Deoxycytidine Transport in the Presence of a Cytidine Deaminase Inhibitor and the Transport of Uracil in Escherichia coli B*

SUMMARY Tetrahydrouridine, a cytidine deaminase inhibitor, prevents periplasmic degradation of deoxycytidine by Escherichia coli B. It does not inhibit deoxycytidine transport and therefore allows an accurate determination of deoxycytidine transport. Data obtained using tetrahydrouridine show that deoxycytidine is transported in E. coli B as the intact nucleoside by an active transport process, with a K, of 6 x 10e6 M. Cytidine and deoxyadenosine inhibit transport competitively, whereas guanosine has no effect on transport. Arsenate or KCN greatly reduces transport. In a mutant resistant to the nucleoside antibiotic, showdomycin, the active transport of deoxycytidine is lost, and residual slow uptake occurs by passive diffusion. Uracil is accumulated in E. coli B by an active transport process with a Km of 5 X 10e7 M. Rapid conversion nucleosides deaminated products and free bases by Escherichia coli difficulties accurate quantitative estimation nucleoside transport in cells,

Data obtained using tetrahydrouridine show that deoxycytidine is transported in E. coli B as the intact nucleoside by an active transport process, with a K, of 6 x 10e6 M.
Cytidine and deoxyadenosine inhibit transport competitively, whereas guanosine has no effect on transport.
Arsenate or KCN greatly reduces transport. In a mutant resistant to the nucleoside antibiotic, showdomycin, the active transport of deoxycytidine is lost, and residual slow uptake occurs by passive diffusion. Uracil is accumulated in E. coli B by an active transport process with a Km of 5 X 10e7 M.
Rapid conversion of nucleosides to deaminated products and free bases by Escherichia coli leads to difficulties in accurate quantitative estimation of nucleoside transport in whole cells, particularly since these conversions may occur in the periplasmic space. Thus, determinations of uricline transport are complicated by a periplasmic degradation of uridine to uracil. This uracil contributes to an unknown extent to observed uptake from uridine (1). In addition, rapid degradation of substrate in the medium prevents measurement of accurate kinetic parameters for transport.
SimiIar problems are associated with quantitation of cytidine and deoxycytidine transport. These nucleosides are not subject to phosphorolysis but are rapidly deaminated and then degraded to the free base. Cytidine deaminase is found in cell-free extracts (2) and is generally considered to be a cytoplasmic enzyme (3). However, its presence in shock fluid from E. coli has been reported by Hochstadt (4). Irrespective of the location of the deaminase, it can be demonstrated that uracil appears rapidly in the medium when cells are incubated for short time periods in the presence of cytidine (3). * This work was supported by Grant CA-02373-18 from the National Cancer Institute, National Institutes of Health.
Use of membrane vesicles in transport determinations eliminates the problem of intracellular degradations.
Hochstadt has reported transport of intact cytidine by E. coli vesicles denuded of periplasmic enzymes (4). However, vesicles tend to be "leaky" as evidenced by permeability to phosphorylated compounds (5), whereas whole cells are generally considered to be impermeable to such compounds, and transport components present in intact cells may be lost in the process of vesicle formation (4).
These difficulties can be circumvented by utilization of compounds that inhibit degradative enzymes but have no effect on the transport system. In the present studies deoxycytidine and cytidine transport were investigated in the presence of H&URD,l a potent inhibitor of cytidine deaminase (2). MATERIALS AND METHODS  (6). The mutant and parent cells used in all experiments were grown in minimal medium (7) in the absence of showdomycin and were harvested as described previously (6).

Materials
Assay for Uptake-Unless otherwise noted, the standard reaction mixture (1 ml) contained a cell suspension of E. coli B or the showdomycin-resistant mutant (equivalent to 0.4 to 0.5 mg dry weight) in Medium A (Davis and Mingioli medium without glucose) (7). The cells were preincubated without substrate for 15 min at 37". When glucose or tetrahydrouridine was added, the addition was made immediately prior to the preincubation period. The reaction was initiated by addition of "C-labeled substrate, and incubation continued at 37" for the indicated time period. Specific activity of [2-%]deoxycytidine varied from 1.4 Ci/mol to 29.7 Ci/mol. Specific activity of [2-%]uracil ranged from 10 Ci/mol to 50 Ci/mol. Heterologous nucleosides, when present, were added together with the substrate. Cells were collected and washed on Millipore filters as described previously (6). Measurements of radioactivity were carried out as described previously (1). Uptake at zero time of incubation was subtracted from the results.

Analysis of Products of Deozycytidine
Uptake in E. coli I3 and of Degradation Products in the Medium-Analysis of the medium was performed as described previously for uridine (1)) with separation of degradation products by descending chromatography in 86yo 1-butanol in water (v/v). Where uridine, uracil, and deoxycytidine

RESULTS
E$ect of Tetrahydrouridine on Cytidim Uptuke-Tetrahydrouridine, added to the standard assay at concentrations ranging from 41 PM to 410 PM, has no effect on uptake of 250 PM [%J4c]cytidine during a 5-min incubation period. Similarly, 205 PM tetrahydrouridine has no effect on uptake of either 200 FM [2-14C]or [ U-14C]uridine during a l-min incubation period. Since uridine has been reported to inhibit cytidine uptake competitively (6), these preliminary data indicate that tetrahydrouridine does not compete for the transport of either of these nucleosides.
Uptake of 250 PM cytidine was found to be linear for 1 min. Fig. 1 shows uptake of various concentrations of cytidine during a 1-min incubation period in the presence of 250 PM tetrahydrouridine, and in the presence or absence of 2500 PM deoxycytidine. Cytidine transport consists of a high affinity saturable component and a component that shows no evidence of saturation at 1 mM cytidine concentration (Fig. l), and a Lineweaver-Burk plot of its uptake is biphasic. The high affinity transport is completely inhibited by 2500 FM deoxycytidine, while the slower transport is not affected. The slower transport may therefore represent either a very low affinity transport system that is not inhibited by deoxycytidine or it may represent passive diffusion.
Deoxycytidine uptake (Fig. l), on the other hand, gives a linear Lineweaver-Burk plot. It is possible that a second very low affinity system for deoxycytidine transport may exist as it does for cytidine. However, the much higher transport rate of deoxycytidine obviates the necessity of taking such a component into account, since it comprises such a minor portion of the total uptake at these concentrations.
Therefore deoxycytidine transport was studied extensively in preference to cytidine transport. E$ect of Tetruh~drour~d~ne on Deozycytidine Uptake-The effect of varying concentrations of tetrahydrouridine on deoxycytidine uptake was determined (Table 1). Tetrahydrouridine appears to inhibit uptake of 0.5 PM deoxycytidine in the presence of glucose, with maximum inhibition at 82 PM tetrahydrouridine. At higher deoxycytidine concentrations, or in the absence of glucose, tetrahydrouridine appears to stimulate deoxycytidine uptake. These results are verified by the data of Table II, which show the distribution of radioactivity from deoxycytidine, intracellularly and in the medium. It is evident (Table II) that a considerable fraction of deoxycytidine is degraded and appears as uracil in the medium, and tetrahydrouridine largely prevents this appearance of degradation products in the medium. At all con- centrations of deoxycytidine, uracil represents the major fraction (95 to 100%) of the degradation products in the medium, the remainder consisting of uridine and deoxyuridine. Intracellular concentrations of uracil and uridine as well as total nucleotides are generally higher in the presence of tetrahydrouridine than in its absence. It is therefore apparent that intracellular breakdown of deoxycytidine is not prevented by 205 PM tetrahydrouridine.
Although tetrahydrouridine prevents the rapid disappearance of deoxycytidine, it does not completely block the appearance of uracil in the medium. It is possible that the release of small molecules by the cold shock effect found by Leder (11) and other Since tetrahydrouridine causes an inhibition of total uptake only at low (0.5 PM) deoxycytidine concentration, it was important to verify that this result is not related to an inhibition of uptake by the analog. Therefore, the uptake at 0.5 PM deoxycytidine was investigated in greater detail. By comparison of the uptake of [2-i4C]-and [W4C]deoxycytidine, the retention of the pyrimidine and deoxyribose moieties of deoxycytidine can be determined. It was found ( Table III) that uptake of the deoxyribose moiety is lower in the absence of tetrahydrouridine than in its presence, whereas uptake of the pyrimidine moiety is greater. Thus, the increased uptake of radioactivity observed at 0.5 PM [i4C]deoxycytidine in the absence of tetrahydrouridine is entirely accounted for by an increase in uptake of the pyrimidine moiety. Uptake of the deoxyribose and pyrimidine moieties is equal in the presence of tetrahydrouridine, indicating that deoxycytidine is transported as the intact nucleoside and substantiating the conclusion that no loss of either pyrimidine or deoxyribose moieties occurs after transport into the cell. Furthermore, the increased uptake of the pyrimidine moiety at 0.5 PM deoxycytidine must represent transport of uracil, which is formed in the periplasmic space only in the absence of tetrahydrouridine. This contribution of uracil transport in the absence of tetrahydrouridine is significant only at low deoxycytidine concentrations and accounts entirely for the apparent inhibition of total uptake by tetrahydrouridine at 0.5 PM deoxycytidine as discussed below.
investigators (12) may account for a fraction of the uracil found in the medium. However, there is no relationship between the concentrations of uracil found in the medium and the uracil concentration in the cell. On the other hand, the low levels of uracil in the medium when tetrahydrouridine is present are in all cases directly related to the extracellular concentration of deoxycytidine and represent only about 1 y0 of deoxycytidine (Table II). It may be concluded that a very low rate of deamination occurs in the periplasmic space even in the presence of tetrahydrouridine. However, this deamination is insignificant in comparison with deamination occurring in the absence of tetrahydrouridine.
Intracellular uracil is evidently not freely diffusable through the cell membrane at the experimental conditions used, since very high intracellular concentrations of uracil can be maintained. At the higher substrate concentrations used, the uptake of deoxycytidine is lower in the absence of tetrahydrouridine than in its presence (Table II). This result can be explained by the rapid disappearance of deoxycytidine in the medium due to peri-Uracil Transport in E. cola' B-The transport of uracil in E. coli B was studied to elucidate further the effect of uracil on total uptake from [14C]deoxycytidine. Fig. 2 (Table IV) shows that the major intracellular component is the nucleotide fraction at all time periods. Uracil can be concentrated intracellularly, and an inside to outside gradient of approximately 9O:l can be maintained after a I-min incubation period.
These data on uracil uptake lead to the conclusion that the apparent inhibition of 0.5 PM deoxycytidine uptake by tetrahydrouridine can be explained by transport of extracellular uracil, which is present in the medium in the absence of tetrahydrouridine. Uracil is transported by an energy-dependent system with a high affinity and at a low maximum velocity (Fig. 2) compared with deoxycytidine transport (Fig. 3). Uracil and deoxycytidine concentrations at 5 s can be calculated from the data in Table II for an initial deoxycytidine concentration of 0.5 PM in the absence of tetrahydrouridine.
These concentrations of uracil and deoxycytidine should result in an uptake of 40 pmol/lO s of deoxycytidine and 49 pmol/lO s of uracil, based on uracil uptake (Fig. 2) and deoxycytidine uptake in the presence of tetrahydrouridine and glucose (Fig. 3). Observed uptake is 41 pmol/lO s of deoxycytidine and 54 pmol/lO s of uracil (Table III). Uracil uptake appears to be inhibit,ed completely at higher deoxycytidine concentrations by the high intracellular uracil concentration resulting from deoxycytidine degradation, since total uptake becomes proportional to the 5-s deoxycytidine concentration.
Deoxycytidine Uptake in the Presence of Tetrahydrouridine- Fig. 3 compares deoxycytidine uptake in E. coli B and a showdo- in the presence of glucose and tetrahydrouridine is very low as compared to the parent strain, and is linear with increase in substrate concentration up to 1 mM substrate.
Kinetic data for deoxycytidine uptake in E. coli B are given in Table V. The K, for deoxycytidine uptake in the absence of glucose is almost IO-fold higher than in the presence of glucose, but the V,,,, is unchanged. A possible explanation for this finding will be presented in the discussion. Effect of energy poisons on deoxycytidine uptake Uptake was assayed by the standard procedure, with the addition of 5 mM glucose and 205 pM HdUrd, except that when arsenate was to be added potassium arsenate was substituted for potassium phosphate in Medium A. This modified buffer was used in the final wash and the incubation medium. KCN concentration was 20 mM, and was added just prior to preincubation.
Deoxycytidine concentration was 0.5 FM. in the presence of glucose, while guanosine has no effect. Concentrations of both substrate and inhibitors after 5-s incubation were calculated and used in all kinetic determinations, since the initial substrate concentration decreases by 10% at lower concentrations during the 10-s incubation period. Table VI shows that uridine and adenosine also inhibit deoxycytidine uptake in E. coli B. Kinetic data for inhibition by these nucleosides were not determined, since they are rapidly degraded both intracellularly and in the periplasmic space, whereas cytidine degradation is prevented by tetrahydrouridine, and deoxyadenosine degradation is slow.2 Heterologous nucleosides do not inhibit uptake in the mutant. E$ect of Energy Poisons on Deoxycytidine Uptake in E. coli B-Addition of either potassium arsenate or KCN greatly reduces uptake of deoxycytidine in the presence of exogenous glucose (Table VII). The uptake in the presence of these inhibitors is onehalf and one-fifth, respectively, of that in the absence of glucose without the addition of an energy poison. Addition of both arsenate and KCN eliminates uptake. DISCUSGION The characteristics of deoxycytidine transport in E. coli B show that tetrahydrouridine does not enter the cell. Lack of a kinase 2 Unpublished data.
for deoxycytidine has been reported previously (13), and we have failed to find any conversion of deoxycytidine to the nucleotide level in a cell-free system. Therefore, recovery of intracellular radioactivity in the nucleotide fraction, as well as in uracil and uridine fractions, can be accounted for only by prior deamination to deoxyuridine.
The fraction of total uptake represented by nucleotides, uridine, and uracil is the same in the presence and absence of tetrahydrouridine.
It is therefore apparent that tetrahydrouridine does not inhibit intracellular deamination of deoxycytidine.
Prior to the discovery of periplasmic cytidine deaminase (4) and nucleoside phosphorylase, it was assumed by other authors (3,14) that uracil found in the medium is formed intracellularly and is subsequently excreted. The data of Table II show that this assumption is incorrect, since tetrahydrouridine greatly reduces the amount of uracil recovered in the medium, even when intracellular uridine and uracil concentrations are considerably higher than those found in the absence of the deaminase inhibitor. The concept that uracil in the medium is the result of periplasmic deamination to deoxyuridine, followed by periplasmic cleavage to uracil and deoxyribose l-phosphate is also supported by the data in Table III. Comparison of transport data from [2-14C]-and [ U-i*C]deoxycytidine shows that the intracellular pyrimidine moiety concentration is higher than the intracellular deoxyribose moiety concentration when tetrahydrouridine is not present. This indicates that uracil, formed in the periplasmic space, is transported into the cell without accompanying deoxyribose transport.
The deaminase inhibitor prevents this periplasmic degradation as evidenced by the equal uptake of the pyrimidine and deoxyribose moieties (Table III). This confirms the conclusion stated previously ("Results") that the small amount of uracil found in the medium when tetrahydrouridine is present must result principally from a very slow rate of periplasmic degradation of deoxycytidine.
Although equality of the intracellular pyrimidine and deoxyribose moieties when tetrahydrouridine is present indicates transport of deoxycytidine before cleavage to base and sugar, it does not preclude entry into the cell as a nucleotide, by a group translocation mechanism. In this case the high deoxycytidine concentration found intracellularly could be the result of dCMP breakdown by phosphatases. However, transport as dCMP cannot occur since a kinase for deoxycytidine is not present in E. coli B. Nucleotide formation requires prior deamination to deoxyuridine. Since, in the presence of tetrahydrouridine, this reaction in the periplasmic space is largely inhibited, while total uptake is generally greater, the possibility of group translocation of deoxycytidine as dUMP is eliminated.
It can be concluded that in the presence of tetrahydrouridine, deoxycytidine is transported as the intact nucleoside, and its accumulation against a gradient occurs without interference by the analog.
Kinetic data for deoxycytidine transport in the presence of tetrahydrouridine indicate the existence of a single mode of transport that is stimulated by an energy source. We have found no evidence of the second transport system proposed by Komatsu and Tanaka (14,15) in E. coli K12 that is retained by their showdomycin-resistant mutant. Lineweaver-Burk plots of uptake are linear in E. coli B, and our showdomycin-resistant mutant appears to have lost the ability to transport deoxycytidine by a mediated process. The stimulatory effect of glucose is consistent with an active transport system in E. coli B, as is the intracellular accumulation of nucleoside to give a 40 : 1 concentration gradient (Table II). Inhibition of transport by energy poisons