Saccharomyces cerevisiae: Experimental

Transcription

1 JouRNAL of BAcrmoLGY, Feb. 97, p American Society for Microbiology Vol., No. Printed in U.S.A. Facilitated Diffusion of Monosaccharides in Saccharomyces cerevisiae: Experimental Investigation of Kinetic Parameters Without the Assumptions of Symmetry CAROLYN M. KALSOW' AND R. J. DOYLE Department of Microbiology, University of Louisville Schools of Medicine and Dentistry, Louisville, Kentucky 00 Received for publication October 97 Until the question of symmetry or asymmetry in the facilitated diffusion of monosaccharides by Saccharomyces cerevisiae is resolved, attempts to study the transport process cannot be based on assumptions of symmetry, such as equal concentrations at equilibrium or kinetic parameters that are equal in opposite directions. The assumptions of symmetry may be circumvented by measuring efflux against water and against various external concentrations of sugar. The measurement of efflux against water eliminates any involvement of influx, and the separate determinations of influx and efflux parameters do not require that the parameters be equal. Furthermore, the use of relative internal concentrations eliminates any necessity of assuming that the equilibrium concentrations are equal. Since the influx and efflux parameters are to be compared, the measurement of influx on effluxing cells allows both sets of parameters to be determined on cells which are physiologically the same. This procedure has been tested by obtaining the kinetic parameters of L-sorbose transport. The validity of these parameters was demonstrated by using them to generate theoretical efflux curves that fit the experimental data and by showing that they give the best fit curve to the relationship of velocity and permeant concentration. Although the question of symmetry remains unanswered, this procedure has opened the way for experimental evaluation of the situation and further investigation of the transport process in yeast. The first paper in this series (8) suggested that the facilitated diffusion of monosaccharides in Saccharomyces cerevisiae might be an asymmetrical process. Recent publications dealing with other cell types (7, ) have also proposed that facilitated diffusion might be asymmetrical. The question of symmetry could be resolved by studying the kinetic parameters of transport with a nonmetabolizable sugar such as L-sorbose. However, experiments to calculate these values have determined the parameters in only one direction or have been based on assumptions of symmetry (, 0,, ), i.e., equal concentrations on either side of the membrane at equilibrium or kinetic parameters that are equal in opposite directions. A IPresent address: Department of Ophthalmology, University of Louisville School of Medicine, Louisville, Ky. 00. survey of the values that have been determined in various laboratories reveals great disparity in the results (, 0,, ). Accordingly, in this paper we present an experimental procedure for studying facilitated diffusion in S. cerevisiae which does not assume symmetry and which may therefore be used to determine the nature of the system whether it be symmetrical or asymmetrical and to investigate further the mechanism of facilitated diffusion. This work was presented in part at the Annual Meeting of the American Society for Microbiology, May 970, Boston, Mass. MATERIALS AND METHODS Calculation of kinetic parameten. The calculation of the kinetic parameters was based on equations of the independent type of derivation (8), i.e., a derivation in which carrier molecules are assumed to

2 VOL., 97 be always available to either side of the membrane at a given time, as opposed to a derivation based upon the conservation of carrier, which takes into account the unavailability of the carrier to both sides of the membrane at one time: ne= -V () SUGAR TRANSPORT IN YEAST Si * Vti Se- Vte Vnet= -() Si + Kt Si + Kte where v is the velocity of transport, S is the permeant concentration, Vt and Kt are kinetic parameters, and the subscripts e and i refer to movement into and out of the cell, respectively. Use of these equations required obtaining a series of concentrations and corresponding unidirectional velocities for each direction so that calculations similar in form to those for enzyme kinetics could be utilized (). The kinetic parameters of efflux were readily determined by loading the cells with sugar and measuring permeant inside of the cell at various time intervals as described below. The velocity for the jth term was then calculated as V(J) = SIJ - S(J+) () Since the sugar was moving from the smaller to the larger volume, it was sufficiently diluted upon efflux so as not to induce a significant influx, and the parameters could then be calculated according to the relationship St Si - Vvtl V S + Kt In tum, the kinetic parameters of influx were determined by measuring the net velocity of transport of sugar against various external sugar concentrations according to equation. The efflux velocity was calculated according to equation from the concentration of sugar inside of the cell, S,', and the previously determined kinetic parameters of efflux, V,, and Kt,. By using equation, the velocity of influx, ve, was then calculated as the difference between the net velocity and the efflux velocity. Once a series of external concentrations and influx velocities had been obtained, the kinetic parameters of influx were calculated according to the relationship - S. Vt. ve () S. + Kte Maintenance and preparation of cells. The strain of S. cerevisiae used in these experiments was obtained from E. Spoerl of the U.S. Army Medical Research Laboratory, Fort Knox, Ky. All incubations were at 8. C in a New Brunswick Psychro-Therm shaking incubator. The culture was maintained on slants. Broth cultures were aerated at 8 rev/min to give a doubling time during exponential growth of. hr. The slants for maintaining the cultures were prepared as suggested by Dr. Spoerl. Nutrient agar (Difco) was suspended in.% NaCl with D-glucose (Difco) and yeast extract (Difco) added to give final concentrations of % and 0.%, respectively. The broth () contained % tryptone (Difco), 0.% yeast extract, 0.% KHP0, and % -glucose. The solutions were prepared so that the sugar and sugar-free portions could be autoclaved separately and then aseptically mixed to give the proper concentrations. The final ph of the medium was.8. By using the following procedures to prepare the cells for transport experiments, physiologically similar cells could be obtained. The growth from a -hr slant was suspended in ml of sterile water and centrifuged in a clinical centrifuge at top speed for min. The cells were washed twice in ml of sterile water before resuspension in ml of sterile water for a direct count. A portion of this suspension was added to 0 ml of preincubated broth to give 0 cells/ml. After hr of incubation, the culture was hr into exponential growth, with approximately 8 x 0 cells/ml. A portion of this culture was then added to 0 ml of fresh, preincubated broth to give 0 cells/ ml. After hr of growth, the culture was hr into stationary phase with 0 cells/ml. A 00-ml amount of the culture was centrifuged at 80 x g at 0 C for min. The cells were washed twice in 00 ml of water and held at room temperature until used in efflux experiments, a time period which did not exceed hr. Transport measurements. The methods used to measure efflux are basically those introduced by Burger et al. () and modified by Kotyk and Kleinzeller (). All transport experiments were conducted in a shaking water bath at 0 C. The washed cells were added to a sugar solution to give 09 cells/ml in a M solution of L-sorbose (Mann Research Laboratories or Pfanstiehl Laboratories). After uptake for 0 min, -ml samples of the cell suspension were centrifuged at 0 C to,00 x g, and the centrifuge was quickly braked. After two washings in ml of water at C, the pellets were placed in an ice bath. There were no distinguishable changes in efflux rates or kinetic parameters of cells held in this manner for periods up to hr, even though there was a slight efflux of L-sorbose. For each batch of cells, five consecutive efflux measurements against various solutions were set up in the following sequence: water, 0 mm L-sorbose, 00 mm L-sorbose, 00 mm L-sorbose, and water. The first (A) and second (B) water effluxes were used to check the stability of the cells held in the ice bath as described above. As the timing was started, the cells were resuspended to ml with water and added to ml of water or sugar solution to yield the desired final concentration of sugar (0, 00, or 00 mm) and of cells (0' cells/ml). At -min intervals, -ml samples were removed, added to 0 ml of water at C above a membrane filter (type RA,. lsm pore size; Millipore Corp.), and filtered. The cells were quickly washed with 0 ml of water at C. This volume of water had been determined to be sufficient to remove extracellular sorbose. The filter was then placed in a graduated centrifuge tube with a given amount of water, and the tube was placed in a boiling-water

3 KALSOW AND DOYLE J. BACTERIOL. bath for 0 min, so that the intracellular L-sorbose would be equilibrated into the resuspending medium. Cells that had sedimented during this incubation were resuspended to avoid entrapment of sugar between the cells. The suspensions were centrifuged at,00 x g at 0 C for 0 min, and the volume in the tube, minus 0. ml for the volume of the filter, was recorded. The supernatant solutions, including control samples of uptake and efflux in water only were assayed for sugar content. Sorbose assay. The cysteine hydrochloride method of Dische and Devi () was used to assay the solutions for sorbose content. Amounts of ml of 7% HSO and 0.0 ml of % cysteine hydrochloride (Calbiochem) were added to 0. ml of the samples or of standard solutions of 0.0 to 0.0 mm L-sorbose. The absorbancy at 0 nm was measured after hr of incubation at room temperature, and was converted to the concentration of sugar from the computed regression line of the standard curve. Although Dische and Devi had suggested an incubation time of hr, we found that maximal absorption at 0 nm developed after hr of incubation. The absorbancy of the control sample was found to be negligible. By expressing the intracellular sugar concentration on a relative basis, millimoles per milliliter of cell suspension, the problems associated with determining absolute internal concentrations were circumvented. To allow comparison with other experiments, this value was divided by the optical density of the cell suspension at 00 nm to give the concentration per volume of cell suspension per unit of optical density of cells (S,'). Optical density was used as a measure of cell density because it exhibited a low coefficient of variation and a sensitivity similar to that of direct counting. At 00 nm, an optical density of 0. was equivalent to 08 cells/ml. Dry weights could not be used because of their low sensitivity and the effect of sorbose in solution on the measurements. Construction of composite graphs. A composite graph of several experiments was produced by vertically aligning data from each experiment with the composite and then sliding the graph horizontally until the points gave the best fit. This procedure is equivalent to adding or subtracting a constant time value to each point in a set. This procedure is legitimate since, according to equation, the velocity of transport is only dependent upon concentration and kinetic parameters and is not influenced by the absolute time of efflux over small time periods. Generation of theoretical transport curves. Theoretical transport of L-sorbose was approximated by sequential calculations of the amount of sugar transport over a small time interval (0.0 sec), and the corresponding correction of the intemal amount of sugar according to equation. The value of the term S.e VtJ(S. + Kt.) was calculated as a constant with S. equal to the appropriate external concentration of sorbose (0, 0, 00, or 00 mm). RESULTS Efflux sponding kinetic parameters. The concentration and velocity corre- values TABLE. Concentration and velocity values for efflux of L-sorbose against water Expt Time Sit a vb(s,'/min) A B A B A B a Si' millimoles of L-sorbose = per milliliter of cell suspension per unit of optical density of cell suspension. I The value of v,j, was calculated according to equation. obtained from the efflux experiments against water (Table ) were used to calculate the kinetic parameters of efflux by the weighted Lineweaver-Burk (WLB) method (7) to give values for = Vti Si'/min and Kti 0. = i 0. Si'. An attempt to estimate the validity of the values for Vti and Kti is shown in Fig., where the theoretical curve for these parameters is superimposed over the composite graph of the

4 VOL., 97 SUGAR TRANSPORT IN YEAST data. However, it was found that there are many pairs of values for Vt and Kt, that can generate theoretical curves also producing a good fit to the data. Therefore, a further check of the values was accomplished by determining the best fitting curve relating v to Si as Z[V,J, - L¾,I where v(j, is the experimental value and O(j, is the expected value for a given set of parameters. This sum of squares value was calculated for the WLB values i standard error, as well as for the values obtained from regression lines of the regular Lineweaver-Buck plot and other nonweighted plots of SJ/v versus St and v versus v/s,. As shown in Table, the WLB gave the best fit curve to the data. Influx kinetic parameters. The data obtained from the efflux experiments against various external concentrations of L-sorbose were used to calculate the influx kinetic parameters (Table ). Because there were only three extemal concentration values, it was decided to obtain an average influx velocity value (v) for each extemal concentration before using the WLB method to calculate the Vte and Kte values of S'/min and 70 ± mm, respectively. These values were also checked by a composite graph (Fig. ) and by determining the best fitting curve from several methods of calculation (Table ). The calculations showed that parameters determined by the WLB method again gave the best fit curve and that parameters giving a better fit curve to the data were obtained by use of average values rather than each influx velocity value independently. DISCUSSION The methods employed in the present study were designed to circumvent the assumptions of symmetry in facilitated diffusion of monosaccharides by yeast. The internal amount of sugar was always expressed as Si' to avoid the problem of determining an absolute internal concentration. Since the only available method for determining intemal sugar space, volume of distribution (), involves the assumption that equilibrium concentrations are equal, these absolute concentrations could not be used to test the equality of the equilibrium concentrations. In these experiments, the efflux and influx parameters had to be determined separately. By measuring the rate of efflux against water, the efflux kinetic parameters were obtained without any measurable involvement of influx. Although Newton's equation for equispaced arguments with associated decreasing differences could have been used successfully to calculate the initial velocity of influx (, 9), Si' Relative Time (min) FIG.. Comparison of the theoretical efflux of L-sorbose (V,, = 0. S,/min; Kt, = 0.7Si') with a composite graph of the experimental data for efflux against water. TABLE. Sum of the squares (SS) of the deviations of the experimental v(j) to the theoretical V(J)a Vt Method of calculation (SEK SS" min) (Si,) Weighted Lineweaver- Burk (WLB) x 0-8 WLB - SE x 0-9 WLB + SE X 0- Regression Lineweaver- Burk x 0- Si/v vs. S, X 0- v vs. v/sa x 0- av(j) = (Si * Vti)/(Si + Kt). SS = Z [V-j_ V(j) ]. at which time the velocity of efflux should be zero, the parameters calculated from such data could not have been used to test the equality of influx and efflux. There is the possibility that cells which have transported M sugar for 0 min are not the same physiologically as those which have not been previously exposed to the sugar. Spoerl () has shown that the total amount of L-sorbose to exit from cells is inversely related to the time of uptake. Although he stated that the exit patterns were similar, the parameters may have changed. Therefore, the measurement of influx velocity as the difference between the efflux velocity and net velocity gave a value which had been determined on cells in the same physiological state. In presenting the case for possible symmetry (8), two basic equations relating transport velocity to concentration of permeant were discussed-the independent type and the conservation of carrier type. The independent type was used in these calculations since it could be rearranged to give a relationship between veloc-

5 KALSOW AND DOYLE J. BACrEIuoL. TABLE. Concentration and velocity values for efflux of L-sorbose against various concentrations of L-sorbose S. Time Vna v0ut Vnc lit. (mm) Expt (min (S( Une t,/ ( St,/ min) min) min) (St,'/ min) (St,'/ Reloatve Time (min) FIG.. Comparison of the theoretical efflux of L-sorbose against various external concentrations of L-sorbose (Vt, 0. S,'/min; K&, 0.7 S,'; V,, = = = 0. S,/min; Kt. = 70 mm) with a composite graph of the experimental data for efflux against the various concentrations. Symbols: 0, 0 mm; x, 00 mm; A, 00 mm. () Expt (mm) (mi) min) (S') (SI/ (S,'/ (S,'/ (Si'/ min) min) min) min) V..t= [S, (J- ) - Si(j+)]/. V v C = (S,. 0. S,'/min)/(S, S,). Ve = V - Vnet TABLE. Sum of the squares (SS) of deviations of the experimental v(j) to the theoretical O(J)a Vt. Method of calculation (S,/ Kt (,, SSb WLB using x 0-7 WLBc x 0-' Regression Lineweaver- Burk using x 0- S,'lv vs. S,' x 0-7 v vs. v/si' using...i.0.8i 97. x 0-' aoj) = (S *0. S,'/min)/(S, Si') - (S.oVte)/(S. + Kt.). SS = Z [V(J) - o(j). All SS values were calculated against the experimental values rather than against each individual experimental point. c Calculation of SS values by use of each point also indicated that using in the weighted Lineweaver- Burk method gave the best fitting curve to the data.

6 VOL., 97 SUGAR TRANSPORT IN YEAST ity and concentration that would lend itself to an analysis giving the kinetic parameters, such as a Lineweaver-Burk plot. The equation of the conservation of carrier type could not be so arranged that the kinetic parameters could be determined from simple measurements of concentration and velocity. Therefore, it should be pointed out that the use of the independent method in this study is not an endorsement of the approach but merely a convenient point of departure for studying the problem of symmetry, as well as for studying the two types of derivations. The kinetic parameters of L-sorbose transport by S. cerevisiae produced by this method appear to be valid. The theoretical curves correlate with the composite graphs of the experimental data. Although it is true that the relationship of the composite and the theoretical curve may show a false correlation as a result of the calculations, it should be remembered that for efflux against water the data used to calculate the parameters (vj and S,) were one calculation removed from the experimental data of the composite graph (S, and time), whereas for efflux against various concentrations of sugar there were several calculations between the data (SA and time) and the figures used to calculate the parameters (ve and Se). The validity of the parameters is further supported by the demonstration that the WLB method gives the best fit curve to the concentration-velocity relationship even though many pairs of numbers may appear to give theoretical curves that fit the composite graphs. However, since the data only cover a small range of concentration and time values and since many pairs of numbers may fit the curves well, the possibility remains that the equations presented here do not accurately represent the relationship that exists between permeant concentration and velocity of transport. The value for Kte is in general agreement with those of mm reported by Wilkins and Cirillo () and 00 mm reported by Sols (), but not with the,00 and mm values reported by Cirillo () and Kotyk (0), respectively. However, if the physiological state of the cell does influence the kinetic parameters, then true comparisons can be made only between physiologically identical cells. Although the efflux and influx Kt values cannot be directly compared since they are calculated in different units, which are related by an unknown factor of the sugar space, their validity and the validity of this approach might be examined by calculating the theoretical 7 sugar space necessary if the cells do indeed exhibit symmetry. Kti was expressed as 0. S,' (millimoles per milliliter of suspension per optical density unit). If there were 0. optical density unit/08 cells, then K, could be expressed as 0. mmole/08 cells, since under experimental conditions there were 08 cells/ ml. If symmetry does exist, Kt. should equal Kti, and a simple proportion of 70 mm 0. mm ml x ml would give a sugar space in 08 cells of.9 x 0-i ml or.9 um of sugar space/cell. If the average volume of a yeast cell was approximately 00 um (), then.9% of the cell must have been available for sugar space. This value varies greatly from the sorbose sugar space of 7% reported by Cirillo (). Our own estimations have also indicated that the volume of our cells was approximately 00,um and the volume of distribution of sorbose space was approximately 0% of the cell volume (unpublished data). Therefore, it may be concluded that symmetry does not exist or that the independant approach is not valid and the conservation of carrier must be taken into account, or it may be concluded that there is intracellular compartmentalization or binding of the sugar () that must be accounted for in the calculations or that some as yet unconsidered factor affects transport. Vt values were calculated in the same units and may therefore be compared. In these experiments, the Vte of L-sorbose was found to be 0. Si'/min and Vt, to be 0. Si'/min. Although the values appear to be similar, more experiments covering a greater range of concentrations and perhaps longer or shorter times of transport are necessary to establish the equality or inequality of the Vt values. However, it must be remembered that an equality of Vt values does not mean that the Kt and S values must be equal. There is also reason to believe that Vt values may be equal in an asymmetric system since in the equilibrium type of independent derivation (8) they contain no reaction constants goveming association or dissociation of carrier and permeant but are dependent on various properties of the membrane. However, if the arguments of Schultz () are correct, the Vt values could be unequal even if they were not influenced by the association and dissociation reaction constants. This would be so if the membrane itself were asymmetrical in nature. It is readily evident that more experiments

7 8 KALSOW AND DOYLE J. BAcroL. are needed to substantiate any claim for symmetry or asymmetry. Various modifications are going to be necessary for investigations involving transport of two sugars or counterflow. Furthermore, since this approach assumes no intracellular compartmentalization or binding, modifications may be necessary to examine specifically or to account for these possibilities. However, the method provides a starting point for the study of symmetry in transport. It may also be used to differentiate the independent type of derivation from that based on conservation of carrier or to measure environmental effects upon transport parameters. Yet, this may be accomplished by using a simple method and only one sugar at a time. ACKNOWLEDGMENTS This work was supported by the National Defense Education Act Training Grant OE , a grant from the Kentucky-Jefferson County Heart Association, and GRS support from the National Institutes of Health. LuTRATURE CITED. Burger, M., L. Hejmova, and A. Kleinzeller. 99. Transport ofsome mono- and di-saccharides into yeast cells. Biochem. J. 7:-.. Cirillo, V. P. 9. The transport of nonfermentable sugars across the yeast cell membrane, p. -. In A. Kleinzeller and A. Kotyk (ed.), Membrane transport and metabolism. Academic Press Inc., London.. Cirillo, V. P. 9. Sugar transport by Saccharomyces cerevisiae protoplasts. J. Bacteriol. 8:-.. Cirillo, V. P. 98. Relationship between sugar structure and competition for the sugar transport system in baker's yeast. J. Bacteriol. :0-.. Conway, E. J., and M. Downey. 90. An outer metabolic region ofthe yeast cell. Biochem. J. 7:7-.. Dische, Z., and A. Devi. 90. A new colorimetric method for the determination of ketohexoses in presence of aldoses, ketoheptoses, and ketopentoses. Biochim. Biophys. Acta 9: Geck, P. 97. Eigenschaften eines asymmetrischen Carrier-Modells f;ir den Zuckertransport am menschlichen Erythrozyten. Biochim. Biophys. Acta : Kalsow, C. M., and R. J. Doyle. 97. Facilitated diffision of monosaccharides in Saccharomyces cerevisiae. I. Theoretical considerations of asymmetry. J. Theor. Biol. :-. 9. Kotyk, A. 97. Mobility of the free and the loaded monosaccharide carrier in Saccharomyces cereuisiae. Biochim. Biophys. Acta : Kotyk, A. 97. Properties of the sugar carrier in baker's yeast. II. Specificity of transport. Folia Microbiol. :-.. Kotyk, A., and A. Kleinzeller. 9. Transport of D- xylose and sugar space in baker's yeast. Folia Microbiol. 8:-.. Riggs, D. S. 9. The mathematical approach to physiological problems. The Williams & Wilkins Co., Baltimore.. Schultz, J. S. 97. Passive asymmetric transport through biological membranes. Biophys. J. :9-9.. Sols, A. 98. Regulation of carbohydrate transport and metabolism in yeast, p In A. K. Mills (ed.), Aspects of yeast metabolism, a Guiness symposium. F. A. Davis Co., Philadelphia.. Spoerl, E. 99. Membrane changes in yeast cells caused by sulfhydryl reagents and accompanied by a selective release of sugar. J. Membrane Biol. : Wilkins, P. O., and V. P. Cirillo. 9. Sorbose counterflow as a measure of intracellular glucose in baker's yeast. J. Bacteriol. 90: Wilkinson, G. N. 9. Statistical estimations in enzyme kinetics. Biochem. J. 80:-.