AMINOAcm COMPOSITION OF Euglena PROTEIN
446
method for determining tryptophan in peptides and proteins. Anal. Biochem. 60, 45-50. 9. Reeck G. 1970. Amino acid compositions of selected proteins, in Sober HA, ed., Handbook of Biochemistry, 2nd ed., CRC Press, Cleveland, Ohio, pp. C-281-93.
10. Rosen H. 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 61, 10-5. 11. Schantz R, Schantz M-L,Duranton H. 1975. Changes in amino acid and peptide composition of Euglena gracilis cells during chloroplast development. Plant Sci. Lett. 5, 313-24.
J. PROTOZOOL. 23(3), 446-449 (1976).
Specificity of the Glucose Transport System in L&hmnita tropica Promastigotes* FRANK W. SCHAEFER, IIIt and ANTONY J. MUILKADAS Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221
SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has aOI .Q of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (a-methyl-D-ghcoside, 1,5-anhydroglucitol) and carbon 3 ( 3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature-they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.
Index Key Words: Leishmania tropica; glucose transport; 2-deoxy-~-glucose;carrier specificity.
I
N Leishmania tropica promastigotes the transport of glucose as measured by the uptake of a nonmetabolizable glucose analog, 2-deoxy-~-glucose( 2-D0G), is a carrier-mediated process characterized by saturation kinetics (8, 14). Both D-glUCOSe and 2-DOG share a common transport carrier as inferred from the fact that the transport of either sugar is competitively inhibited by the other. The initial rates of uptake of 2-DOG and D-glucose are essentially identical and intracellular 2-DOG readily exchanges with extracellular D-glucose which further support the involvement of a common carrier for both sugars. Other glucose analogs such as a-methyl-D-g!ucoside (a-MG) and 3-0-methylD-glucose (3-0-MG) are not transported by the cells ( 14). In the present paper we attempt to establish a correlation between the structure of various sugars and their ability to combine with the glucose carrier. If several sugars are transported by the same route, they should compete with each other for the same carrier. Thus, in competition experiments, the degree of competitive inhibition of 2-DOG uptake by other sugars and sugar derivatives was taken as a measure of the affinity of the glucose carrier for such substrates. It is demonstrated that the carrier for D-glucose in L. tropica is shared by a few other hexose sugars and sugar derivatives in the &form but has no affinity for the L-isomer of glucose. A preliminary report of some of our data was presented earlier ( 12). MATERIALS AND METHODS The Organism Leishmania tropica stock cultures originally obtained from Ms. L. Norman, National Center for Disease Control, Atlanta, Georgia, were maintained on NIH blood agar slants overlaid with Locke’s solution ( 16). For experimental purposes, the cells were
* This investigation was supported by a grant from the Research Council of the University of Cincinnati. t Present address: Department of Biology, College of Science, University of Notre Dame, Notre Dame, Indiana 46556. To whom reprint requests should be directed.
*
grown in a monophasic, cell-free, liquid medium (13). Usually 50 ml media in 250-ml Erlenmeyer flasks were inoculated with promastigotes and incubated at 26 C in a New Brunswick incubator shaker at 140 rpm. Cells were harvested when the cultures reached a cell density of 2.0 X 10s cells/ml. The cells were removed by centrifugation at 1,500 g for 10 min at 4 C and washed twice with 0.85% (w/v) NaCl solution to remove adhering nutrients. Uptake Experiments
The uptake of [U-14C]2-deoxy-~-glucose was measured by the rapid filtration technic described in previous communications (8, 14, 15). Twice-washed promastigotes were resuspended in the basal salts solution used in the growth medium to give a final cell concentration equivalent to 0.35 mg dry wt/ml. Uptake experiments were carried out with 10 ml of cell suspensions in 50-ml Erlenmeyer flasks. The suspensions were equilibrated at 30 C for 20 min before [U-W]2-DOG was added to them. The concentrations of the labeled sugar and of other additives in various experiments are indicated in the Results section. After adding isotopic 2-DOG, 1-ml samples were removed at appropriate intervals, filtered immediately through 0.8 pm membrane filters (Millipore Corp., Bedford, Mass.) and washed with 10 ml basal salts solution (5.2 gm sodium chloride, 0.5 gm potassium chloride, 10.3 gm disodium hydrogen phosphate and 1 liter glass distilled water at pH 7.0). The filters with the cells were transferred to scintillation vials containing 10 ml Bray’s (1) scintillation fluid and radioactivity determined in a Packard Liquid Scintillation Spectrometer Model 3310. The results are expressed as nanomoles of sugarlmg cells. The apparent K,,,for the transport of 2-DOG was determined graphically by the method of Lineweaver & Burk (6). The values of K, for different sugars which competitively inhibited 2-DOG uptake were calculated from Lineweaver-Burk plots using the equation of Brown & Romano (2) : K, = I/(Kp/Km- 1) where I represents the concentration of the inhibitor, and Kp and K, are values calculated from the points where the lines obtained in the presence and absence of the
L. tropica:
1.1
1.0
THE
GLUCOSETRANSPORT SYSTEM
447
I .
Y
z
-I
0.9
'
0.8
'
!/RT Xld'
1 I
0 -1 0
0
10 20 1/S (MM)
30
0
10 20 30 40 0 10 20 30 40 I l S [2-DOG (uM)] 3
2
Fig. 1. Arrhenius plot of uptake rates between 26 and 32 C. K, velocity of uptake expressed as nmoles/mg/2 min; R, universal gas constant; T, absolute temperature. Figs. 2, 3. Lineweaver-Burk plots of the inhibition of 2-deoxy-~glucose(2-DOG) by various sugars. Cells were incubated with [v-uC] 2-DOG (0.2 pCi/pmole) at the indicated concentrations. Up.take of 2-DOG was followed in the absence or presence of the inhibiting sugars, each at a 0.1 mM concentration. The results are given as the initial rates of uptake expressed as nanomoles/mg/min, at each 2-DOG 0 , 2-DOG; 0-0, 2-DOG N-acetyl glucosamine. 3. Inhibiconcentration. 2. Inhibition by N-acetyl glucosamine. 00 , 2-DOG alone; 00, 2tion by galactose (A), glucosamine (B), mannose (C), and fructose (D). In all plots, 0DOG inhibiting sugar.
+
+
inhibitor intercept the X axis. These points represent negative reciprocals of Kp and K,,,respectively. Source of Chemicals [14C]2-DOG was purchased from International Chemical and Nuclear Co., Irvhe, California. a-methyl D-glucoside, 3-0-methyl glucose and 1,5-anhydro-~-gluutolwere obtained from Schwanl Mann, Orangeburg, New York. All other sugars were purchased from Sigma Chemical Co., St. Louis, Missouri and Nutritional Biochemicals, Cleveland, Ohio. RESULTS The Glucose Carrier.-Maximal uptake occurred at 30-32 C beyond which the rate of uptake dropped precipitously. An Arrhenius plot of the rates at 26 to 32 C is shown in Fig. 1. From the slope of this plot, a heat of activation of 10.2 kcal/
mole !&DOG, and a Qlo of 2 for the system were obtained. These values are in the range expected of enzyme mediated reactions and further support the idea of carrier involvement in 2-DOG entry into the cell which we reported earlier (14). Optimal rates of uptake were observed at around pH 7.0. Specificity of the Carrier.-The results of competition experiments in which the uptake of 2-DOG was determined in the presence of several other sugars and sugar derivatives are shown in Table 1. None of the pentose sugars tested (D-ribose, D-arabinose, D-xylose and D-lyxose) had any effect on 2-DOG uptake. The frequently used glucose analogs, a-MG and 3-0-MG or the L-isomer of D-glucose did not inhibit 2-DOG uptake. Similarly, 1,5-anhydro-n-glucitol ( 1-deoxy-D-glucose) , fucose, rhamnose and sorbose were without appreciable effect on the uptake of this glucose analog. O n the other hand, galactose, glucosamine, N-acetyl glucosamine, mannose, and fructose lowered its uptake significantly. It is evident from Lineweaver-Burk plots
448
L. tropica:
THE
GLUCOSETRANSPORT SYSTEM
TABLE1. Effect of different sugars on the uptake of 2-DOG.'
sugar D-glucose D-glUCOSamine N-acetyl glucosamine D-mannose D-galactose D-fructoset
Carbon (9) at which substituents differ from cl-D-glUCOpyranose structure
n
Z Percent inhibition
Changes
-
-
2
2-amino-2-deoxy 2-N-acetyl-2-deoxy
2 2 4 1,2 and 5
98 44
52
2 axial OH 4 axial OH 1-deoxy, 2 axial OH,
+
63 37
L-glucoset
1 and 5
a-methylaglucoside 3-0-methyl glucose 1,5-anhydro-~glucitol Drhamnose L-sorboset
1
5-(C&OH OH) (anomdric) 1 CHsOH, 50H (anomeric) a-1-0-methyl
3
3-0-methyl
NI
1
1-deoxy
NI
3 and 6 1 and 5
~
72 NIO NI
NI 6-deoxy, 3 axial OH 1-deoxy, 5NI (CHnOH OH) D-fucose NI 4 and 6 4 axial OH, 6-deoxy D-ribose* 3 NI 3 axial OH D-arabinose 2 and 3 2 axial OH, 3 NI axial OH D-XylOSe* 5 NI 5-deoxy D-lyXOSe* NI 2 2 axial OH * Cell suspensions, prepared as described in Materials and Methods, were incubated with 0.1 mM ~C]2-deoxy-~glucose (0.2 pCi/pmole). Nonisotopic sugars tested as competitors were added simultaneously with labeled 2-DOG. All sugars were used at a concentration of 1 mM. The results represent inhibition of uptake during a 10-min incubation period. ?Sugars in the 1C conformation; *sugars in the Cl-~-xylopyranose conformation; % NI, no inhibition.
-10
+
0 10 2 0 3 0 4 0 I/S (MM)
Fig. 4. Lineweaver-Burk plots of the inhibition of z-deoxy-~glucose uptake by glycerol. Cells were incubated with WIZ-DOG (0.2 pCi/pmole) at the indicated concentrations. Uptake of the 0 ) and presence sugar was followed in the absence ( 0(0 0 ) of 1 mM glycerol.
~~~~~
of the inhibition of 2-DOG uptake by all these sugars that they act as competitive inhibitors (Figs. 2, 3). We have already reported competitive reciprocal inhibition of 2-DOG uptake by D-glucose (8, 14). The values of apparent K4 for individual sugars that competitively inhibited 2-DOG uptake were calculated from these data (Table 2). Inhibition of 2-DOG Uptake by Glycerol.-It was thought that addition of glycerol, which is structurally unrelated to glucose, might stimulate the uptake of 2-DOG by providing additional energy, but that it would not compete with 2-DOG for uptake. Accordingly, experiments were carried out in which the uptake of 2-DOG was followed in cells incubated with glycerol for various periods of time ranging from zero to 15 min before the addition of the sugar. In all instances, glycerol proved to be a strong inhibitor of 2-DOG uptake. In experiments using 0.1 mM 2-DOG, the addition of 1 mM glycerol simultaneously with 2-DOG resulted in 90% inhibition. It is evident from Lineweaver-Burk plots of 2-DOG uptake in the presence and absence of glycerol that glycerol acts as a noncompetitive inhibitor (Fig. 4). Cells grown in a medium supplemented with 20 mM glycerol transported 2-DOG nearly as well as the control cells (Fig. 5). Uptake of 2-DOG in both types of cells, however, was severely inhibited by the addition of glycerol to the reaction mixture. Apparently, glycerol does not prevent the synthesis of the glucose carrier (repression) but only affects the overall function of the carrier already present.
DISCUSSION In this and previous publications (8, 14) we have reported that glucose is taken up by L. tropica promastigotes by a mediated transport mechanism. D-glucose and 2-DOG share the same transport carrier which, however, has no affinity for the closely related glucose analogs a-MG and 3-0-MG or for the L-isomer of glucose since they are not transported nor do they inhibit 2DOG uptake. The glucose carrier thus has a certain degree of stereospecificity. The stereochemical requirements for glucose transport in L. tropica resemble those of Trypanosoma gambiense in which Southworth & Read (17) reported that a-MG and 3-0-MG failed to inhibit glucose uptake. Although these investigators did not study the uptake of these glucose analogs (a-MG and 3-0-MG), their inability to inhibit glucose uptake indicates that as in L. tropica, the glucose transport system in T . gambiense has no affinity for them. Glucose uptake in Trypanosoma lewisi (11) and Trypanosoma equiperdum ( 10) is, however, inhibited by 3-0-MG. Since most sugars in solution exist either as open chains or as
TABLE 2. Znhibition of 2-DOG uptake by L. tropica. Sugar Dglucose Dfructose D-mannose N-acetyl glucosamine Dglucosamine Dgalactose
Apparent Kc (mu) 0.08 0.13 0.26
0.46 2.0 2.63 Promastigotes were incubated with labeled 2-deoxy-~-glucose (0.2 pCi/pmole) . Nonisotopic sugars were added at a concentration of 0.1 mM.
L. tropica:
2
4 6 8 1 TIME (MINI
THE
GLUCOSETRANSPORT SYSTEM
0
Fig. 5. Effect of glycerol on 2-DOG uptake. 00, uptake of 2-DOG by cells grown in the absence of glycerol; 00, uptake of 2-DOG by cells grown in the presence of 20 mM glycerol; AA, uptake of 2-DOG in the presence of 1 mM glycerol by cells grown in the absence of glycerol; Aa,uptake of 2-DOG in the presence of 1 mM glycerol by cells grown in the presence of 20 mM glycerol. a variety of furanose and pyranose ring structures ( 3 ) , interpretation of the relationship of transport activity with the steric properties of particular sugars is difficult. D-glucose in solution is known to occur almost entirely in the C1 glucopyranose form (5). Thus it is conceivable that other sugars which in solution come closest to the C1 pyranose form would be the most likely to interact with the glucose transport system. This in fact is true in the case of L. tropica; all sugars that competitively inhibit 2-DOG uptake (glucosamine, N-acetyl glucosamine, mannose, galactose and fructose) are capable of existing in the pyranose form. Aldohexoses differing from glucose by substitutions or changes in configuration at carbon 2 (2-DOG, glucosamine, N-acetyl glucosamine and mannose) act as competitive inhibitors of 2-DOG uptake. Apparently, the extent of changes at carbon 2 in these sugars does not eliminate the ability of the glucose carrier to recognize and bind them. The fact that both glucosamine and N-acetyl glucosamine act as competitive inhibitors of 2-DOG uptake suggests that substitutions at carbon 2 which affect the length of the side chain at least to a limited extent can be tolerated without loss of carrier recognition. Changes a t carbon 1 (a-MG, 1,5-anhydroglucitol) and carbon 3 (3-0-MG) result in loss of carrier affinity since these sugars do not compete with 2-DOG for uptake. As in T. lewisi ( 11) and T. equiperdum ( l o ) , the glucose transport system in L. tropica is subject to competitive inhibition by the keto sugar fructose. Carbon 4 does not seem to contribute substantially to specificity, since 2-DOG uptake is competitively inhibited by galactose which differs from glucose at carbon 4. This is not unique to L. tropica, being true also of T. lewisi (11). In fact, galactose has been shown to bind with the glucose carrier and is presumably taken up by the glucose transport system in a number of eukaryotes ( 7 ) and prokaryotes (9). Fucose, a structural analog of galactose, however, does not affect 2-DOG uptake in L. tropica. Inhibition of hexose transport by the 3-carbon compound, glycerol, in L. tropica (present study) was also observed in T. gambiense (17) and T . equiperdum (10). I n T . gambiense, Southworth & Read ( 17) postulated a common carrier for glucose
449
and glycerol, but in T . equiperdum the 2 were found to have separate transport sites ( 10). Examination of the initial rates of 2-DOG uptake as a function of increasing concentrations of the sugar in the presence and absence of glycerol (Fig. 4 ) suggested a noncompetitive mode of inhibition in L. tropica and that glucose and glycerol do not share the same carrier. Glycerol does not affect the K,,,for 2-DOG (0.083 mM) but lowers the V,,,,, (from 3.1 nmoles/mg/min to 1.3 nmoles/mg/min). Inhibition by glycerol is thus not caused by competition for a common carrier nor by changes in the affinity of the carrier. It is possible that some product of glycerol metabolism has a regulatory role and exerts an inhibitory effect on glucose uptake. An alternative possibility is that glycerol enhances the rate of efflux of intracellular sugar causing an apparent reduction in net uptake. This would be analogous to the situation in Escherichia coli, where a number of metabolizable substrates were shown to increase the efflux of a nonmetabolizable sugar, a-MG (4). Cells grown in the presence of 20 mM glycerol transport 2-DOG just as well as those grown without glycerol; this finding rules out repression of the glucose transport system by glycerol. ACKNOWLEDGEMENTS We thank Dr. Geetha Bhat, Department of Chemistry, University of Cincinnati, for helpful comments in the preparation of this manuscript. The technical help of Michael Simon and Peggy Lepley is gratefully acknowledged. REFERENCES 1. Bray GA. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-85. 2. Brown CE, Romano AH. 1969. Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans. J. Bacteriol. 100, 1198-203. 3. Cirillo VP. 1968. Relationship between sugar structure and competition for the sugar transport system in baker's yeast. J. Bacteriol. 95, 603-11. 4. Hagihira H, Wilson TH, Lin EC. 1963. Studies on the glucose transport system in Escherichia coli with a-methylglucoside as substrate. Biochim. Biophys. Acta 78, 505-15. 5. Isbell HS, Tipson RS. 1959. A nomenclature for conformations of pyranoid sugars and derivatives. Science 130, 793-4. 6. Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J. A m . Chem. SOC.56, 658-66. 7. Mark C, Romano AH. 1971. Properties of the hexose transport systems of Aspergillus nidulans. Biochim. Biophys. Acta 249, 216-26. 8. Mukkada AJ, Schaefer FW 111, Simon MW, Neu C. 1974. Delayed in uitro utilization of glucose by Leishmania tropica promastigotes. J . Protozool. 21, 393-7. 9. Rogers D, Yu S. 1962. Substrate specificity of a glucose permease of Escherichia coli. J. Bacteriol. 84, 877-81. 10. Ruff MD, Read CP. 1974. Specificity of carbohydrate transport in Trypanosoma equiperdum. Parasitology 68, 103-15. 11. Sanchez G, Read CP. 1969. Carbohydrate transport in Trypanosoma lewisi. Comp. Biochem. Physiol. 28, 93 1-7. 12. Schaefer FW 111, Mukkada AJ. 1974. Characteristics and specificity of hexose transport in Leishmania tropica promastigotes. Proc. 3rd Znt. Congr. Parasitol., Munich, 1974. Facta Publications, H. Egermann, Vienna, 3, 1461. , Bell EJ, Etges FJ. 1970. Leishmania tropica: 13. chemostatic cultivation. Exp. Parasitol. 28, 465-72. , Martin E, Mukkada AJ. 1974. The glucose trans14. port system in Leishmania tropica promastigotes. J . Protozool. 21, 592-6. 15. Simon MW, Rusnak JM, Mukkada AJ. 1976. Toxicity of bilirubin to Leishmania tropica promastigotes. Exp. Parasitol. 39, 51-8. 16. Sorouri P. 1955. The nuclear cytology of Leishmania tropica. J. Morphol. 97, 393-414. 17. Southworth GC, Read CP. 1970. Specificity of sugar transport in Trypanosoma gambiense. J . Protozool. 17, 396-9.
446
method for determining tryptophan in peptides and proteins. Anal. Biochem. 60, 45-50. 9. Reeck G. 1970. Amino acid compositions of selected proteins, in Sober HA, ed., Handbook of Biochemistry, 2nd ed., CRC Press, Cleveland, Ohio, pp. C-281-93.
10. Rosen H. 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 61, 10-5. 11. Schantz R, Schantz M-L,Duranton H. 1975. Changes in amino acid and peptide composition of Euglena gracilis cells during chloroplast development. Plant Sci. Lett. 5, 313-24.
J. PROTOZOOL. 23(3), 446-449 (1976).
Specificity of the Glucose Transport System in L&hmnita tropica Promastigotes* FRANK W. SCHAEFER, IIIt and ANTONY J. MUILKADAS Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221
SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has aOI .Q of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (a-methyl-D-ghcoside, 1,5-anhydroglucitol) and carbon 3 ( 3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature-they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.
Index Key Words: Leishmania tropica; glucose transport; 2-deoxy-~-glucose;carrier specificity.
I
N Leishmania tropica promastigotes the transport of glucose as measured by the uptake of a nonmetabolizable glucose analog, 2-deoxy-~-glucose( 2-D0G), is a carrier-mediated process characterized by saturation kinetics (8, 14). Both D-glUCOSe and 2-DOG share a common transport carrier as inferred from the fact that the transport of either sugar is competitively inhibited by the other. The initial rates of uptake of 2-DOG and D-glucose are essentially identical and intracellular 2-DOG readily exchanges with extracellular D-glucose which further support the involvement of a common carrier for both sugars. Other glucose analogs such as a-methyl-D-g!ucoside (a-MG) and 3-0-methylD-glucose (3-0-MG) are not transported by the cells ( 14). In the present paper we attempt to establish a correlation between the structure of various sugars and their ability to combine with the glucose carrier. If several sugars are transported by the same route, they should compete with each other for the same carrier. Thus, in competition experiments, the degree of competitive inhibition of 2-DOG uptake by other sugars and sugar derivatives was taken as a measure of the affinity of the glucose carrier for such substrates. It is demonstrated that the carrier for D-glucose in L. tropica is shared by a few other hexose sugars and sugar derivatives in the &form but has no affinity for the L-isomer of glucose. A preliminary report of some of our data was presented earlier ( 12). MATERIALS AND METHODS The Organism Leishmania tropica stock cultures originally obtained from Ms. L. Norman, National Center for Disease Control, Atlanta, Georgia, were maintained on NIH blood agar slants overlaid with Locke’s solution ( 16). For experimental purposes, the cells were
* This investigation was supported by a grant from the Research Council of the University of Cincinnati. t Present address: Department of Biology, College of Science, University of Notre Dame, Notre Dame, Indiana 46556. To whom reprint requests should be directed.
*
grown in a monophasic, cell-free, liquid medium (13). Usually 50 ml media in 250-ml Erlenmeyer flasks were inoculated with promastigotes and incubated at 26 C in a New Brunswick incubator shaker at 140 rpm. Cells were harvested when the cultures reached a cell density of 2.0 X 10s cells/ml. The cells were removed by centrifugation at 1,500 g for 10 min at 4 C and washed twice with 0.85% (w/v) NaCl solution to remove adhering nutrients. Uptake Experiments
The uptake of [U-14C]2-deoxy-~-glucose was measured by the rapid filtration technic described in previous communications (8, 14, 15). Twice-washed promastigotes were resuspended in the basal salts solution used in the growth medium to give a final cell concentration equivalent to 0.35 mg dry wt/ml. Uptake experiments were carried out with 10 ml of cell suspensions in 50-ml Erlenmeyer flasks. The suspensions were equilibrated at 30 C for 20 min before [U-W]2-DOG was added to them. The concentrations of the labeled sugar and of other additives in various experiments are indicated in the Results section. After adding isotopic 2-DOG, 1-ml samples were removed at appropriate intervals, filtered immediately through 0.8 pm membrane filters (Millipore Corp., Bedford, Mass.) and washed with 10 ml basal salts solution (5.2 gm sodium chloride, 0.5 gm potassium chloride, 10.3 gm disodium hydrogen phosphate and 1 liter glass distilled water at pH 7.0). The filters with the cells were transferred to scintillation vials containing 10 ml Bray’s (1) scintillation fluid and radioactivity determined in a Packard Liquid Scintillation Spectrometer Model 3310. The results are expressed as nanomoles of sugarlmg cells. The apparent K,,,for the transport of 2-DOG was determined graphically by the method of Lineweaver & Burk (6). The values of K, for different sugars which competitively inhibited 2-DOG uptake were calculated from Lineweaver-Burk plots using the equation of Brown & Romano (2) : K, = I/(Kp/Km- 1) where I represents the concentration of the inhibitor, and Kp and K, are values calculated from the points where the lines obtained in the presence and absence of the
L. tropica:
1.1
1.0
THE
GLUCOSETRANSPORT SYSTEM
447
I .
Y
z
-I
0.9
'
0.8
'
!/RT Xld'
1 I
0 -1 0
0
10 20 1/S (MM)
30
0
10 20 30 40 0 10 20 30 40 I l S [2-DOG (uM)] 3
2
Fig. 1. Arrhenius plot of uptake rates between 26 and 32 C. K, velocity of uptake expressed as nmoles/mg/2 min; R, universal gas constant; T, absolute temperature. Figs. 2, 3. Lineweaver-Burk plots of the inhibition of 2-deoxy-~glucose(2-DOG) by various sugars. Cells were incubated with [v-uC] 2-DOG (0.2 pCi/pmole) at the indicated concentrations. Up.take of 2-DOG was followed in the absence or presence of the inhibiting sugars, each at a 0.1 mM concentration. The results are given as the initial rates of uptake expressed as nanomoles/mg/min, at each 2-DOG 0 , 2-DOG; 0-0, 2-DOG N-acetyl glucosamine. 3. Inhibiconcentration. 2. Inhibition by N-acetyl glucosamine. 00 , 2-DOG alone; 00, 2tion by galactose (A), glucosamine (B), mannose (C), and fructose (D). In all plots, 0DOG inhibiting sugar.
+
+
inhibitor intercept the X axis. These points represent negative reciprocals of Kp and K,,,respectively. Source of Chemicals [14C]2-DOG was purchased from International Chemical and Nuclear Co., Irvhe, California. a-methyl D-glucoside, 3-0-methyl glucose and 1,5-anhydro-~-gluutolwere obtained from Schwanl Mann, Orangeburg, New York. All other sugars were purchased from Sigma Chemical Co., St. Louis, Missouri and Nutritional Biochemicals, Cleveland, Ohio. RESULTS The Glucose Carrier.-Maximal uptake occurred at 30-32 C beyond which the rate of uptake dropped precipitously. An Arrhenius plot of the rates at 26 to 32 C is shown in Fig. 1. From the slope of this plot, a heat of activation of 10.2 kcal/
mole !&DOG, and a Qlo of 2 for the system were obtained. These values are in the range expected of enzyme mediated reactions and further support the idea of carrier involvement in 2-DOG entry into the cell which we reported earlier (14). Optimal rates of uptake were observed at around pH 7.0. Specificity of the Carrier.-The results of competition experiments in which the uptake of 2-DOG was determined in the presence of several other sugars and sugar derivatives are shown in Table 1. None of the pentose sugars tested (D-ribose, D-arabinose, D-xylose and D-lyxose) had any effect on 2-DOG uptake. The frequently used glucose analogs, a-MG and 3-0-MG or the L-isomer of D-glucose did not inhibit 2-DOG uptake. Similarly, 1,5-anhydro-n-glucitol ( 1-deoxy-D-glucose) , fucose, rhamnose and sorbose were without appreciable effect on the uptake of this glucose analog. O n the other hand, galactose, glucosamine, N-acetyl glucosamine, mannose, and fructose lowered its uptake significantly. It is evident from Lineweaver-Burk plots
448
L. tropica:
THE
GLUCOSETRANSPORT SYSTEM
TABLE1. Effect of different sugars on the uptake of 2-DOG.'
sugar D-glucose D-glUCOSamine N-acetyl glucosamine D-mannose D-galactose D-fructoset
Carbon (9) at which substituents differ from cl-D-glUCOpyranose structure
n
Z Percent inhibition
Changes
-
-
2
2-amino-2-deoxy 2-N-acetyl-2-deoxy
2 2 4 1,2 and 5
98 44
52
2 axial OH 4 axial OH 1-deoxy, 2 axial OH,
+
63 37
L-glucoset
1 and 5
a-methylaglucoside 3-0-methyl glucose 1,5-anhydro-~glucitol Drhamnose L-sorboset
1
5-(C&OH OH) (anomdric) 1 CHsOH, 50H (anomeric) a-1-0-methyl
3
3-0-methyl
NI
1
1-deoxy
NI
3 and 6 1 and 5
~
72 NIO NI
NI 6-deoxy, 3 axial OH 1-deoxy, 5NI (CHnOH OH) D-fucose NI 4 and 6 4 axial OH, 6-deoxy D-ribose* 3 NI 3 axial OH D-arabinose 2 and 3 2 axial OH, 3 NI axial OH D-XylOSe* 5 NI 5-deoxy D-lyXOSe* NI 2 2 axial OH * Cell suspensions, prepared as described in Materials and Methods, were incubated with 0.1 mM ~C]2-deoxy-~glucose (0.2 pCi/pmole). Nonisotopic sugars tested as competitors were added simultaneously with labeled 2-DOG. All sugars were used at a concentration of 1 mM. The results represent inhibition of uptake during a 10-min incubation period. ?Sugars in the 1C conformation; *sugars in the Cl-~-xylopyranose conformation; % NI, no inhibition.
-10
+
0 10 2 0 3 0 4 0 I/S (MM)
Fig. 4. Lineweaver-Burk plots of the inhibition of z-deoxy-~glucose uptake by glycerol. Cells were incubated with WIZ-DOG (0.2 pCi/pmole) at the indicated concentrations. Uptake of the 0 ) and presence sugar was followed in the absence ( 0(0 0 ) of 1 mM glycerol.
~~~~~
of the inhibition of 2-DOG uptake by all these sugars that they act as competitive inhibitors (Figs. 2, 3). We have already reported competitive reciprocal inhibition of 2-DOG uptake by D-glucose (8, 14). The values of apparent K4 for individual sugars that competitively inhibited 2-DOG uptake were calculated from these data (Table 2). Inhibition of 2-DOG Uptake by Glycerol.-It was thought that addition of glycerol, which is structurally unrelated to glucose, might stimulate the uptake of 2-DOG by providing additional energy, but that it would not compete with 2-DOG for uptake. Accordingly, experiments were carried out in which the uptake of 2-DOG was followed in cells incubated with glycerol for various periods of time ranging from zero to 15 min before the addition of the sugar. In all instances, glycerol proved to be a strong inhibitor of 2-DOG uptake. In experiments using 0.1 mM 2-DOG, the addition of 1 mM glycerol simultaneously with 2-DOG resulted in 90% inhibition. It is evident from Lineweaver-Burk plots of 2-DOG uptake in the presence and absence of glycerol that glycerol acts as a noncompetitive inhibitor (Fig. 4). Cells grown in a medium supplemented with 20 mM glycerol transported 2-DOG nearly as well as the control cells (Fig. 5). Uptake of 2-DOG in both types of cells, however, was severely inhibited by the addition of glycerol to the reaction mixture. Apparently, glycerol does not prevent the synthesis of the glucose carrier (repression) but only affects the overall function of the carrier already present.
DISCUSSION In this and previous publications (8, 14) we have reported that glucose is taken up by L. tropica promastigotes by a mediated transport mechanism. D-glucose and 2-DOG share the same transport carrier which, however, has no affinity for the closely related glucose analogs a-MG and 3-0-MG or for the L-isomer of glucose since they are not transported nor do they inhibit 2DOG uptake. The glucose carrier thus has a certain degree of stereospecificity. The stereochemical requirements for glucose transport in L. tropica resemble those of Trypanosoma gambiense in which Southworth & Read (17) reported that a-MG and 3-0-MG failed to inhibit glucose uptake. Although these investigators did not study the uptake of these glucose analogs (a-MG and 3-0-MG), their inability to inhibit glucose uptake indicates that as in L. tropica, the glucose transport system in T . gambiense has no affinity for them. Glucose uptake in Trypanosoma lewisi (11) and Trypanosoma equiperdum ( 10) is, however, inhibited by 3-0-MG. Since most sugars in solution exist either as open chains or as
TABLE 2. Znhibition of 2-DOG uptake by L. tropica. Sugar Dglucose Dfructose D-mannose N-acetyl glucosamine Dglucosamine Dgalactose
Apparent Kc (mu) 0.08 0.13 0.26
0.46 2.0 2.63 Promastigotes were incubated with labeled 2-deoxy-~-glucose (0.2 pCi/pmole) . Nonisotopic sugars were added at a concentration of 0.1 mM.
L. tropica:
2
4 6 8 1 TIME (MINI
THE
GLUCOSETRANSPORT SYSTEM
0
Fig. 5. Effect of glycerol on 2-DOG uptake. 00, uptake of 2-DOG by cells grown in the absence of glycerol; 00, uptake of 2-DOG by cells grown in the presence of 20 mM glycerol; AA, uptake of 2-DOG in the presence of 1 mM glycerol by cells grown in the absence of glycerol; Aa,uptake of 2-DOG in the presence of 1 mM glycerol by cells grown in the presence of 20 mM glycerol. a variety of furanose and pyranose ring structures ( 3 ) , interpretation of the relationship of transport activity with the steric properties of particular sugars is difficult. D-glucose in solution is known to occur almost entirely in the C1 glucopyranose form (5). Thus it is conceivable that other sugars which in solution come closest to the C1 pyranose form would be the most likely to interact with the glucose transport system. This in fact is true in the case of L. tropica; all sugars that competitively inhibit 2-DOG uptake (glucosamine, N-acetyl glucosamine, mannose, galactose and fructose) are capable of existing in the pyranose form. Aldohexoses differing from glucose by substitutions or changes in configuration at carbon 2 (2-DOG, glucosamine, N-acetyl glucosamine and mannose) act as competitive inhibitors of 2-DOG uptake. Apparently, the extent of changes at carbon 2 in these sugars does not eliminate the ability of the glucose carrier to recognize and bind them. The fact that both glucosamine and N-acetyl glucosamine act as competitive inhibitors of 2-DOG uptake suggests that substitutions at carbon 2 which affect the length of the side chain at least to a limited extent can be tolerated without loss of carrier recognition. Changes a t carbon 1 (a-MG, 1,5-anhydroglucitol) and carbon 3 (3-0-MG) result in loss of carrier affinity since these sugars do not compete with 2-DOG for uptake. As in T. lewisi ( 11) and T. equiperdum ( l o ) , the glucose transport system in L. tropica is subject to competitive inhibition by the keto sugar fructose. Carbon 4 does not seem to contribute substantially to specificity, since 2-DOG uptake is competitively inhibited by galactose which differs from glucose at carbon 4. This is not unique to L. tropica, being true also of T. lewisi (11). In fact, galactose has been shown to bind with the glucose carrier and is presumably taken up by the glucose transport system in a number of eukaryotes ( 7 ) and prokaryotes (9). Fucose, a structural analog of galactose, however, does not affect 2-DOG uptake in L. tropica. Inhibition of hexose transport by the 3-carbon compound, glycerol, in L. tropica (present study) was also observed in T. gambiense (17) and T . equiperdum (10). I n T . gambiense, Southworth & Read ( 17) postulated a common carrier for glucose
449
and glycerol, but in T . equiperdum the 2 were found to have separate transport sites ( 10). Examination of the initial rates of 2-DOG uptake as a function of increasing concentrations of the sugar in the presence and absence of glycerol (Fig. 4 ) suggested a noncompetitive mode of inhibition in L. tropica and that glucose and glycerol do not share the same carrier. Glycerol does not affect the K,,,for 2-DOG (0.083 mM) but lowers the V,,,,, (from 3.1 nmoles/mg/min to 1.3 nmoles/mg/min). Inhibition by glycerol is thus not caused by competition for a common carrier nor by changes in the affinity of the carrier. It is possible that some product of glycerol metabolism has a regulatory role and exerts an inhibitory effect on glucose uptake. An alternative possibility is that glycerol enhances the rate of efflux of intracellular sugar causing an apparent reduction in net uptake. This would be analogous to the situation in Escherichia coli, where a number of metabolizable substrates were shown to increase the efflux of a nonmetabolizable sugar, a-MG (4). Cells grown in the presence of 20 mM glycerol transport 2-DOG just as well as those grown without glycerol; this finding rules out repression of the glucose transport system by glycerol. ACKNOWLEDGEMENTS We thank Dr. Geetha Bhat, Department of Chemistry, University of Cincinnati, for helpful comments in the preparation of this manuscript. The technical help of Michael Simon and Peggy Lepley is gratefully acknowledged. REFERENCES 1. Bray GA. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1, 279-85. 2. Brown CE, Romano AH. 1969. Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans. J. Bacteriol. 100, 1198-203. 3. Cirillo VP. 1968. Relationship between sugar structure and competition for the sugar transport system in baker's yeast. J. Bacteriol. 95, 603-11. 4. Hagihira H, Wilson TH, Lin EC. 1963. Studies on the glucose transport system in Escherichia coli with a-methylglucoside as substrate. Biochim. Biophys. Acta 78, 505-15. 5. Isbell HS, Tipson RS. 1959. A nomenclature for conformations of pyranoid sugars and derivatives. Science 130, 793-4. 6. Lineweaver H, Burk D. 1934. The determination of enzyme dissociation constants. J. A m . Chem. SOC.56, 658-66. 7. Mark C, Romano AH. 1971. Properties of the hexose transport systems of Aspergillus nidulans. Biochim. Biophys. Acta 249, 216-26. 8. Mukkada AJ, Schaefer FW 111, Simon MW, Neu C. 1974. Delayed in uitro utilization of glucose by Leishmania tropica promastigotes. J . Protozool. 21, 393-7. 9. Rogers D, Yu S. 1962. Substrate specificity of a glucose permease of Escherichia coli. J. Bacteriol. 84, 877-81. 10. Ruff MD, Read CP. 1974. Specificity of carbohydrate transport in Trypanosoma equiperdum. Parasitology 68, 103-15. 11. Sanchez G, Read CP. 1969. Carbohydrate transport in Trypanosoma lewisi. Comp. Biochem. Physiol. 28, 93 1-7. 12. Schaefer FW 111, Mukkada AJ. 1974. Characteristics and specificity of hexose transport in Leishmania tropica promastigotes. Proc. 3rd Znt. Congr. Parasitol., Munich, 1974. Facta Publications, H. Egermann, Vienna, 3, 1461. , Bell EJ, Etges FJ. 1970. Leishmania tropica: 13. chemostatic cultivation. Exp. Parasitol. 28, 465-72. , Martin E, Mukkada AJ. 1974. The glucose trans14. port system in Leishmania tropica promastigotes. J . Protozool. 21, 592-6. 15. Simon MW, Rusnak JM, Mukkada AJ. 1976. Toxicity of bilirubin to Leishmania tropica promastigotes. Exp. Parasitol. 39, 51-8. 16. Sorouri P. 1955. The nuclear cytology of Leishmania tropica. J. Morphol. 97, 393-414. 17. Southworth GC, Read CP. 1970. Specificity of sugar transport in Trypanosoma gambiense. J . Protozool. 17, 396-9.
