Vol. 136, No. 3
JouRNAL OF BACTERIOLOGY, Dec. 1978, p. 924-928 0021-9193/78/0136-0924$02.00/0 Copyright @) 1978 American Society for Microbiology
Printedin U.S.A.
Specificity of Nucleoside Transport in Neurospora crassa DEBRA DUNAWAY-MARIANO1 AND JANE M. MAGILL2* Department of Chemistry' and Department of Biochemistry and Biophysics,2 Texas A & M University, College Station, Texas 77843
Received for publication 10 August 1978
The specificity of nucleoside uptake in germinating conidia of Neurospora crassa was investigated by examining the kinetics of [2-14C]uridine and [8-14C]adenosine uptake in the wild-type, ad-8, and ud-1 pyr-1 strains. The results obtained strongly indicate that nucleoside transport in N. crassa is mediated solely by a general transport system which accepts both purine and pyrimidine nucleosides. Studies directed at characterizing the specificity of the transport system indicate that general structural features of the nucleoside which enhance its efficiency in binding to the transport system include: (i) a purine or pyrimidine as the heterocyclic ring, (ii) an unfimctionalized ribose or 2'-deoxyribose as the sugar unit, (iii) a fl-configuration about the anomeric carbon, (iv) the absence of substituents at C8 in the purine series and at C5 and C6 in the pyrimidine series, (v) the presence of a C5-C6 double bond in the pyrimidine series, and (vi) the absence of a charge on the heterocycic ring. Nucleoside analogs are becoming increasingly important as a class of therapeutic agents which vary widely in the biological systems they can affect, as well as in their particular biochemical targets. The design of nucleoside derivatives for biological testing is aided by the ability to make reliable predictions of the rate of entry of the nucleoside analog into various cell types. To this end, emphasis in recent years has been placed on characterization of the mode and specificity of nucleoside transport (1, 7). Earlier studies (9) of nucleoside transport in germinating Neurospora crassa conidia suggested that two transport systems may be operative, one which transports purine and pyrimidine nucleosides and another which specifically transports purine nucleosides. More recent investigations (6) in this area have indicated that nucleoside uptake by germinating conidia of the ad-8 strain is mediated by a single transport system which accepts both purine and pyrimidine nucleosides. No evidence of a transport system specific for purine nucleosides was found in that mutant strain. In the present study, nucleoside transport in germinating conidia of wild-type, ad-8, and udI pyr-1 strains of N. crassa was examined. Evidence for the existence of a single nucleoside transport system in germinating wild-type conidia is presented, and the structural features of the nucleoside substrates required for uptake are described. MATERIALS AND METHODS Chemicals. [8-14C]adenosine and [2-14C]uridine were purchased from New England Nuclear Corp. The
nucleosides a-D-cytidine and 4-thiouridine were obtained from P-L Biochemicals; 1-benzylinosine, N6(A2-isopentenyl)adenosine, and 2',3',5'-triacetyluridine were obtained from Aldrich Chemical Co.; 3-deazauridine and 6-methyluridine were a kind gift from the National Cancer Institute. 3-Isoadenosine (5) and N4methylcytidine (4) were prepared by known methods. The remainder of the nucleosides used were purchased from Sigma Chemical Co. Neurospora strains. Wild-type N. crassa strain 74A and the ad-8 strain were used in all experiments to test the inhibition of [8-14C]adenosine uptake. These strains, together with the mutant strain ud-1 pyr-1, were obtained from the Fungal Genetics Stock Center, Humboldt College, Arcata, Calif. All strains were maintained on slants of Vogel minimal medium (14) supplemented with 200 mg of adenine or uracil per liter. Transport assay. Conidia from 5- to 7-day cultures were harvested in Vogel liquid minimal medium (14) containing 200 mg of adenine or uracil per liter (pH 6.0). The conidial suspension was filtered through sterile cheesecloth and incubated with stirring at 30°C for 5 h. The conidia were then suspended in Vogel minimal medium at a concentration of 4 x 10' conidia per ml (about 250 ug/ml, dry weight). After 1 h of incubation in Vogel minimal medium, 2.0 ml of conidial suspension was added to tubes containing [8-14C]adenosine, Vogel minimal medium, and the nucleoside to be tested. The rate of adenosine uptake was found to be linear for 40 min at 2MuM [8-_4C]adenosine under these conditions. After an incubation period of 5 min (during which time efflux was negligible), the tube contents were rapidly filtered through a fiber glass filter (Reeve-Angel Div., Whatman Inc.) and immediately washed with 10 ml of Vogel minimal medium (0WC). The filters containing the conidial residue were dried, added to 10 ml of Omnifluor liquid scintillation fluid (New England Nuclear Corp.), and then counted 924
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SPECIFICITY OF NUCLEOSIDE TRANSPORT
VOL. 136, 1978
with a Beckman liquid scintillation counter. The apparent Ki values for the nucleoside inhibitors were determined from Dixon plots by using linear regression analysis to determine the line of best fit in each case.
RESULTS Initial efforts at characterizing the specificity of nucleoside transport in germinating conidia of wild-type N. crassa were focused on the kinetics of purine and pyrimidine nucleoside uptake in conidia germinated to the stage where the activities of the purine-specific and common nucleoside transport system were proposed (9) to overlap maximally. Accordingly, the initial velocities of [8-14C]adenosine and [2-'4C]uridine transport were determined at several nucleoside concentrations. The results obtained are represented in the Lineweaver-Burk plots shown in Fig. 1. From these plots the apparent Km values for uridine and adenosine were determined to be 12 and 8 uM, respectively. Uridine and adenosine were tested as inhibitors of [8-14C]adenosine and [2-14C]uridine transport, respectively. Both nucleosides behaved as inhibitors. From the Dixon plots shown in Fig. 2, the apparent Ki values of adenosine and uridine were determined to be 11 and 10 ,LM, respectively. Additional evidence in support of a single transport system is provided from investigations of the mutant strain ud-1 pyr-1, which was reported (15) to lack uridine transport capacity.
Accordingly, the rates of [8-'4C]adenosine and [2-14C]uridine uptake by this mutant were measured. As can be seen in Fig. 3, uridine transport was negligible. Importantly, the adenosine uptake by this mutant was also insignificant. Specificity of nucleoside transport. The specificity of nucleoside transport in germinated conidia of N. crassa was investigated by measuring the degree of inhibition of [8-'4C]adenosine uptake by structural analogs. The apparent Ki values of the inhibitors, determined from Dixon plots, are reported in Table 1. All nucleoside analogs showing inhibition behaved as competitive inhibitors. This was indicated by the fact that, when the data were plotted as slope (from the Dixon plot) versus 1/[S], a straight line through the origin was obtained (11). DISCUSSION Previous studies (9) have demonstrated that nucleoside uptake by germinated conidia of N. crassa strain 74A is an energy-dependent, mediated process. Two different types of transport systems were reported (9) to be operating in the germinating conidia, one specific for purine nucleosides and the other a general system, transporting both purine and pyrimidine nucleosides. In more recent investigations (6) with germinated conidia of the ad-8 strain of N. crassa, we found evidence for only a single transport system.
a 4.5-
0
I
ao |
v
, 0
1.5
a(* -0.1
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01
0.2
1/ (ADEN OS IN
03
0.4
-0.02
0
002
o04
0.08
0.06
E),q(M)
-I
1/
UR IDINE () M I,
FIG. 1. Lineweaver-Burk plots of initial velocities of transport of [8-14C]adenosine (1.0 M&Ci/umol) (a) and [2-'4Cluridine (1.0 ACi/prnol) (b) uptake by germinating conidia of wild-type strain 74A. The apparent Km values are approximately 8 for adenosine and 12 pzM for uridine. Velocity is expressed as nanomoles of adenosine or uridine transported in 5 min by 8 x 106 conidia.
J. BACTERIOL.
DUNAWAY-MARIANO AND MAGILL
926
a
12D 0
X
V 10
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0/ /V /X K 4.0
0
-10
10
0
20
30
40
0 -8 8 16 LA BE LED AODENO SINE. (UN E IM (1U R I IDNI
24
32
M
FIG. 2. Dixon plots of initial velocities of transport of 4 (0) and 8 AiM (0) [8-'4C]adenosine (1.0 liCi/pmol) in the presence of unlabeled adenosine (a) and uridine (b) by germinating conidia ofwild-type strain 74A. The apparent K, values are 11 uM for adenosine and 10 uU for uridine. Velocity is expressed as nanomoles of adenosine or uridine transported in 5 min by 8 x 106 conidia.
The present studies were designed to determine the number of different transport systems operative in germinating conidia of wild-type N. crassa strain 74A. In particular, the kinetics of adenosine and uridine transport in wild-type conidia of strain 74A, germinated to the stage where the activities of the purine-specific and common nucleoside transport systems were proposed (9) to overlap maximally, were investigated. Results from this study have shown that the apparent Km for adenosine uptake is exceptionally close to the value of the apparent Ki for adenosine as an inhibitor of uridine transport. Similarly, the apparent Km for uridine transport is very close to the value of the apparent Ki C= determined for uridine as an inhibitor of adeno2 6 2 0/ 4£ sine uptake. Furthermore, under identical conditions, uridine and adenosine display the same efficiency in inhibiting [8-14C]adenosine transport. These results strongly suggest that a single transport system, transporting both purine and pyrimidine nucleosides, is operative in these conidia and that a purine-specific transport system is not. This proposal is further evidenced by the observed inability of the germinating conidia of FIG. 3. Time course of [8.14C]adenosine (0) and [2Y1C]uridine (A) uptake by 8 x 106 germninating the ud-l pyr-1 strain to transport both adenosine and uridine. conidia of wild-type strain 74Aand [8-14C]adenosine Specificity of nucleoside transport in N. (t4) uptake by 8 x 106 germi(0) and [2.'4C]uridine nating conidia of strain ud-1 pyr-1. The adenosine crasa. The specificity of nucleoside transport in N. crassa was examined by testing nucleoside and uridine concentrations used were 10 W. 3.0
0--ft ~ ~ ~
~
~
SPECIFICITY OF NUCLEOSIDE TRANSPORT
VOL. 136, 1978
TABLE 1. Apparent Ki values for nucleoside analogs as inhibitors of [8-14C]adenosine uptakea Inhibitor
Strain
Apparent K5 (AIM)
Showdomycin Virazole Purine riboside 7-Deazaadenosine 1-Methylguanosine 2'-Deoxyadenosineb Guanosineb Inosineb 6-Mercaptopurine riboside 6-Mercaptoguanosine 6-Methylaminopurine riboside 6-Dimethylaminopurine riboside
ad-8A 74A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A 74A
345 132 36 10 22 15 8 26 24 19 29 49
N6-(A2-isopentenyl)adenosine Cytidine N4-Methylcytidine
74A 53 ad-8A 8 74A 20 Uridineb 11 ad-8A 4-Thiouridine 74A 10 3-Deazauridine 14 74A ad-8A 12 3-Methyluridine Pseudouridine 74A 58 422 Thymidineb ad-8A ad-8A 140 5-Methyl-2'-deoxycytidine 74A 5-Bromo-2'-deoxyuridine 108 5-Fluorouridine ad-8A 18 2'-Deoxyuridineb ad-8A 7 2'-Deoxyguanosine ad-8A 28 2'-Deoxyinosineb 43 ad-8A ad-8A 3'-Deoxyadenosine 845 5'-Azacytidine ad-8A 38 a All apparent Ki values listed were obtained from Dixon plots. The concentration range was 4 to 40 jtM for all inhibitors. Adenine, ,B-D-ribose, AMP, 3'-isoadenosine, 7-methylguanosine, 1-benzylinosine, 8-bromoguanosine, 6-methyluridine, and 2',3',5'-triacetyluridine showed no inhibition when tested using strain 74A. 8-Mercaptoguanosine, 8-bromoadenosine, xanthosine, dihydrouridine, arabinocytidine, 2',3'-isopropylideneuridine, a-cytidine, and 6-azauridine showed no inhibition when tested using strain ad-8A. b The apparent Ki has been reported in an earlier paper (6).
components and nucleoside analogs for their ability to inhibit the uptake of [8-_4C]adenosine. The failure of adenine, ,B-D-ribose, and AMP to inhibit [8-_4C]adenosine uptake indicated that the system under study is in fact specific for nucleosides. The nature of the sugar unit of the nucleoside was found to be quite important in determining the affinity of the nucleosides examined; only those possessing unsubstituted f8D-ribose or 8i-D-2'-deoxyribose sugar moieties were effective inhibitors (Table 1). Furthermore, of the nucleosides tested, those having a complete purine or pyrimidine heterocyclic ring dis-
927
played the highest binding affinity. For example, showdomycin (3-f3-D-ribofuranosyl- 1,2,4-triazole-3-carboxamide) and virazole, which may be viewed as nucleosides having incomplete purine rings, gave only moderate to poor inhibition. To determine the structural features of the heterocyclic base of the nucleoside important for transport, a series of strategically modified purine and pyrimidine nucleosides was examined for their ability to inhibit the uptake of [8'4C]adenosine by the germinating conidia. The relatively small apparent Ki values measured for 9-,8-D-purine riboside, 4-thiouridine, 7-deazaadenosine, 1-methylguanosine, and 3-deazauridine indicate that hydrogen bonding or any other specific interaction between base substituents or endocycic nitrogen atoms and the binding site of the transport system is not required for association. The nucleosides 7-methylguanosine, 6-azauridine, and xanthosine, all of which possess a charge undetI the experimental conditions, showed little or no inhibitory properties. The relatively low apparent K, values determined for 7-deazaadenosine, 6-mercaptopurine riboside, 6-mercaptoguanosine, 3-deazauridine, 4-thouridine, 5-azacytidine, and the naturally occurring nucleosides indicate that the electronic nature of the 7, 6, and 3 positions of the purine ring and of the 5, 4, and 3 positions of the pyrimidine ring is not an important determinant of the binding affinity of the nucleoside. The steric requirements of the binding site for the 6 position of the purine ring and the 3 and 4 positions of the pyrimidine of the purine and pyrimidine nucleosides are judged to be lax, on the basis of the observed low apparent Ki values of 6-methylaminopurine riboside, 6-dimethylaminopurine riboside, N6-(A2-isopentenyl)adenosine, 3-methyluridine, and N4-methylcytidine. In contrast, the binding properties of the purine nucleosides are sensitive to large substituents at the 8 position, and similarly, those of pyrimidine nucleosides are sensitive to large substituents at the 5 or 6 positions, as indicated by the observed lack of inhibition of adenosine transport by 8-bromoguanosine, 8-bromoadenosine, 8-mercaptoguanosine, 6-methylcytidine, 5bromo-2'-deoxyuridine, and thymidine. Whereas substitution at the 5 position of the pyrimidines appears to be a direct steric effect, the lack of inhibition observed for the 8-substituted purine nucleosides and 6-methylcytidine probably derives from the syn conformer, which predominates in these systems (10, 13), not having the proper geometry for efficient binding. Interestingly, binding properties of the pyrimidine nucleosides are also lost when the C5-C6 double bond is reduced, as in dihydrouridine.
DUNAWAY-MARIANO AND MAGILL 928 The loss of ring planarity (8) or aromaticity upon saturation of the C5-C6 double bond may explain this result. It may be inferred from the observed broad specificity of the transport system toward pyrimidine and purine nucleosides of the "natural" series and nucleosides possessing modified heterocyclic moieties that binding is not dependent on the presence of any specific group on the heterocyclic ring. This feature, taken with the apparent inability of the transport system to bind nucleosides having nonplanar or saturated heterocyclic units or nucleosides which possess a charge on the heterocycic ring, seems to suggest that binding might involve, in part, a s-sr or stacking interaction of the heterocycle of the nucleoside with a transport protein. The nucleoside transport system in N. crassa appears to be similar in many respects to the systems found in human erythrocytes by Cass and Paterson (2, 3) and in rabbit leukocytes by Taube and Berlin (12). In both of these mammalian systems, a single carrier mediates transport of both purine and pyrimidine ribosides and deoxyribosides. As in N. crassa, the system 'in rabbit leukocytes does not bind xanthosine, which is charged at physiological pH, or a nucleoside having an incomplete purine ring (4amino 5-imidazo carboxamide riboside). In addition, the transport systems in N. crassa, human erythrocytes, and rabbit leukocytes all appear to be specific for nucleosides having an unfunctionalized ribose or deoxyribose as the sugar unit and a fl-configuration about the anomeric carbon. At present, the only observed significant difference in the specificities of the N. crassa system versus these mammalian systems appears to be in the behavior toward thymidine. The mammalian systems bind thymidine efficiently, whereas the N. crassa nucleoside transport system binds thymidine and 5methylcytidine very poorly, indicating a difference in tolerance for substituents at the C5 pOsition of the pyrimidine ring. ACKNOWLEDGMENTS This research was supported in part by the Texas Agricultural Experiment Station. The authors thank P. S. Mariano
J. BACTERIOL. for helpful discussions and partial financial support by his Camille and Henry Drefus Foundation Teacher-Scholar Grant and C. W. Magill for providing the facilities for this research. The technical assistance of Paulette Carona and Ellen Edwards is also gratefully acknowledged. LITERATURE CITED 1. Berlin, R. D., and J. M. Oliver. 1975. Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. Int. Rev. Cytol. 42:287-336. 2. Cass, C. E., and A. R. P. Paterson. 1972. Mediated transport of nucleosides in human erythrocytes. J. Biol. Chem. 247:3314-3320. 3. Cass, C. E., and A. R. P. Paterson. 1973. Mediated transport of nucleosides by human erythrocytes: specificity toward purine nucleosides as permeants. Biochim. Biophys. Acta 291:734-736. 4. Fox, J. J., D. V. Praag, L. Wempen, I. L. Doerr, L. Cheong, J. E. Knoll, M. L. Eidinoff, A. Bendich, and G. B. Brown. 1959. Thiation of nucleosides. J. Am. Chem. Soc. 81:178-187. 5. Lauren, R. A., W. Grimm, and N. S. Leonard. 1968. p. 160-162. In W. W. Zarbach and R. S. Tipson (ed.), Synthetic procedures in nucleic acid chemistry, vol. 1. John Wiley & Sons, Inc., New York. 6. Magill, J. M., R. R. Spencer, and C. W. Magill. 1974. Relationship between [8-14C]adenosine transport and growth inhibition in Neurospora crassa strain ad-8. J. Bacteriol. 119:202-206. 7. Pateson, A. R. P., S. C. Kim, D. Bernard, and C. E. Cass. 1975. Transport of nucleosides. Ann. N. Y. Acad. Sci. 255:402-410. 8. Rohrer, D. C., and M. Sundarlingam. 1970. Stereochemistry of nucleic acids and their constituents. Acta Crystallogr. (Sect. B) 26:546-553. 9. Schlitz, J. R., and K. D. Terry. 1970. Nucleoside uptake during the germination of Neurospora crassa conidia. Biochim. Biophys. Acta 209:278-288. 10. Schweizer, M. P., J. T. Witowski, and R. K. Robbins. 1971. Nuclear magnetic resonance determination of syn and anti conformations in pyrimidine nucleosides. J. Am. Chem. Soc. 93:277-279. 11. Segel, I. 1975. Enzyme kinetics. John Wiley & Sons, Inc., New York. 12. Taube, R. A., and R. D. Berlin. 1972. Membrane transport of nucleosides in rabbit polymorphonuclear leukocytes. Biochim. Biophys. Acta 255:6-18. 13. Tavale, S. S., and H. M. Sobell. 1970. Crystal and molecular structure of 8-bromoguanosine and 8-bromoadenosine, two purine nucleosides in the syn conformation. J. Mol. Biol. 48:109-123. 14. Vogel, J. H. 1964. Distribution of lysine among fungi: evoluationary implications. Am. Nat. 98:435-466. 15. Williams, L. G., and H. K. Mitchell. 1969. Mutants affecting thymidine metabolism in Neurospora crassa. J. Bacteriol. 100:383-389.
JouRNAL OF BACTERIOLOGY, Dec. 1978, p. 924-928 0021-9193/78/0136-0924$02.00/0 Copyright @) 1978 American Society for Microbiology
Printedin U.S.A.
Specificity of Nucleoside Transport in Neurospora crassa DEBRA DUNAWAY-MARIANO1 AND JANE M. MAGILL2* Department of Chemistry' and Department of Biochemistry and Biophysics,2 Texas A & M University, College Station, Texas 77843
Received for publication 10 August 1978
The specificity of nucleoside uptake in germinating conidia of Neurospora crassa was investigated by examining the kinetics of [2-14C]uridine and [8-14C]adenosine uptake in the wild-type, ad-8, and ud-1 pyr-1 strains. The results obtained strongly indicate that nucleoside transport in N. crassa is mediated solely by a general transport system which accepts both purine and pyrimidine nucleosides. Studies directed at characterizing the specificity of the transport system indicate that general structural features of the nucleoside which enhance its efficiency in binding to the transport system include: (i) a purine or pyrimidine as the heterocyclic ring, (ii) an unfimctionalized ribose or 2'-deoxyribose as the sugar unit, (iii) a fl-configuration about the anomeric carbon, (iv) the absence of substituents at C8 in the purine series and at C5 and C6 in the pyrimidine series, (v) the presence of a C5-C6 double bond in the pyrimidine series, and (vi) the absence of a charge on the heterocycic ring. Nucleoside analogs are becoming increasingly important as a class of therapeutic agents which vary widely in the biological systems they can affect, as well as in their particular biochemical targets. The design of nucleoside derivatives for biological testing is aided by the ability to make reliable predictions of the rate of entry of the nucleoside analog into various cell types. To this end, emphasis in recent years has been placed on characterization of the mode and specificity of nucleoside transport (1, 7). Earlier studies (9) of nucleoside transport in germinating Neurospora crassa conidia suggested that two transport systems may be operative, one which transports purine and pyrimidine nucleosides and another which specifically transports purine nucleosides. More recent investigations (6) in this area have indicated that nucleoside uptake by germinating conidia of the ad-8 strain is mediated by a single transport system which accepts both purine and pyrimidine nucleosides. No evidence of a transport system specific for purine nucleosides was found in that mutant strain. In the present study, nucleoside transport in germinating conidia of wild-type, ad-8, and udI pyr-1 strains of N. crassa was examined. Evidence for the existence of a single nucleoside transport system in germinating wild-type conidia is presented, and the structural features of the nucleoside substrates required for uptake are described. MATERIALS AND METHODS Chemicals. [8-14C]adenosine and [2-14C]uridine were purchased from New England Nuclear Corp. The
nucleosides a-D-cytidine and 4-thiouridine were obtained from P-L Biochemicals; 1-benzylinosine, N6(A2-isopentenyl)adenosine, and 2',3',5'-triacetyluridine were obtained from Aldrich Chemical Co.; 3-deazauridine and 6-methyluridine were a kind gift from the National Cancer Institute. 3-Isoadenosine (5) and N4methylcytidine (4) were prepared by known methods. The remainder of the nucleosides used were purchased from Sigma Chemical Co. Neurospora strains. Wild-type N. crassa strain 74A and the ad-8 strain were used in all experiments to test the inhibition of [8-14C]adenosine uptake. These strains, together with the mutant strain ud-1 pyr-1, were obtained from the Fungal Genetics Stock Center, Humboldt College, Arcata, Calif. All strains were maintained on slants of Vogel minimal medium (14) supplemented with 200 mg of adenine or uracil per liter. Transport assay. Conidia from 5- to 7-day cultures were harvested in Vogel liquid minimal medium (14) containing 200 mg of adenine or uracil per liter (pH 6.0). The conidial suspension was filtered through sterile cheesecloth and incubated with stirring at 30°C for 5 h. The conidia were then suspended in Vogel minimal medium at a concentration of 4 x 10' conidia per ml (about 250 ug/ml, dry weight). After 1 h of incubation in Vogel minimal medium, 2.0 ml of conidial suspension was added to tubes containing [8-14C]adenosine, Vogel minimal medium, and the nucleoside to be tested. The rate of adenosine uptake was found to be linear for 40 min at 2MuM [8-_4C]adenosine under these conditions. After an incubation period of 5 min (during which time efflux was negligible), the tube contents were rapidly filtered through a fiber glass filter (Reeve-Angel Div., Whatman Inc.) and immediately washed with 10 ml of Vogel minimal medium (0WC). The filters containing the conidial residue were dried, added to 10 ml of Omnifluor liquid scintillation fluid (New England Nuclear Corp.), and then counted 924
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SPECIFICITY OF NUCLEOSIDE TRANSPORT
VOL. 136, 1978
with a Beckman liquid scintillation counter. The apparent Ki values for the nucleoside inhibitors were determined from Dixon plots by using linear regression analysis to determine the line of best fit in each case.
RESULTS Initial efforts at characterizing the specificity of nucleoside transport in germinating conidia of wild-type N. crassa were focused on the kinetics of purine and pyrimidine nucleoside uptake in conidia germinated to the stage where the activities of the purine-specific and common nucleoside transport system were proposed (9) to overlap maximally. Accordingly, the initial velocities of [8-14C]adenosine and [2-'4C]uridine transport were determined at several nucleoside concentrations. The results obtained are represented in the Lineweaver-Burk plots shown in Fig. 1. From these plots the apparent Km values for uridine and adenosine were determined to be 12 and 8 uM, respectively. Uridine and adenosine were tested as inhibitors of [8-14C]adenosine and [2-14C]uridine transport, respectively. Both nucleosides behaved as inhibitors. From the Dixon plots shown in Fig. 2, the apparent Ki values of adenosine and uridine were determined to be 11 and 10 ,LM, respectively. Additional evidence in support of a single transport system is provided from investigations of the mutant strain ud-1 pyr-1, which was reported (15) to lack uridine transport capacity.
Accordingly, the rates of [8-'4C]adenosine and [2-14C]uridine uptake by this mutant were measured. As can be seen in Fig. 3, uridine transport was negligible. Importantly, the adenosine uptake by this mutant was also insignificant. Specificity of nucleoside transport. The specificity of nucleoside transport in germinated conidia of N. crassa was investigated by measuring the degree of inhibition of [8-'4C]adenosine uptake by structural analogs. The apparent Ki values of the inhibitors, determined from Dixon plots, are reported in Table 1. All nucleoside analogs showing inhibition behaved as competitive inhibitors. This was indicated by the fact that, when the data were plotted as slope (from the Dixon plot) versus 1/[S], a straight line through the origin was obtained (11). DISCUSSION Previous studies (9) have demonstrated that nucleoside uptake by germinated conidia of N. crassa strain 74A is an energy-dependent, mediated process. Two different types of transport systems were reported (9) to be operating in the germinating conidia, one specific for purine nucleosides and the other a general system, transporting both purine and pyrimidine nucleosides. In more recent investigations (6) with germinated conidia of the ad-8 strain of N. crassa, we found evidence for only a single transport system.
a 4.5-
0
I
ao |
v
, 0
1.5
a(* -0.1
0
01
0.2
1/ (ADEN OS IN
03
0.4
-0.02
0
002
o04
0.08
0.06
E),q(M)
-I
1/
UR IDINE () M I,
FIG. 1. Lineweaver-Burk plots of initial velocities of transport of [8-14C]adenosine (1.0 M&Ci/umol) (a) and [2-'4Cluridine (1.0 ACi/prnol) (b) uptake by germinating conidia of wild-type strain 74A. The apparent Km values are approximately 8 for adenosine and 12 pzM for uridine. Velocity is expressed as nanomoles of adenosine or uridine transported in 5 min by 8 x 106 conidia.
J. BACTERIOL.
DUNAWAY-MARIANO AND MAGILL
926
a
12D 0
X
V 10
0
0/ /V /X K 4.0
0
-10
10
0
20
30
40
0 -8 8 16 LA BE LED AODENO SINE. (UN E IM (1U R I IDNI
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M
FIG. 2. Dixon plots of initial velocities of transport of 4 (0) and 8 AiM (0) [8-'4C]adenosine (1.0 liCi/pmol) in the presence of unlabeled adenosine (a) and uridine (b) by germinating conidia ofwild-type strain 74A. The apparent K, values are 11 uM for adenosine and 10 uU for uridine. Velocity is expressed as nanomoles of adenosine or uridine transported in 5 min by 8 x 106 conidia.
The present studies were designed to determine the number of different transport systems operative in germinating conidia of wild-type N. crassa strain 74A. In particular, the kinetics of adenosine and uridine transport in wild-type conidia of strain 74A, germinated to the stage where the activities of the purine-specific and common nucleoside transport systems were proposed (9) to overlap maximally, were investigated. Results from this study have shown that the apparent Km for adenosine uptake is exceptionally close to the value of the apparent Ki for adenosine as an inhibitor of uridine transport. Similarly, the apparent Km for uridine transport is very close to the value of the apparent Ki C= determined for uridine as an inhibitor of adeno2 6 2 0/ 4£ sine uptake. Furthermore, under identical conditions, uridine and adenosine display the same efficiency in inhibiting [8-14C]adenosine transport. These results strongly suggest that a single transport system, transporting both purine and pyrimidine nucleosides, is operative in these conidia and that a purine-specific transport system is not. This proposal is further evidenced by the observed inability of the germinating conidia of FIG. 3. Time course of [8.14C]adenosine (0) and [2Y1C]uridine (A) uptake by 8 x 106 germninating the ud-l pyr-1 strain to transport both adenosine and uridine. conidia of wild-type strain 74Aand [8-14C]adenosine Specificity of nucleoside transport in N. (t4) uptake by 8 x 106 germi(0) and [2.'4C]uridine nating conidia of strain ud-1 pyr-1. The adenosine crasa. The specificity of nucleoside transport in N. crassa was examined by testing nucleoside and uridine concentrations used were 10 W. 3.0
0--ft ~ ~ ~
~
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SPECIFICITY OF NUCLEOSIDE TRANSPORT
VOL. 136, 1978
TABLE 1. Apparent Ki values for nucleoside analogs as inhibitors of [8-14C]adenosine uptakea Inhibitor
Strain
Apparent K5 (AIM)
Showdomycin Virazole Purine riboside 7-Deazaadenosine 1-Methylguanosine 2'-Deoxyadenosineb Guanosineb Inosineb 6-Mercaptopurine riboside 6-Mercaptoguanosine 6-Methylaminopurine riboside 6-Dimethylaminopurine riboside
ad-8A 74A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A ad-8A 74A
345 132 36 10 22 15 8 26 24 19 29 49
N6-(A2-isopentenyl)adenosine Cytidine N4-Methylcytidine
74A 53 ad-8A 8 74A 20 Uridineb 11 ad-8A 4-Thiouridine 74A 10 3-Deazauridine 14 74A ad-8A 12 3-Methyluridine Pseudouridine 74A 58 422 Thymidineb ad-8A ad-8A 140 5-Methyl-2'-deoxycytidine 74A 5-Bromo-2'-deoxyuridine 108 5-Fluorouridine ad-8A 18 2'-Deoxyuridineb ad-8A 7 2'-Deoxyguanosine ad-8A 28 2'-Deoxyinosineb 43 ad-8A ad-8A 3'-Deoxyadenosine 845 5'-Azacytidine ad-8A 38 a All apparent Ki values listed were obtained from Dixon plots. The concentration range was 4 to 40 jtM for all inhibitors. Adenine, ,B-D-ribose, AMP, 3'-isoadenosine, 7-methylguanosine, 1-benzylinosine, 8-bromoguanosine, 6-methyluridine, and 2',3',5'-triacetyluridine showed no inhibition when tested using strain 74A. 8-Mercaptoguanosine, 8-bromoadenosine, xanthosine, dihydrouridine, arabinocytidine, 2',3'-isopropylideneuridine, a-cytidine, and 6-azauridine showed no inhibition when tested using strain ad-8A. b The apparent Ki has been reported in an earlier paper (6).
components and nucleoside analogs for their ability to inhibit the uptake of [8-_4C]adenosine. The failure of adenine, ,B-D-ribose, and AMP to inhibit [8-_4C]adenosine uptake indicated that the system under study is in fact specific for nucleosides. The nature of the sugar unit of the nucleoside was found to be quite important in determining the affinity of the nucleosides examined; only those possessing unsubstituted f8D-ribose or 8i-D-2'-deoxyribose sugar moieties were effective inhibitors (Table 1). Furthermore, of the nucleosides tested, those having a complete purine or pyrimidine heterocyclic ring dis-
927
played the highest binding affinity. For example, showdomycin (3-f3-D-ribofuranosyl- 1,2,4-triazole-3-carboxamide) and virazole, which may be viewed as nucleosides having incomplete purine rings, gave only moderate to poor inhibition. To determine the structural features of the heterocyclic base of the nucleoside important for transport, a series of strategically modified purine and pyrimidine nucleosides was examined for their ability to inhibit the uptake of [8'4C]adenosine by the germinating conidia. The relatively small apparent Ki values measured for 9-,8-D-purine riboside, 4-thiouridine, 7-deazaadenosine, 1-methylguanosine, and 3-deazauridine indicate that hydrogen bonding or any other specific interaction between base substituents or endocycic nitrogen atoms and the binding site of the transport system is not required for association. The nucleosides 7-methylguanosine, 6-azauridine, and xanthosine, all of which possess a charge undetI the experimental conditions, showed little or no inhibitory properties. The relatively low apparent K, values determined for 7-deazaadenosine, 6-mercaptopurine riboside, 6-mercaptoguanosine, 3-deazauridine, 4-thouridine, 5-azacytidine, and the naturally occurring nucleosides indicate that the electronic nature of the 7, 6, and 3 positions of the purine ring and of the 5, 4, and 3 positions of the pyrimidine ring is not an important determinant of the binding affinity of the nucleoside. The steric requirements of the binding site for the 6 position of the purine ring and the 3 and 4 positions of the pyrimidine of the purine and pyrimidine nucleosides are judged to be lax, on the basis of the observed low apparent Ki values of 6-methylaminopurine riboside, 6-dimethylaminopurine riboside, N6-(A2-isopentenyl)adenosine, 3-methyluridine, and N4-methylcytidine. In contrast, the binding properties of the purine nucleosides are sensitive to large substituents at the 8 position, and similarly, those of pyrimidine nucleosides are sensitive to large substituents at the 5 or 6 positions, as indicated by the observed lack of inhibition of adenosine transport by 8-bromoguanosine, 8-bromoadenosine, 8-mercaptoguanosine, 6-methylcytidine, 5bromo-2'-deoxyuridine, and thymidine. Whereas substitution at the 5 position of the pyrimidines appears to be a direct steric effect, the lack of inhibition observed for the 8-substituted purine nucleosides and 6-methylcytidine probably derives from the syn conformer, which predominates in these systems (10, 13), not having the proper geometry for efficient binding. Interestingly, binding properties of the pyrimidine nucleosides are also lost when the C5-C6 double bond is reduced, as in dihydrouridine.
DUNAWAY-MARIANO AND MAGILL 928 The loss of ring planarity (8) or aromaticity upon saturation of the C5-C6 double bond may explain this result. It may be inferred from the observed broad specificity of the transport system toward pyrimidine and purine nucleosides of the "natural" series and nucleosides possessing modified heterocyclic moieties that binding is not dependent on the presence of any specific group on the heterocyclic ring. This feature, taken with the apparent inability of the transport system to bind nucleosides having nonplanar or saturated heterocyclic units or nucleosides which possess a charge on the heterocycic ring, seems to suggest that binding might involve, in part, a s-sr or stacking interaction of the heterocycle of the nucleoside with a transport protein. The nucleoside transport system in N. crassa appears to be similar in many respects to the systems found in human erythrocytes by Cass and Paterson (2, 3) and in rabbit leukocytes by Taube and Berlin (12). In both of these mammalian systems, a single carrier mediates transport of both purine and pyrimidine ribosides and deoxyribosides. As in N. crassa, the system 'in rabbit leukocytes does not bind xanthosine, which is charged at physiological pH, or a nucleoside having an incomplete purine ring (4amino 5-imidazo carboxamide riboside). In addition, the transport systems in N. crassa, human erythrocytes, and rabbit leukocytes all appear to be specific for nucleosides having an unfunctionalized ribose or deoxyribose as the sugar unit and a fl-configuration about the anomeric carbon. At present, the only observed significant difference in the specificities of the N. crassa system versus these mammalian systems appears to be in the behavior toward thymidine. The mammalian systems bind thymidine efficiently, whereas the N. crassa nucleoside transport system binds thymidine and 5methylcytidine very poorly, indicating a difference in tolerance for substituents at the C5 pOsition of the pyrimidine ring. ACKNOWLEDGMENTS This research was supported in part by the Texas Agricultural Experiment Station. The authors thank P. S. Mariano
J. BACTERIOL. for helpful discussions and partial financial support by his Camille and Henry Drefus Foundation Teacher-Scholar Grant and C. W. Magill for providing the facilities for this research. The technical assistance of Paulette Carona and Ellen Edwards is also gratefully acknowledged. LITERATURE CITED 1. Berlin, R. D., and J. M. Oliver. 1975. Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. Int. Rev. Cytol. 42:287-336. 2. Cass, C. E., and A. R. P. Paterson. 1972. Mediated transport of nucleosides in human erythrocytes. J. Biol. Chem. 247:3314-3320. 3. Cass, C. E., and A. R. P. Paterson. 1973. Mediated transport of nucleosides by human erythrocytes: specificity toward purine nucleosides as permeants. Biochim. Biophys. Acta 291:734-736. 4. Fox, J. J., D. V. Praag, L. Wempen, I. L. Doerr, L. Cheong, J. E. Knoll, M. L. Eidinoff, A. Bendich, and G. B. Brown. 1959. Thiation of nucleosides. J. Am. Chem. Soc. 81:178-187. 5. Lauren, R. A., W. Grimm, and N. S. Leonard. 1968. p. 160-162. In W. W. Zarbach and R. S. Tipson (ed.), Synthetic procedures in nucleic acid chemistry, vol. 1. John Wiley & Sons, Inc., New York. 6. Magill, J. M., R. R. Spencer, and C. W. Magill. 1974. Relationship between [8-14C]adenosine transport and growth inhibition in Neurospora crassa strain ad-8. J. Bacteriol. 119:202-206. 7. Pateson, A. R. P., S. C. Kim, D. Bernard, and C. E. Cass. 1975. Transport of nucleosides. Ann. N. Y. Acad. Sci. 255:402-410. 8. Rohrer, D. C., and M. Sundarlingam. 1970. Stereochemistry of nucleic acids and their constituents. Acta Crystallogr. (Sect. B) 26:546-553. 9. Schlitz, J. R., and K. D. Terry. 1970. Nucleoside uptake during the germination of Neurospora crassa conidia. Biochim. Biophys. Acta 209:278-288. 10. Schweizer, M. P., J. T. Witowski, and R. K. Robbins. 1971. Nuclear magnetic resonance determination of syn and anti conformations in pyrimidine nucleosides. J. Am. Chem. Soc. 93:277-279. 11. Segel, I. 1975. Enzyme kinetics. John Wiley & Sons, Inc., New York. 12. Taube, R. A., and R. D. Berlin. 1972. Membrane transport of nucleosides in rabbit polymorphonuclear leukocytes. Biochim. Biophys. Acta 255:6-18. 13. Tavale, S. S., and H. M. Sobell. 1970. Crystal and molecular structure of 8-bromoguanosine and 8-bromoadenosine, two purine nucleosides in the syn conformation. J. Mol. Biol. 48:109-123. 14. Vogel, J. H. 1964. Distribution of lysine among fungi: evoluationary implications. Am. Nat. 98:435-466. 15. Williams, L. G., and H. K. Mitchell. 1969. Mutants affecting thymidine metabolism in Neurospora crassa. J. Bacteriol. 100:383-389.
