfides AKIO IWASHIMA,* YASUO WAKABAYASHI, AND YOSHITSUGU NOSE Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto, Japan Received for publication 19 May 1977
Both thiamine disulfide and O-benzoyl thiamine disulfide, which are thiolform derivatives of thiamine, strongly inhibited thiamine transport in Saccharomyces cerevisiae. The inhibition appeared to be due to a high affinity of the analogs for yeast cell membranes, in which thiamine transport component(s) may be integrated. Previous studies have shown that transport of thiamine across cell membranes ofSaccharomyces cerevisiae is mediated by a specific transport system (5, 10). The transport process is pH, temperature, and energy dependent, and it displays structural specificity and saturation kinetics (apparent Km of 0.18 ,uM). Several thiamine derivatives, such as pyrithiamine, are also taken up by yeast similarly to thiamine and appear to act as competitive inhibitors (6, 7). During a further investigation into the structural specificity of the thiamine transport system in S. cerevisiae, we found that disulfide derivatives of thiamine, particularly 0-benzoylthiamine disulfide (BTDS), strongly inhibit the uptake of the vitamin by whole yeast cells. In this communication we present data suggesting that thiamine disulfides specifically inhibit yeast thiamine transport by interacting with some transport component(s), which appears to be firmly attached to the cell membrane, of the thiamine transport system in S. cerevisiae. Initial rates of thiamine uptake by S. cerevisiae, which was obtained as a clonal isolate of commercial bakers' yeast (Oriental Yeast Co., Ltd., Osaka, Japan) as previously reported (7), in the presence of thiamine disulfides at various concentrations are shown in Table 1. Both thiamine disulfide, prepared as described by Kawasaki et al. (8), and BTDS (a gift from Tanabe Seiyaku Co., Ltd., Osaka, Japan) markedly inhibited thiamine uptake by yeast cells, and the inhibition by the latter was much stronger than that by the former. The extent of inhibition of thiamine transport by BTDS was not significantly increased even if the analog was added to the cell suspensions during preincubation, and the inhibition could be reversed by washing the cells with 0.05 M potassium phosphate buffer (pH 5.0). These findings would rule out the possibility that BTDS re-
acts with some transport component(s) to yield a covalently bound thiamine derivative, as indicated in an affinity label for yeast biotin transport component(s) with biotinyl-p-nitrophenyl ester (1). The kinetics of thiamine transport shows that both thiamine disulfide and BTDS competitively inhibit thiamine uptake, with apparent K{ values of 57 nM for thiamine disulfide and 1.8 nM for BTDS (Fig. 1). Since the K{ value for pyrithiamine, which is known as one of the most potent antimetabolites of thiamine, is 0.11 ,uM, the affinity of BTDS to the yeast thiamine transport system appears to be approximately 60-fold higher than that of pyrithiamine. Suzuoki (10) showed that thiamine propyl disulfide cannot be taken up by bakers' yeast cells unless it is converted to thiamine by reduction. Subsequently, it was also demonstrated with Escherichia coli (2) that thiamine propyl disulfide is not directly taken up by the cells, but is first reduced to thiamine on the outer surface of the cell membrane (probably by a nonenzymatic process), which is then transported via the uptake system specific for thiamine. Table 2 shows intracellular thiamine levels after incubation of yeast cells with BTDS. At a concentration of 0.1 uM, BTDS inhibited thiamine uptake 91.8%, whereas incubation of the cells with BTDS at the same concentration only caused a slight elevation of the intracellular thiamine level, which corresponds to 1.1% of the amount of thiamine transported. The thiamine level still remained at 3.1% even with incubation with 1 ,uM BTDS. These results indicate that the inhibition of thiamine transport by BTDS is not due to an intracellular accumulation of thiamine and consequent regulation, but appears to be due to specific competition between thiamine and BTDS on the cell membrane before they enter into the cells. An earlier investigation has shown that pyrithiamine
1013
1014
NOTES
J. BACTERIOL.
TABLE 1. Inhibition of thiamine transport by thiamine disulfidesa Thiamine uptake Addition (,uM) (nmol/mg [dry wt] per 2 min) None ...................... 12.0 Thiamine disulfide (0.25) ..... 7.90 Thiamine disulfide (0.5) ...... 4.71 Thiamine disulfide (1.0) ...... 3.17 BTDS (0.005) ................ 6.63 BTDS (0.01) ................. 4.96 BTDS (0.02) ................. 2.93 a Five milliliters of yeast cell suspension (73 ,ug [dry weight]/mi) in 0.05 M potassium phosphate buffer (pH 5.0) containing 0.1 M glucose was preincubated for 15 min at 37°C and then exposed simultaneously to 1 ,uM ['4C]thiamine (15.5 mCi/ mmol) and various concentrations of thiamine disulfide or BTDS as indicated, followed by further incubation at 37°C for 2 min. The uptake of [14C]thiamine was measured as previously described (5).
3
-
Thiamine disulfid
2
BTDS
is taken up by the same transport system as thiamine in S. cerevisiae (7). The uptake of pyrithiamine was also strongly inhibited by BTDS (Table 3), suggesting that the inhibitory effect of BTDS is exerted on a common transport system for thiamine and pyrithiamine in
yeast.
In the yeast thiamine transport reaction, the presence of the intact pyrimidine moiety of the thiamine molecule seems to be important, at least for recognition by the transport component(s) on the cell membrane, as judged by the facts that, whereas thiamine analogs, such as oxythiamine, in which the pyrimidine moiety is modified do not appreciably affect thiamine transport, modification of the thiazole moiety inhibits transport greatly, as shown in pyrithiamine and dimethialium (5). However, the remarkable inhibitory action of BTDS, which exceeds by an order of magnitude that of pyrithiamine, cannot be ascribed only to its structural analogy to thiamine, but it is supposed that there is some additional mechanism(s) in the inhibition induced by BTDS. One of the distinct chemical properties of
both thiamine disulfide and BTDS compared with thiamine and pyrithiamine is their greater solubility in organic solvents. For instance, the partition coefficients at pH 6.6 (25°C) of thiamine, thiamine disulfide, and BTDS have been reported to be 0.02, 2.6, and 550 for n-butanol and below 0.0001, 0.004, and 28 for benzene, respectively (3). Thus, it was suggested that the inhibitory action of these TABLE 2. Intracellular thiamine leuels of yeast cells after incubation with thiamine and BTDSa Addition (,uM)
I
1 I/S (xIO7M)
2
FIG. 1. Demonstration of competitive inhibition of thiamine transport by thiamine disulfide and BTDS. After preincubation for 15 min at 37°C, yeast cell suspensions (18 pg [dry weight]/mg) were incubated for 1 min with varying concentrations of [14C]thiamine in the presence of 0.2 ,M thiamine disulfide or 4 nM BTDS. Abscissa, Reciprocal thiamine concentration; ordinate, thiamine uptake, in nmollmg (dry weight) per min.
Intracellular thiamine (nmollmg [dry wt])
None ...................... 0.12 Thiamine (1.0) ............... 7.66 Thiamine (1.0), BTDS (0.1) ... 0.74 BTDS (0.1) .................. 0.20 BTDS (1.0) .................. 0.35 a Intracellular thiamine amount was determined as previously described (7) after incubation for 2 min at 37°C. TABLE 3.
EffectofBTDSonuptakeofpyrithiaminea Addition (uM)
Pyrithiamine uptake
None ....... 100 BTDS (0.01) ... .... 32.9 BTDS (0.1) ....... 6.3 a The uptake of pyrithiamine was assayed as previously described (7). BTDS was added to yeast cell suspensions simultaneously with 1 ,tM pyrithia-
mine.
VOL. 131, 1977
thiamine disulfides probably depends on their high solubility to lipid components in the yeast cell membranes. The inhibition of thiamine disulfides, furthermore, appeared to be specific for thiamine transport in S. cerevisiae, since BTDS did not affect the uptake of pyridoxine, which can be taken up by yeast cells (9) and has a similar structure to the pyrimidine moiety of thiamine, at 10-fold concentrations of the vitamin (data not shown). On the other hand, with E. coli K-12 BTDS did not inhibit thiamine transport, even at a 10-fold concentration of thiamine, which may reflect the difference in lipid composition between the two microorganisms. The transport component(s) responsible for such inhibition by thiamine disulfides in S. cerevisiae is still obscure, but it seems to be present firmly attached to the cell membrane, since thiamine-binding activity (4) found in soluble fractions of sonic extracts of yeast cells (6) was not affected by BTDS at the concentration that is inhibitory for yeast thiamine transport. LITERATURE CITED 1. Becker, J. M., M. Wilchek, and E. Katchalski. 1971.
NOTES
2.
3.
4.
5. 6.
7. 8.
9.
10.
1015
Irreversible inhibition of biotin transport in yeast by biotinyl-p-nitrophenyl ester. Proc. Natl. Acad. Sci. U.S.A. 68:2604-2607. Fujii, A., T. Kawasaki, and Y. Nose. 1970. Uptake of thiamine propyl disulfide in Escherichia coli. J. Vitaminol. 16:39-44. Harada, K., K. Kohno, I. Daira, I. Saito, and I. Utsumi. 1965. Studies on thiamine disulfide. XIII. Blood thiamine levels by the intravenous administration of O-acylthiamine disulfide and some properties of 0butyroylthiamine disulfide. Vitamins 32:464-471. Iwashima, A., A. Matsuura, and Y. Nose. 1971. Thiamine-binding protein ofEscherichia coli. J. Bacteriol. 108:1419-1421. Iwashima, A., H. Nishino, and Y. Nose. 1973. Carriermediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta 330:222-234. Iwashima, A., and Y. Nose. 1976. Regulation of thiamine transport in Saccharomyces cerevisiae. J. Bacteriol. 128:855-857. Iwashima, A., Y. Wakabaywahi, and Y. Nose. 1975. Thiamine transport mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta 413:243-247. Kawaski, C., T. Horio, and I. Daira. 1963. Formation of thiochrome. VI. Formation of thiamine disulfide and thiochrome from thiamine by ferricyanide and its dependency to pH of the reaction media. Vitamins 26:298-302. Shane, B., and E. E. Snell. 1976. Transport and metabolism of vitamin B,, in the yeast Saccharomyces carlsbergensis 4228. J. Biol. Chem. 251:1042-1051. Suzuoki, J. 1955. Thiamine uptake by yeast cells. J. Biochem. (Tokyo) 42:27-39.
