BiochemicalGenetics, VoL 16, Nos. 9/10, 1978
Genetic Control of Nucleoside Transport in Sheep Erythrocytes S. M. Jarvis 1 and J. D. Young ~
Received 9 Feb. 1978--Final20 Apr. 1978
Nucleoside transport in sheep erythrocytes is under the genetic control of two allelomorphic genes (Nu I and Nui), where Nu I codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene (Nu i) coding for the presence of the transport system. Kinetic and inhibitor experiments show that the high-affinity transport system is not present in heterozygous erythrocytes, demonstrating that the Nu ~gene is completely dominant over the N u i gene. It is suggested that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus. KEY WORDS: nucleoside; transport; sheep; erythrocyte. INTRODUCTION The major route for nucleoside transport across the erythrocyte membrane is by facilitated diffusion, and studies of the h u m a n erythrocyte suggest the presence of a single broad-specificity transport system for both purine and pyrimidine nucleosides (e.g,, see Berlin and Oliver, 1975). In contrast to h u m a n erythrocytes, sheep erythrocytes are generally impermeable to nucleosides. There is, however, a small number of animals whose erythrocytes rapidly transport nucleosides (Young, 1978). This variation in permeability between sheep is due to the presence of a high-affinity nucleoside transport system. The characteristics of this system are very similar to those of nucleoside transport in human erythrocytes, although h u m a n cells have a higher S. M. J. is the recipient of an MRC postgraduate studentship. 1ARC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, England. 1035 0006-2928/78/1000-1035505.00/0 © 1978 Plenum Publishing Corporation
1036
Jarvis and Young
maximum velocity. A comparison of the intracellular ATP concentrations of nucleoside-permeable and -impermeable sheep erythrocytes suggests that this transport system may play a significant role in the energy metabolism of the cell (see also Young, 1978). The present investigation demonstrates that the variation in nucleoside transport in sheep erUthrocytes is genetically controlled. MATERIALS AND METHODS Whole blood samples were obtained from adult sheep by jugular venipuncture into heparinized evacuated tubes. All animals were maintained under standard husbandry conditions. Materials
Uniformly labeled [14C]inosine and [14C]uridine were obtained from the Radiochemical Centre, Amersham, Bucks., U.K. [~4C]Uridine was purified on Polygrarn 0.25-mm silica gel thin-layer chromatography plates (Camlab, Cambridge, U.K.) using butan-l-ol saturated with water as solvent. Nonradioactive inosine and uridine were purchased from Sigma (London) Chemical Co. Ltd., Kingston-upon-Thames, Surrey, U.K. Nitrobenzylthioinosine (6-[4-nitrobenzyl)thio]-9-fl-I~-ribofuranosylpurine, NBMPR) was a generous gift from Professor A. R. P. Paterson, University of Alberta Cancer Research Unit, Edmonton, Canada. Nucleoside Transport Studies
Erythrocytes were washed three times with 10 vol of a medium containing 140 mM NaC1, 5 mM KC1, 20 mM tris-HC1 (pH 7.2 at 37 C), 2 mM MgCI2, 0.1 mU diaminoethanetetraacetic acid (disodium salt), and 5 mM glucose. The buffy coat was discarded. The hemoglobin content of cell suspensions was determined by absorbance measurements of lysates (1 : 50 dilution with water) at 540 nm and correlated with the volume of cells in the original suspension using an experimentally determined extinction coefficient for packed erythrocytes of 290 cm- 1. There was no significant difference in the value of this coefficient for nucleoside-permeable and -impermeable cells. Initial rates of nucleoside uptake were measured at 37 C as previously described (Young, 1978) by mixing 0.2 ml of prewarmed washed erythrocytes (hematocrit approximately 20%) with 0.2 ml of prewarmed medium containing the appropriate 14C-labeled nucleoside (0.1-0.5 #Ci//~mole). In the case of inhibitor studies this solution also contained NBMPR (final concentration
Nucleoside Transport in Sheep Erythrocytes
1037
0.01-1.25 #M). At predetermined time intervals, incubations were stopped by the addition of 1 ml ice-cold medium, and the cells were rapidly washed four times with 1 ml of ice-cold medium using an Eppendorf 3200 microcentrifuge (10 sec, 15,000g). The washed packed cells were lysed with 0.5 ml of 0.5% (v/v) Triton X-100 in water, and 0.5 ml of 5~ (w/v) trichloroacetic acid was added. The precipitate was removed by centrifugation (30 sec, 15,000g), and 0.9 ml of the supernatant was transferred to 7 ml of scintillation fluid (0.1 g, 1,4-bis[2-(5phenyloxazolyl)]benzene (POPOP), 5 g of 2,5-diphenyloxazole (PPO), and 480 ml of Triton X-100 in 1 liter of toluene). Samples were counted for radioactivity in a Packard Tricarb scintillation counter with quench correction. Incubation times (3 rain and 30 rain for nucleoside-permeable and -impermeable cells, respectively) were chosen such that the maximum intracellular concentration did not e'xceed 20~ of the corresponding extracellular concentration. Control experiments established that time courses were linear. RESULTS In a survey of 225 sheep comprising several different breeds it was found that 5~ of the animals were nucleoside permeable (Young, 1978). The Finnish Landrace breed, with eight nucleoside-permeable animals from a total of 95, had the highest frequency of nucleoside-permeable sheep. The pedigrees of these eight Finnish Landrace animals are shown in Fig. 1A. The numbers refer to the initial rates of inosine transport (mmoles per liter of cells per hour at 5 mM extracellular inosine). The permeability characteristics of erythrocytes from individual animals remained constant during repeated analyses over a period of 2 years. The nucleoside-permeable sheep were the progeny of two nucleoside-impermeable rams and six ewes, two of which were themselves nucleoside permeable. Both rams, when mated to other nucleoside-impermeable ewes, produced nucleoside-impermeable offspring (data not shown). Figure 1B shows the results of an experimental series of crosses in which a nucleoside-permeable ram was mated to two nucleoside-permeable and five unrelated nucleoside-impermeable ewes. The progeny from the nucleosidepermeable x permeable matings were all nucleoside permeable whereas the other progeny were nucleoside impermeable. These inheritance data, although limited, demonstrate that nucleoside transport in sheep erythrocytes is under genetic control, nucleo side impermeability behaving as if dominant to nucleoside permeability. The results are consistent with the involvement of two allelomorphic genes (Nu ~ and Nu'), where Nu I codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene coding for the presence of this system (Nu'). Thus nucleoside-impermeable animals are either homozygous
Jarvis and Young
1038
A
0-04
0"17 2'0
2'0 0,12
2-2 0"05
2.4 2-6
2"3
0"05
B
2.1 1-6 2"3 1.8 1-5 2.4
0.070"05
0-020'03
0.030.04
0-14
0-04
Fig. 1. Inheritance of nucleoside transport variation in sheep erythrocytes. A: Family pedigrees of nucleoside-permeable Finnish Landrace sheep. B: Results of an experimental series of crosses in which a nucleoside-permeable ram from A was mated to two nucleoside-permeable ewes (also from A) and five unrelated nucleoside-impermeable ewes. Nucleoside-impermeable rams and ewes are identified as [] and o, respecti?ely. Nucleoside-permeable animals are represented by [] and o. The numbers refer to initial inosine uptake rates in mmole per liter of cells per hour (5 mM extracellular inosine). Three of the original ewes in A and a number of the subsequent offspring were not available for typing.
(Nu I, Nu t) or heterozygous (Nu I, Nu') for the gene specifying nucleoside impermeability whereas nucleoside-permeable animals are homozygous (Nu i, Nu i) for the gene coding for nucleoside permeability. To test for complete dominance of the Nu I gene, the rate, inhibitor susceptibility, and kinetic characteristics of nucleoside transport in heterozygous cells were investigated in detail. Presumed heterozygotes were identified as nucleoside-impermeable animals which either gave rise to nucleoside-permeable offspring or had one nucleoside-permeable parent. Figure 2 compares the inosine permeability (at 5 m i extracellular inosine) of erythrocytes from 14 presumed heterozygotes with that of cells from 28 randomly selected nucleoside-impermeable animals [0.055 +0.008 (14) and 0.063+0.008 (28)
Nucleoside Transport in Sheep Erythrocytes
1039
15
10
I
I
!
I
0
0,1
0.2
1'4
Nucleoside
Ill
I I - " q F'I I
1'8
I 2-2
Nucleoside-permeable
-
impermeable
Uptake
(mmol/litre
cells
p e r h)
Fig. 2. Inosine uptake by erythrocytes from presumed heterozygotes, randomly selected nucleoside-impermeableanimals and nucleoside-permeable
sheep. Inosineuptake was measuredat 5 mMextracellularinosine.Presumed heterozygotes(hatched areas) were identifiedas described in the text. mmole per liter of cells per hour (mean_+ SEM (n) for the heterozygous and random nucleoside-impermeable groups, respectively (difference not significant by Student's t test)]. For comparison, nucleoside-permeable sheep gave an uptake rate of 1.93 _+0.08 (13) mmoles per liter of cells per hour. In another experiment the uridine permeability (at 1 mM extracellular uridine) of erythrocytes from eight presumed heterozygotes was compared with that of cells from ten randomly selected nucleoside-impermeable animals and six nucleosidepermeable sheep. As with inosine, there was no significant difference in uptake rate between the two nucleoside-impermeable groups [0.039 + 0.004 (8) and 0.037 ± 0.003 (10) mmole per liter of cells per hour for the heterozygous and random groups, respectively]. The nucleoside-permeable animals gave an uptake rate of 2.14 ±_0.13 (6) mmoles per liter of cells per hour. S-Substituted 6-thiopurine ribonucleosides are potent inhibitors of nucleoside transport in a variety of cell types (e.g., see Berlin and Oliver, 1975). Figure 3 shows the effect of NBMPR (0.01-1.25 #M) on uridine uptake (extracellular concentration 1 raM) by heterozygous nucleoside-impermeable erythrocytes and cells from a nucleoside-permeable animal. NBMPR was an effective inhibitor of uridine uptake by nucleoside-permeable erythrocytes (50% inhibition at 0.025 #M). In contrast, uridine uptake by heterozygous cells was not inhibited, even at high NBMPR concentrations. Figure 4 shows the concentration dependence of uridine uptake (0.1-7.6 raM) by erythrocytes from a presumed heterozygote compared with cells from
1040
Jarvis and Young
A e-
~- 2.4
0"048
¢o
u
0
%
1"6
O
0
O_o oo
0"032
o o
E E ® 0"8 .,:¢
0"016
% ~
e
~
,
,
0"2
0-4
e ,
0
1-25
NBMPR (pM)
Fig. 3. Effect of NBMPR on uridine uptake by heterozygous nucleoside-impermeable and nucleoside-permeable erythrocytes. Uridine uptake at an extracellular concentration of 1 mM was determined as described in the text. [14C]Uridine and N BMPR were added simultaneously to heterozygous nucleoside-impermeable (o) (right-hand ordinate) and nucleosidepermeable (e) (left-hand ordinate) erythrocytes.
a nucleoside-permeable animal. Uptake by the heterozygote was linear (0.036 mmole per liter of cells per hour per mM) compared with the saturable uptake curve given by the nucleoside-permeable erythrocytes (apparent Km 0.48 mM, Vmax3.94 mmoles per liter of cells per hour). Figure 4 also shows that in the presence of NBMPR (25 gM) the concentration dependence of uridine uptake by nucleoside-permeable cells was linear, in contrast to the saturable curve observed in its absence. The magnitude of this linear transport (0.032 mmole per liter of cells per hour per mM) was similar to that of nucleoside-impermeable cells. DISCUSSION The present results demonstrate that nucleoside permeability in sheep erythrocytes is under the simple genetic control of two allelomorphic genes (Nu ~, Nu'), with the gene for nucleoside impermeability (Nu t) behaving as if dominant to the gene for nucleoside permeability (Nu'). Thus nucleosideimpermeable animals are either homozygous or heterozygous for the gene specifying nucleoside impermeability. The genetic hypothesis predicts that nucleoside-impermeable animals
Nucleoside Transport in Sheep Erythroeytes
1041
A
i
21
0-24 0.16
E ®
oi 0
° , 2 4 Uridine (mM)
o 6
Fig. 4. Concentration dependence of uridine uptake by heterozygous nucleoside-impermeable and nucleoside-permeable erythrocytes. The saturable uptake curve by nucleoside-permeable erythrocytes (o) (left-hand ordinate) was fitted as v (mmoles per liter of cells per hour) = 3.94s/(0.48 +s), where s is the extracellular uridine concentration (mM). The Vmax (3.94 mmoles per liter of cells per hour) and Km (0.48 mM) values were determined from linear regression analysis of s/v vs. s. Uptake by heterozygous cells (o) and nucleoside-permeable cells in the presence of 25/aM NBMPR (m) (both right-hand ordinate) was linear (0.036 and 0.032 mmole per liter of cells per hour per n ~ , respectively).
which either give rise to nucleoside-permeable offspring or have one nucleoside-permeable parent must be heterozygous for the Nu i gene. The inosine and uridine permeability of such presumed heterozygotes was not significantly different from that of a random group of nucleoside-impermeable animals. Since the majority of the latter group must have been homozygous for the Nu 1 gene, the data suggest that the nucleoside transport rates of heterozygous (Nu 1, Nu') and homozygous (Nu 1, Nu I) nucleoside-impermeable cells are the same. Previous studies have shown that the concentration dependence of inosine and uridine uptake by nucleoside-permeable erythrocytes conforms to simple Michaelis-Menten kinetics, with apparent Km values of 0.26 and 0.47 raM, respectively (Young, 1978). In contrast, nucleoside-impermeable cells show a slow linear concentration dependence. The present results confirm the saturable nature of uridine uptake by nucleoside-permeable erythrocytes (apparent Km 0.48 mM) and further demonstrate that heterozygous cells show a linear concentration dependence. Furthermore, uridine uptake by such
1042
Jarvis and Young
heterozygous cells is not affected by NBMPR, a potent inhibitor of nucleoside transport systems in a variety of cells including nucleoside-permeable sheep erythrocytes. These transport studies therefore demonstrate that the highaffinity nucleoside transport route is not functional, even in a very low activity, in heterozygous cells. Thus the Nu t gene is completely dominant over the Nu i gene. In the presence of NBMPR at a concentration a thousandfold above its Ki value, the concentration dependence of uridine uptake by nucleoside-permeable erythrocytes was linear and similar in magnitude to that of nucleosideimpermeable cells, demonstrating that nucleoside-permeable erythrocytes possess both the high- and low-affinity uptake routes. This eliminates the possibility that the Nu I gene codes for a modified carrier which has a high Km and is insensitive to NBMPR, but rather suggests that the gene codes for the functional absence of the nucleoside transport system. The initial rate (v) of nucleoside uptake by permeable erythrocytes is therefore more precisely described by the relationship v=
Vmax " S
-+k's Km+ s
rather than by the simple Michaelis-Menten equation alone. However, for both uridine and inosine, k ~ Vmax/Km SOthat the Michaelis-Menten equation is a good approximation to the experimental data, and allowance for the linear uptake component has only a small effect on the estimated kinetic constants for the high-affinity transport system. Transport by the low-affinity route probably represents simple diffusion through the lipid bilayer (Young, 1978). It is interesting that nucleoside impermeability behaves as if dominant to nucleoside permeability. This possibly suggests that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus that either is directly capable of modifying the expression of the structural gene or indirectly influences nucleoside transport by coding for the synthesis of a specific inhibitor of the transport system. Indeed, the genetic control of nucleoside transport in sheep erythrocytes may be analogous to that responsible for the regulation of potassium transport in sheep erythrocytes, where the L blood group antigen acts as an inhibitor of the potassium pump (see Ellory, 1977, for a recent review). It is hoped that further investigation of the genetic control of nucleoside transport variation in sheep erythrocytes will help elucidate the molecular mechanism by which nucleosides cross the cell membrane. ACKNOWLEDGMENTS
We wish to thank Mr. D. A. Fincham for his skilled and enthusiastic help. We
Nucleoside Transport in Sheep Erythrocytes
1043
a r e also g r a t e f u l to Dr. E. M. T u c k e r for h e r c o n t i n u e d a d v i c e a n d e n c o u r a g e ment.
REFERENCES Berlin, R. D., and Oliver, J. M. (1975). Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. Int. Rev. Cytol. 42:49. Ellory, J. C. (1977). Ion transport in ruminant red cells. In Transport in Red Cells, Ellory, J. C., and Lew, V. L. (eds.), Transport in Red Cells, Academic Press, London. Young, J. D. (1978). Nucleoside transport in sheep erythrocytes: Genetically controlled transport variation and its influence on erythrocyte ATP concentrations. J. Physiol. 277:325.
Genetic Control of Nucleoside Transport in Sheep Erythrocytes S. M. Jarvis 1 and J. D. Young ~
Received 9 Feb. 1978--Final20 Apr. 1978
Nucleoside transport in sheep erythrocytes is under the genetic control of two allelomorphic genes (Nu I and Nui), where Nu I codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene (Nu i) coding for the presence of the transport system. Kinetic and inhibitor experiments show that the high-affinity transport system is not present in heterozygous erythrocytes, demonstrating that the Nu ~gene is completely dominant over the N u i gene. It is suggested that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus. KEY WORDS: nucleoside; transport; sheep; erythrocyte. INTRODUCTION The major route for nucleoside transport across the erythrocyte membrane is by facilitated diffusion, and studies of the h u m a n erythrocyte suggest the presence of a single broad-specificity transport system for both purine and pyrimidine nucleosides (e.g,, see Berlin and Oliver, 1975). In contrast to h u m a n erythrocytes, sheep erythrocytes are generally impermeable to nucleosides. There is, however, a small number of animals whose erythrocytes rapidly transport nucleosides (Young, 1978). This variation in permeability between sheep is due to the presence of a high-affinity nucleoside transport system. The characteristics of this system are very similar to those of nucleoside transport in human erythrocytes, although h u m a n cells have a higher S. M. J. is the recipient of an MRC postgraduate studentship. 1ARC Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, England. 1035 0006-2928/78/1000-1035505.00/0 © 1978 Plenum Publishing Corporation
1036
Jarvis and Young
maximum velocity. A comparison of the intracellular ATP concentrations of nucleoside-permeable and -impermeable sheep erythrocytes suggests that this transport system may play a significant role in the energy metabolism of the cell (see also Young, 1978). The present investigation demonstrates that the variation in nucleoside transport in sheep erUthrocytes is genetically controlled. MATERIALS AND METHODS Whole blood samples were obtained from adult sheep by jugular venipuncture into heparinized evacuated tubes. All animals were maintained under standard husbandry conditions. Materials
Uniformly labeled [14C]inosine and [14C]uridine were obtained from the Radiochemical Centre, Amersham, Bucks., U.K. [~4C]Uridine was purified on Polygrarn 0.25-mm silica gel thin-layer chromatography plates (Camlab, Cambridge, U.K.) using butan-l-ol saturated with water as solvent. Nonradioactive inosine and uridine were purchased from Sigma (London) Chemical Co. Ltd., Kingston-upon-Thames, Surrey, U.K. Nitrobenzylthioinosine (6-[4-nitrobenzyl)thio]-9-fl-I~-ribofuranosylpurine, NBMPR) was a generous gift from Professor A. R. P. Paterson, University of Alberta Cancer Research Unit, Edmonton, Canada. Nucleoside Transport Studies
Erythrocytes were washed three times with 10 vol of a medium containing 140 mM NaC1, 5 mM KC1, 20 mM tris-HC1 (pH 7.2 at 37 C), 2 mM MgCI2, 0.1 mU diaminoethanetetraacetic acid (disodium salt), and 5 mM glucose. The buffy coat was discarded. The hemoglobin content of cell suspensions was determined by absorbance measurements of lysates (1 : 50 dilution with water) at 540 nm and correlated with the volume of cells in the original suspension using an experimentally determined extinction coefficient for packed erythrocytes of 290 cm- 1. There was no significant difference in the value of this coefficient for nucleoside-permeable and -impermeable cells. Initial rates of nucleoside uptake were measured at 37 C as previously described (Young, 1978) by mixing 0.2 ml of prewarmed washed erythrocytes (hematocrit approximately 20%) with 0.2 ml of prewarmed medium containing the appropriate 14C-labeled nucleoside (0.1-0.5 #Ci//~mole). In the case of inhibitor studies this solution also contained NBMPR (final concentration
Nucleoside Transport in Sheep Erythrocytes
1037
0.01-1.25 #M). At predetermined time intervals, incubations were stopped by the addition of 1 ml ice-cold medium, and the cells were rapidly washed four times with 1 ml of ice-cold medium using an Eppendorf 3200 microcentrifuge (10 sec, 15,000g). The washed packed cells were lysed with 0.5 ml of 0.5% (v/v) Triton X-100 in water, and 0.5 ml of 5~ (w/v) trichloroacetic acid was added. The precipitate was removed by centrifugation (30 sec, 15,000g), and 0.9 ml of the supernatant was transferred to 7 ml of scintillation fluid (0.1 g, 1,4-bis[2-(5phenyloxazolyl)]benzene (POPOP), 5 g of 2,5-diphenyloxazole (PPO), and 480 ml of Triton X-100 in 1 liter of toluene). Samples were counted for radioactivity in a Packard Tricarb scintillation counter with quench correction. Incubation times (3 rain and 30 rain for nucleoside-permeable and -impermeable cells, respectively) were chosen such that the maximum intracellular concentration did not e'xceed 20~ of the corresponding extracellular concentration. Control experiments established that time courses were linear. RESULTS In a survey of 225 sheep comprising several different breeds it was found that 5~ of the animals were nucleoside permeable (Young, 1978). The Finnish Landrace breed, with eight nucleoside-permeable animals from a total of 95, had the highest frequency of nucleoside-permeable sheep. The pedigrees of these eight Finnish Landrace animals are shown in Fig. 1A. The numbers refer to the initial rates of inosine transport (mmoles per liter of cells per hour at 5 mM extracellular inosine). The permeability characteristics of erythrocytes from individual animals remained constant during repeated analyses over a period of 2 years. The nucleoside-permeable sheep were the progeny of two nucleoside-impermeable rams and six ewes, two of which were themselves nucleoside permeable. Both rams, when mated to other nucleoside-impermeable ewes, produced nucleoside-impermeable offspring (data not shown). Figure 1B shows the results of an experimental series of crosses in which a nucleoside-permeable ram was mated to two nucleoside-permeable and five unrelated nucleoside-impermeable ewes. The progeny from the nucleosidepermeable x permeable matings were all nucleoside permeable whereas the other progeny were nucleoside impermeable. These inheritance data, although limited, demonstrate that nucleoside transport in sheep erythrocytes is under genetic control, nucleo side impermeability behaving as if dominant to nucleoside permeability. The results are consistent with the involvement of two allelomorphic genes (Nu ~ and Nu'), where Nu I codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene coding for the presence of this system (Nu'). Thus nucleoside-impermeable animals are either homozygous
Jarvis and Young
1038
A
0-04
0"17 2'0
2'0 0,12
2-2 0"05
2.4 2-6
2"3
0"05
B
2.1 1-6 2"3 1.8 1-5 2.4
0.070"05
0-020'03
0.030.04
0-14
0-04
Fig. 1. Inheritance of nucleoside transport variation in sheep erythrocytes. A: Family pedigrees of nucleoside-permeable Finnish Landrace sheep. B: Results of an experimental series of crosses in which a nucleoside-permeable ram from A was mated to two nucleoside-permeable ewes (also from A) and five unrelated nucleoside-impermeable ewes. Nucleoside-impermeable rams and ewes are identified as [] and o, respecti?ely. Nucleoside-permeable animals are represented by [] and o. The numbers refer to initial inosine uptake rates in mmole per liter of cells per hour (5 mM extracellular inosine). Three of the original ewes in A and a number of the subsequent offspring were not available for typing.
(Nu I, Nu t) or heterozygous (Nu I, Nu') for the gene specifying nucleoside impermeability whereas nucleoside-permeable animals are homozygous (Nu i, Nu i) for the gene coding for nucleoside permeability. To test for complete dominance of the Nu I gene, the rate, inhibitor susceptibility, and kinetic characteristics of nucleoside transport in heterozygous cells were investigated in detail. Presumed heterozygotes were identified as nucleoside-impermeable animals which either gave rise to nucleoside-permeable offspring or had one nucleoside-permeable parent. Figure 2 compares the inosine permeability (at 5 m i extracellular inosine) of erythrocytes from 14 presumed heterozygotes with that of cells from 28 randomly selected nucleoside-impermeable animals [0.055 +0.008 (14) and 0.063+0.008 (28)
Nucleoside Transport in Sheep Erythrocytes
1039
15
10
I
I
!
I
0
0,1
0.2
1'4
Nucleoside
Ill
I I - " q F'I I
1'8
I 2-2
Nucleoside-permeable
-
impermeable
Uptake
(mmol/litre
cells
p e r h)
Fig. 2. Inosine uptake by erythrocytes from presumed heterozygotes, randomly selected nucleoside-impermeableanimals and nucleoside-permeable
sheep. Inosineuptake was measuredat 5 mMextracellularinosine.Presumed heterozygotes(hatched areas) were identifiedas described in the text. mmole per liter of cells per hour (mean_+ SEM (n) for the heterozygous and random nucleoside-impermeable groups, respectively (difference not significant by Student's t test)]. For comparison, nucleoside-permeable sheep gave an uptake rate of 1.93 _+0.08 (13) mmoles per liter of cells per hour. In another experiment the uridine permeability (at 1 mM extracellular uridine) of erythrocytes from eight presumed heterozygotes was compared with that of cells from ten randomly selected nucleoside-impermeable animals and six nucleosidepermeable sheep. As with inosine, there was no significant difference in uptake rate between the two nucleoside-impermeable groups [0.039 + 0.004 (8) and 0.037 ± 0.003 (10) mmole per liter of cells per hour for the heterozygous and random groups, respectively]. The nucleoside-permeable animals gave an uptake rate of 2.14 ±_0.13 (6) mmoles per liter of cells per hour. S-Substituted 6-thiopurine ribonucleosides are potent inhibitors of nucleoside transport in a variety of cell types (e.g., see Berlin and Oliver, 1975). Figure 3 shows the effect of NBMPR (0.01-1.25 #M) on uridine uptake (extracellular concentration 1 raM) by heterozygous nucleoside-impermeable erythrocytes and cells from a nucleoside-permeable animal. NBMPR was an effective inhibitor of uridine uptake by nucleoside-permeable erythrocytes (50% inhibition at 0.025 #M). In contrast, uridine uptake by heterozygous cells was not inhibited, even at high NBMPR concentrations. Figure 4 shows the concentration dependence of uridine uptake (0.1-7.6 raM) by erythrocytes from a presumed heterozygote compared with cells from
1040
Jarvis and Young
A e-
~- 2.4
0"048
¢o
u
0
%
1"6
O
0
O_o oo
0"032
o o
E E ® 0"8 .,:¢
0"016
% ~
e
~
,
,
0"2
0-4
e ,
0
1-25
NBMPR (pM)
Fig. 3. Effect of NBMPR on uridine uptake by heterozygous nucleoside-impermeable and nucleoside-permeable erythrocytes. Uridine uptake at an extracellular concentration of 1 mM was determined as described in the text. [14C]Uridine and N BMPR were added simultaneously to heterozygous nucleoside-impermeable (o) (right-hand ordinate) and nucleosidepermeable (e) (left-hand ordinate) erythrocytes.
a nucleoside-permeable animal. Uptake by the heterozygote was linear (0.036 mmole per liter of cells per hour per mM) compared with the saturable uptake curve given by the nucleoside-permeable erythrocytes (apparent Km 0.48 mM, Vmax3.94 mmoles per liter of cells per hour). Figure 4 also shows that in the presence of NBMPR (25 gM) the concentration dependence of uridine uptake by nucleoside-permeable cells was linear, in contrast to the saturable curve observed in its absence. The magnitude of this linear transport (0.032 mmole per liter of cells per hour per mM) was similar to that of nucleoside-impermeable cells. DISCUSSION The present results demonstrate that nucleoside permeability in sheep erythrocytes is under the simple genetic control of two allelomorphic genes (Nu ~, Nu'), with the gene for nucleoside impermeability (Nu t) behaving as if dominant to the gene for nucleoside permeability (Nu'). Thus nucleosideimpermeable animals are either homozygous or heterozygous for the gene specifying nucleoside impermeability. The genetic hypothesis predicts that nucleoside-impermeable animals
Nucleoside Transport in Sheep Erythroeytes
1041
A
i
21
0-24 0.16
E ®
oi 0
° , 2 4 Uridine (mM)
o 6
Fig. 4. Concentration dependence of uridine uptake by heterozygous nucleoside-impermeable and nucleoside-permeable erythrocytes. The saturable uptake curve by nucleoside-permeable erythrocytes (o) (left-hand ordinate) was fitted as v (mmoles per liter of cells per hour) = 3.94s/(0.48 +s), where s is the extracellular uridine concentration (mM). The Vmax (3.94 mmoles per liter of cells per hour) and Km (0.48 mM) values were determined from linear regression analysis of s/v vs. s. Uptake by heterozygous cells (o) and nucleoside-permeable cells in the presence of 25/aM NBMPR (m) (both right-hand ordinate) was linear (0.036 and 0.032 mmole per liter of cells per hour per n ~ , respectively).
which either give rise to nucleoside-permeable offspring or have one nucleoside-permeable parent must be heterozygous for the Nu i gene. The inosine and uridine permeability of such presumed heterozygotes was not significantly different from that of a random group of nucleoside-impermeable animals. Since the majority of the latter group must have been homozygous for the Nu 1 gene, the data suggest that the nucleoside transport rates of heterozygous (Nu 1, Nu') and homozygous (Nu 1, Nu I) nucleoside-impermeable cells are the same. Previous studies have shown that the concentration dependence of inosine and uridine uptake by nucleoside-permeable erythrocytes conforms to simple Michaelis-Menten kinetics, with apparent Km values of 0.26 and 0.47 raM, respectively (Young, 1978). In contrast, nucleoside-impermeable cells show a slow linear concentration dependence. The present results confirm the saturable nature of uridine uptake by nucleoside-permeable erythrocytes (apparent Km 0.48 mM) and further demonstrate that heterozygous cells show a linear concentration dependence. Furthermore, uridine uptake by such
1042
Jarvis and Young
heterozygous cells is not affected by NBMPR, a potent inhibitor of nucleoside transport systems in a variety of cells including nucleoside-permeable sheep erythrocytes. These transport studies therefore demonstrate that the highaffinity nucleoside transport route is not functional, even in a very low activity, in heterozygous cells. Thus the Nu t gene is completely dominant over the Nu i gene. In the presence of NBMPR at a concentration a thousandfold above its Ki value, the concentration dependence of uridine uptake by nucleoside-permeable erythrocytes was linear and similar in magnitude to that of nucleosideimpermeable cells, demonstrating that nucleoside-permeable erythrocytes possess both the high- and low-affinity uptake routes. This eliminates the possibility that the Nu I gene codes for a modified carrier which has a high Km and is insensitive to NBMPR, but rather suggests that the gene codes for the functional absence of the nucleoside transport system. The initial rate (v) of nucleoside uptake by permeable erythrocytes is therefore more precisely described by the relationship v=
Vmax " S
-+k's Km+ s
rather than by the simple Michaelis-Menten equation alone. However, for both uridine and inosine, k ~ Vmax/Km SOthat the Michaelis-Menten equation is a good approximation to the experimental data, and allowance for the linear uptake component has only a small effect on the estimated kinetic constants for the high-affinity transport system. Transport by the low-affinity route probably represents simple diffusion through the lipid bilayer (Young, 1978). It is interesting that nucleoside impermeability behaves as if dominant to nucleoside permeability. This possibly suggests that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus that either is directly capable of modifying the expression of the structural gene or indirectly influences nucleoside transport by coding for the synthesis of a specific inhibitor of the transport system. Indeed, the genetic control of nucleoside transport in sheep erythrocytes may be analogous to that responsible for the regulation of potassium transport in sheep erythrocytes, where the L blood group antigen acts as an inhibitor of the potassium pump (see Ellory, 1977, for a recent review). It is hoped that further investigation of the genetic control of nucleoside transport variation in sheep erythrocytes will help elucidate the molecular mechanism by which nucleosides cross the cell membrane. ACKNOWLEDGMENTS
We wish to thank Mr. D. A. Fincham for his skilled and enthusiastic help. We
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a r e also g r a t e f u l to Dr. E. M. T u c k e r for h e r c o n t i n u e d a d v i c e a n d e n c o u r a g e ment.
REFERENCES Berlin, R. D., and Oliver, J. M. (1975). Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. Int. Rev. Cytol. 42:49. Ellory, J. C. (1977). Ion transport in ruminant red cells. In Transport in Red Cells, Ellory, J. C., and Lew, V. L. (eds.), Transport in Red Cells, Academic Press, London. Young, J. D. (1978). Nucleoside transport in sheep erythrocytes: Genetically controlled transport variation and its influence on erythrocyte ATP concentrations. J. Physiol. 277:325.
