Inhibition of glucose transport in Saccharomyces cerevisiae by uranyl ions

Journal of Biotechnology, 27 (1992) 75-84 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

75

BIOTEC 00846

Inhibition of glucose transport in Saccharomyces cereuisiae by uranyl ions G . F . F u h r m a n n , D. Storch, H . - P . B o d e a n d B. V61ker Department of Pharmacology and Toxicology, Philipps Universitiit Marburg, Germany (Received 17 July 1991; revision accepted 14 January 1992)

Summary Transport of glucose into Saccharomyces cerevisiae cells occurs by two transport processes. The first transport is mathematically characterized by facilitated diffusion and the second by diffusion kinetics. Both transport processes are effectively blocked by uranyl ions. Facilitated diffusion of glucose demonstrates a mixed type of inhibition by uranyl ions; Vm decreases and K m increases. The K i value for Vm is 43.78 x 106 molecules uranyl per cell and the Hill slope 1.64. These values are comparable with the data on uranyl inhibition of glucose consumption by Rothstein, Frenkel and Larrabee (1948) with a K i value of 27.21 x 106 molecules uranyl per cell and a Hill slope of 2.51. By considering uranyl chemistry a dimeric complex of uranyl might act as a molecule similar to fl-D-glucopyranose at the binding site of the carrier and by inhibition of the glucose translocation step. The effects on glucose diffusion seem to be more complex. At low concentrations of uranyl a significant inhibition occurs, whereas at higher uranyl concentrations a slight increase is observed. This dual behaviour of uranyl could be due to effects on two different diffusion pathways.

Saccharomyces cerecisiae; Glucose transport; Uranyl ion

Correspondence to: G.F. Fuhrmanu, Department of Pharmacology and Toxicology,Philipps-Universitat Marburg, Karl-von-Frisch-Strasse,3550 Marburg, Germany.

76 Introduction

In 1940 Booij reported that uranyl ions inhibited anaerobic degradation of glucose in Saccharomyces cerevisiae. In 1948 Rothstein and Larrabee presented conclusive evidence that these ions acted at the cell surface by blocking most probably the entrance of glucose into the cell, because inhibition could be completely reversed by complexing uranyl ions with citrate or phosphate. Uranyl ions in low concentrations inhibited the anaerobic consumption of glucose to a maximum of about 90% (Rothstein et al., 1948). The relationship between inhibition and the initial uranyl concentration involved a family of S-shaped curves, one for each yeast concentration. As the yeast concentration was raised, a higher initial uranyl concentration was required to attain a given inhibition. The evidence based on the inhibition curves and on uranyl ion binding by the cells was taken as an indication that the heavy metal formed a dissociable complex with certain active 'groups' on the cell surface. The dissociation constant of this complex was determined to be about 20 × 10 6 per cell, and the total number of such 'groups' was calculated to be 45 × 10 6 per cell. The percentage inhibition of glucose transport by uranyl was thought to be equal to the active 'groups' that are complexed at the cell surface. This indicated that the active 'groups' are necessary for transport and subsequent glucose metabolism and that the rate was first-order relative to the concentration of active 'groups'. In addition to the uranyl complex associated with this inhibition, there was another complex formed with other cell groups, which were not involved in glucose transport amounting to 25 × 10 6 groups per cell and demonstrating a higher dissociation constant than that for the complex associated with glucose consumption inhibition. These studies with uranyl ions on inhibition of glucose consumption in S. cerevisiae of Rothstein and co-workers in 1948 were far in advance of the knowledge of the existence of glucose transporters in S. cerevisiae. In this study we reinvestigated the effect of uranyl ions on glucose transport in S. cerevisiae with modern transport techniques, such as fast initial glucose uptake, and we will give some further experimental evidence that the active 'groups' titrated by uranyl ions at the surface of these cells are indeed associated with the glucose carriers of the plasma membrane.

Materials and Methods

Strain and growth conditions. Saccharomyces cerevisiae H 1022 ( E T H Zi~rich) was grown with 2% peptone, 1% yeast extract and 2% glucose in batch cultures at 30°C under aeration. At the end of the exponential growth phase all glucose was consumed. In order to initiate high-affinity glucose uptake a low glucose concentration of 0.1% glucose was added for 2 h incubation (Fuhrmann and VSlker, 1992, this issue).

77

Initial glucose uptake experiments. Before measuring glucose uptake the cells were washed three times in distilled water and three times in 0.2 M MES buffer, pH 5. The cytocrit was adjusted to 10% cells. Kinetic studies for glucose transport were conducted in the concentration range of 0.5-200 mM (0.075 to 6 /~Ci p.mol -j D-[U-14C]glucose) and a cell density of 50 mg wet weight per ml. Glucose uptake was started by addition of 0.1 ml radioactively labeled glucose solution to 0.1 mi of 10% cells at 25°C. Exactly after 5 s uptake was stopped by addition of 10 ml ice cold 200 mM sorbitol solution, cells were filtered on a glass fiber filter (GF 92, Schleicher and Schuell, Dassel, FRG) and rinsed with two 10 ml portions of ice cold distilled water. The filters with cells were immersed into 5 ml scintillation cocktail (Rotiscint eco plus, Roth, Karlsruhe, FRG) and counted in a Beckman LS 6000IC scintillation counter. Analys& of transport kinetics. Experimental data were analysed by computer assisted nonlinear regression analysis using GraphPAD software (Motulsky, 19851989).

Materials. Uranyl nitrate hexahydrate p.a., peptone from casein and yeast extract were purchased from E. Merck, Darmstadt, FRG. D-[U-14C]glucose, specific activity 300 mCi mmol-l, was obtained from Amersham Buchler, Braunschweig, FRG. MES (morpholinoethane sulfonic acid) p.a. was purchased from SERVA, Heidelberg, FRG. All other chemicals were of analytical grade quality.

Results

The effect of cell concentration on inhibition of glucose consumption by uranyl nitrate In Fig. 1 the experimental data of Rothstein et al. (1948) were recalculated. At first the data points of glucose uptake inhibition by uranyl from 10 to 54 mg ml-1 of yeast concentrations were digitized. The concentration points at 3.6 and 5.2 mg m1-1 were not taken, because the method of glucose determination used to measure the rates of glucose disappearance from the medium was claimed not to be exact enough at the low yeast concentrations. The experiments of Rothstein et al. (1948) were performed with distilled water washed, starved Baker's yeast (Standard Brands, Inc.) adjusted to pH 3.5 with HC1. The percentage inhibition was calculated for each experiment from disappearance of glucose (100 to 200 mM glucose) in the presence and absence of uranyl nitrate over a time period of 30 min with the higher yeast concentrations and 120 rain with the lower yeast concentrations. The final glucose concentration was reported to be never lower than 40 mM, so that during the experiment maximal rates of disappearance were maintained. The data points of six experiments with different yeast concentrations were calculated in molecules uranyl per cell and the logarithm was plotted against percent inhibition (Fig. 1). The mean curve matching the data points was fitted by computer assisted nonlinear regression analysis (GraphPAD) demonstrating perfectly as Rothstein et al. (1948) have stated, that the inhibition of glucose uptake

78 0---

co

O

•

8

9

50

a~ o

o~

100--0

log (UO~ per" c e l l )

x ~0s

Fig. 1. Experimental points were taken from Rothstein et al. (1948), Fig. 1. The effect of yeast concentration on the inhibition of glucose consumption by uranyl nitrate. Cell concentrations: o, 1 0 m g m l - l ; o , 20mgml-1; zx,30mgml i; A, 40 mg ml-I; O, 4 7 m g m l - t ; a n d 0, 54mgml t. 1 mg cells are 107 cells. by uranyl is a f u n c t i o n o f the total n u m b e r o f y e a s t surface g r o u p s p r e s e n t . This is also obvious f r o m t h e small s t a n d a r d d e v i a t i o n of the m e a n dissociation c o n s t a n t o f t h e six single curves ( T a b l e 1), with (27.21 + 3.91) x 106 m o l e c u l e s u r a n y l p e r cell a n d t h a t of the Hill slope, with 2.51 + 0.44.

Inhibition of glucose uptake by uranyl nitrate in initial uptake experiments I n Fig. 2 initial glucose u p t a k e (5 s) was a n a l y s e d with a n d w i t h o u t uranyl. In a c o n c e n t r a t i o n d e p e n d e n t m a n n e r uranyl ions significantly d e c r e a s e d t h e r a t e o f glucose u p t a k e at glucose c o n c e n t r a t i o n s t e s t e d f r o m 0.5 to 200 mM. T h e u p t a k e of glucose can be d e s c r i b e d by two p r o c e s s e s ( F u h r m a n n a n d V61ker, 1992), a f a c i l i t a t e d diffusion ( M i c h a e l i s - M e n t e n kinetics) a n d a diffusion ( f i r s t - o r d e r kinetics). T h e l a t t e r p r o c e s s is obvious from t h e l i n e a r slope of the r a t e of glucose u p t a k e at high glucose c o n c e n t r a t i o n s (50 to 200 mM). U r a n y l ions in increasing c o n c e n t r a t i o n s r e d u c e t h e slope a n d thus the diffusion o f glucose into t h e cells.

TABLE 1 Inhibition of glucose consumption by uranyl

S. cerevisiae

K i×

10 6 molecules per cell

'Hill' slope

r 2

(mg ml- l) 10 20 30 40 47 54

33.79 22.93 23.70 26.41 28.29 28.13

2.05 2.87 2.36 2.24 2.34 3.22

0.987 0.992 0.988 0.993 0.999 0.978

mean +_SD:

27.21 _+3.91

2.51 + 0.44

79

Y

120~ 1101 1004

901 8o ~

70i 60i 501

40 30 2010 0

o

5b

1do

i50

200

mN glucose Fig. 2. Inhibition of glucose uptake by uranyl ions. Initial uptake experiments 5 s, 5% cells and 25°C. Concentration of uranyl nitrate: o, zero; o, 10; O, 15; ~, 20; A, 30; and A, 50 /zM. Mean of four experiments and standard deviation.

Fig. 3 shows the effects o f u r a n y l ions on t h e f a c i l i t a t e d diffusion p r o c e s s in low glucose c o n c e n t r a t i o n s (0.5 to 10 m M ) in a L i n e w e a v e r - B u r k plot. W i t h i n c r e a s i n g uranyl c o n c e n t r a t i o n s the i n t e r c e p t s at t h e x-axis a n d the y-axis a r e c h a n g e d , p o i n t i n g to a m i x e d type o f inhibition. T h e d o t t e d lines in the g r a p h r e p r e s e n t the effect w i t h o u t c o n t r i b u t i o n o f diffusion. C o n t r i b u t i o n o f diffusion is within t h e r e g i o n o f s t a n d a r d d e v i a t i o n a n d thus negligible at low glucose c o n c e n t r a t i o n s . In T a b l e 2 t h e v a l u e s for Vm, K m a n d K d a r e given as m e a n + SD. All t h r e e p a r a m e t e r s a r e c h a n g e d by uranyl ions, Vm a n d Ko a r e d e c r e a s e d , w h e r e a s K m i n c r e a s e d with t h e u r a n y l c o n c e n t r a t i o n .

6 '

.5

IC

.4

E o E II 2z~

2.0

1..5 -1.0 -0.5 0.0 0.5 1.0 1/5 (S = mM glucose)

1.5

2.0

Fig. 3. Graphical analysis by Lineweaver-Burk of the experiment in Fig. 2, but only at the low concentrations of glucose of 0.5, 1, 2, 5 and 10 raM. Dotted lines represent transport without contribution of diffusion.

80 TABLE 2 Inhibition of glucose uptake by uranyl No. of exp.

uranyl ions (p.M)

V~I1 (nmolmg l min i)

Km (raM)

Kd (/xlmg l min ')

5 4 4 4 6 2 2

0 15 20 30 50 100 200

14.52_+ 1.51 13.19 + 1.52 8.88 + 1.24 9.33 -+ 1.41 7.95 + 3.53 1.92 0.75

0.62_+0.34 1.76 + 0.48 1.37 -+0.05 2.11 + 0.18 4.58-+ 1.21 4.58 3.63

0.452_+0.144 o. 184 + 0.046 o. 180 -+0.057 o. 128 _+0.019 0.103 _+0.019 0.153 o. 150

Effects of uranyl ions in inhibition of glucose uptake, transport and diffusion In o r d e r to show the c o m p l i c a t e d kinetics of glucose u p t a k e by uranyl i n h i b i t i o n the p r o p o r t i o n of s a t u r a t i o n kinetics a n d diffusion has b e e n calculated with the p a r a m e t e r s given in T a b l e 2 for a g r a d i e n t of 30 m M glucose outside a n d negligible glucose c o n c e n t r a t i o n inside the cell u n d e r c o n d i t i o n s of effective metabolism. Fig. 4 shows the flux resulting from t r a n s p o r t ( o p e n squares), the flux by diffusion ( o p e n triangles) a n d the total glucose u p t a k e (closed circles) in d e p e n d e n c e o n the uranyl c o n c e n t r a t i o n . F u r t h e r m o r e uranyl i n h i b i t i o n of glucose u p t a k e at high glucose c o n c e n t r a t i o n s for the e x p e r i m e n t s of R o t h s t e i n et al. (1948) is c o m p a r e d in Fig. 5 with the i n h i b i t o r y effects in Vm a n d K d in fast u p t a k e experiments. In o r d e r to calculate a sigmoidal curve from the K d values only uranyl c o n c e n t r a t i o n s up to 5 0 / x M were

E

rOl \|

10

r

20

40

60

BO 100 ~20 140 160 1BO 200 #N U02 ++

Fig. 4. Construction of glucose uptake in nmol glucose per rain per mg cells at a glucose concentration gradient of 30 mM vs. uranyl ion concentration. • sum of facilitated diffusion and diffusion transport;

D, facilitated diffusion; and A, diffusion transport. For construction the parameters given in Table 2 are taken.

81

O"

co

50 ~J

E

o

100 -0

]og (UO2 per c e l l )

x 106

Fig. 5. Comparison between inhibitory effects of uranyl on Vm, K d and glucose consumption. For glucose consumption the m e a n curve of Rothstein et al. (1948) is taken (dotted line), K a values (closed triangles) and Vm values (closed circles) taken from Table 2. The 50% value for K a is 13.61 and for Vm 43.78x106 molecules uranyl per cell. The Hill slope for K a was 0.87 and for Vm 1.64. M e a n + S D (Table 2).

taken, because at higher uranyl concentrations K d increases slightly. The uranyl concentration is normalized as molecules uranyl per cell.

Discussion

Uranyl ions inhibit a reaction at the cell surface of Saccharomyces cerevisiae concerned with the uptake of glucose. This inhibition is not caused by a blockage of any of the enzymes in the glycolytic pathway (Passow et al., 1961). For example, uranyl ions do not inhibit the endogenous CO 2 production of internally stored carbohydrates, nor do they inhibit the reverse path, the formation of glycogen from ethanol. Therefore uranyl can be used as a tool to characterize the mechanism which is responsible for the passage of sugar through the surface (Rothstein, 1954). This statement of Rothstein is still of importance in our days. The reversible binding of uranyl ions to the cell surface was expressed in terms of the mass law. Furthermore the uranyl ion binding at 100% glucose uptake inhibition was calculated from the best fitted line to be 45 x 106 molecules per cell (Rothstein et al., 1948) and the dissociation constant was about 20 × 106 uranyl molecules per cell. Since the maximal uranyl binding was determined to be 65 x 106 molecules per cell (Rothstein and Larrabee, 1948) there are about 20 x 106 surface groups not related to glucose transport. We digitized the experimental data points of Rothstein et al. (1948) on uranyl inhibition of glucose consumption at saturation of the facilitated transport (glucose concentration in the medium was always above 40 mM) and replotted the data in percent inhibition vs the logarithm of number of uranyl molecules per cell. The points scatter only slightly around the calculated mean sigmoidal curve. The

82 steepness of the curve predicts a cooperativity of more than 2 (Hill slope). By analysing the six experiments at different cell concentrations (Table 1) it is obvious that normalization in number of uranyl molecules per cell leads in all experiments to the same result. The mean of the dissociation constant ( K i value) was calculated to be 27.21 × 106 molecules per cell _+3.91. This value is close to about 20 × 10 6 estimated by Rothstein et al. (1948). The Hill slope was 2.51 _+ 0.44; this value would predict that 2 to 3 molecules of uranyl would react with the glucose transporter. In order to use uranyl ions more excessively as tools in the investigation of glucose transport in Saccharomyces cereL,isiae, we examined the effects of uranyl in initial glucose uptake experiments. The philosophy behind this kind of experiments is, that for the short uptake period of 5 s at 25°C, the attainable glucose inside concentration is negligible and does not create a considerable glucose effiux (Fuhrmann and V61ker, 1992). In addition inside glucose will be consumed by metabolism. As demonstrated in a great number of experiments, transport of glucose in S. ceretJisiae occurs by facilitated diffusion and by diffusion (Fuhrmann et al., 1989 and Fuhrmann and V61ker, 1992). T h e r e are many indications that the carriers are proteins similar to the family of glucose transporters for example in human red cells (Celenza et al., 1988; Kruckeberg and Bisson, 1990; Wrede et al., 1992). The cells were pretreated with 0.1% glucose to induce high-affinity glucose transports and a Vm around 15 nmol glucose per minute per mg cells (Fuhrmann and V61ker, 1992). Table 2 shows the kinetic analysis of the experiments depicted in Figs. 2 and 3. The pretreatment with a low glucose concentration of 0.1% caused a high affinity with a K m value of about 1 mM, a Vm of 14.5 nmol glucose per min per mg cells and a K~ for diffusion of 0.45 #zl per min per mg cells. The addition of uranyl ions affected both transport processes, the facilitated diffusion (Michaelis-Menten kinetics) and the diffusion. In facilitated diffusion the half saturation constant ( K m) increases and Vm decreases (Fig. 3 and Table 2). This kind of inhibition has been described as mixed inhibition (Webb, 1963). It might be that the presence of uranyl prevents carrier mediated translocation of glucose but also interferes to some extent with the binding of glucose at the carrier site itself. In Fig. 2 and Table 2 it is shown that also the K d values for diffusion of glucose into the cell are reduced. Both processes are affected differently by uranyl ions. Already at the lowest uranyl concentrations tested the K d values decrease significantly, whereas K m and Vm are only slightly affected. This different behaviour of K d and Vm is depicted in Figs. 4 and 5. In Fig. 5 a comparison with the experiments of Rothstein et al. (1948) on glucose consumption is given. The inhibitory concentrations of uranyl ions for the Kd (diffusion) and Vm cover the region of inhibition in glucose consumption. This is remarkable, because different strains of Saccharomyces cerevisiae in different states of growth have been used. For example, well starved cells were prepared by Rothstein et al. (1948) and we used freshly grown cells, glucose pretreated. Also the transport studies differ considerably, under the first condition a high glucose concentration is continuously used up by the cells over a long time period and in our experiments initial glucose uptake is measured.

83 The effects of uranyl on diffusion seem to be rather complicated, since this inhibitory effect at low uranyl concentrations disappears at high uranyl concentrations and diffusion increases slightly. Therefore two effects of uranyl ions on the diffusion must be considered, namely a decrease and an increase in diffusion. It should be mentioned that also an increase in potassium efflux has been observed by uranyl ions, which might point to a general toxic effect of the heavy metal at the permeability membrane barrier (Fuhrmann, unpublished results). The surface-associated binding of uranyl ions is consistent with the view that a complexation of the positively charged metal ion with negatively charged reactive sites at the cell surface occurs (Theuvenet and Borst-Pauwels, 1976). Such negatively charged groups are provided by phosphoryl and carboxyl groups (Rothstein et al., 1948; Rothstein and Larrabee, 1948; Rothstein, 1954; Passow et al., 1961; Strandberg et al., 1981). In respect to the amino acid composition of putative glucose carriers in Saccharomyces cerevisiae (Celenza et al., 1988; Kruckeberg and Bisson, 1990; Wrede et al., 1992) the strong binding of uranyl with glutamic acid and aspartic acid is especially interesting (Strandberg et al., 1981). As with other heavy metals the chemistry of uranyl in aqueous solution is rather complex (Gmelin Handbook of Inorganic Chemistry, 1984). For example in hydrolysed solutions of uranyl a prominent diffraction appears, due to U - U distances around 3.86 A, which are in favour of a dinuclear complex. In these complexes, the U atoms are joined by double hydroxo bridges. A representative example for a dimeric complex is [(UO2)2(OH)2(NO3)2(H20)3]H20. Also trimeric complexes have been observed. By comparing size and O-distances of such dimeric uranyl complexes with /3-D-glucopyranose (D-glucose), a close similarity in structure is obvious. Therefore, the hypothesis has been advanced that the dimeric form of uranyl could be the species which interferes with the glucose binding at the carrier site a n d / o r with the translocation step of glucose. Such an interaction is consistent with a Hill slope of about 2. Hill slopes of 1.6 (Fig. 5) and of 2.5 (Fig. 1 and Table 1) have been calculated. This small difference could be due to the different times required for the formation of the uranyl complexes in the experiments. To summarize uranyl ions inhibit facilitated diffusion of glucose into Saccharomyces cerevisiae cells by a kinetic type of mixed inhibition. In addition a clearcut inhibitory effect in glucose diffusion is seen at low concentrations of uranyl ions. However, at higher uranyl ion concentrations diffusion increases slightly. As a model for inhibition a predominantly dimeric form of uranyl could be the inhibitor at the carrier site, which is consistent with a Hill slope of around 2. o

Acknowledgment Supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

84

References Booij, H.L. (1940) Influence of metal ions on anaerobic fermentation. Rec. Trav. Bot. Neerl. 37, 1-12. Celenza, J.L., Marshall-Carlson, L. and Carlson, M. (1988) The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc. Natl. Acad. Sci. USA 80, 2130-2134. Fuhrmann, G.F., V61ker, B., Sander, S. and Potthast, M. (1989) Kinetic analysis and simulation of glucose transport in plasma membrane vesicles of glucose-repressed and derepressed Saccharomyces cerel~,isiae cells. Experientia 45, 1018-1023. Fuhrmann, G.F. and V61ker, B. (1992) Regulation of glucose transport in Saccharornyces cereuisiae. J. Biotechnol. 27, 1-15. Gmelin Handbook of Inorganic Chemistry (1984) 8th Edn, U Supplement D1. Springer Verlag, Berlin, pp. 108-110. Kruckeberg, A.L. and Bisson, LF. (1990) The HXT2 gene of Saccharomyces cerecisiae is required for high affinity glucose transport. Mol. Cell. Biol. 10, 5903-5913. Motulsky, H.J. (1985-1989) GraphPAD software, San Diego CA. USA. Passow, H., Rothstein, A. and Clarkson, T.W. (1961) The general pharmacology of the heavy metals. Pharmacol. Rev. 13, 185-224. Rothstein, A. and Larrabee, C. (1948) The relationship of the cell surface to metabolism. II. The cell surface of yeast as the site of inhibition of glucose metabolism by uranium. J. Cell. Comp. Physiol. 32, 247-260. Rothstein, A., Frenkel, A. and Larrabee, C. (1948) The relationship of the cell surface to metabolism. III. Certain characteristics of the uranium complex with the cell surface groups of yeast. J. Cell. Comp. Physiol. 32, 261-274. Rothstein. A. (1954) The enzymology of the cell surface. Protoplasmatologia II. Cytoplasma, E. Cytoplasma-Oberflfiche. In: Heilbrunn, L.V. and Weber, F. (Eds.). Wien, Springer-Verlag, pp. 1-77. Strandberg, G.W., Shumate If, S.E. and Parrot, J.R. (1981) Microbial cells as biosorbents for heavy metals: Accumulation of uranium by Saccharomyces cereuisiae and Pseudornonas aeruginosa. Appl. Environ. Microbiol. 41,237-245. Theuvenet, A.P.R., Borst-Pauwels, G.W.F.H. (1976) Surface charge and the kinetics of two-site mediated ion-translocation. The effects of UP22+ and La 3+ upon Rb+-uptake into yeast cells. Bioelectrochem. Bioenerg. 3, 230-240. Webb, J.L. (1963) Enzyme and metabolic inhibitors, Vol. 1, Academic Press, New York and London. Wrede, C., V61ker, B., Kiintzel, H. and Fuhrmann, G.F. (1992) Kinetic analysis of glucose transport in wild-type and transporter-deficient Saccharornyces cerevisiae strains under glucose repression and depression. J. Biotechnol. 27, 47-57.