ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 187, No. 1, April 15, pp. 264-271, 1978

UMP Pyrophosphorylase Partial

WILLIAM Department

of Tetrahymena

Purification

pyriformis

and Properties’

PLUNKETT2

AND

of Zoology,

Uniuersity

of Massachusetts,

Received

November

1, 1977; revised

J. G. MONER Amherst, December

Massachusetts

01003

12, 1977

UMP pyrophosphorylase (EC 2.4.2.9, UMPpyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyrifomzis GL-7. It was found to have a molecular weight of 36,060, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40°C. above which irreversible inactivation began to occur. The apparent K, values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 gru, respectively. The pyrophosphorylase exhibited a pyrimkhne base specificity for uracil, although 5fluorouracil was utilized by the enzyme. Neither cytosine, erotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylaee activity, UTP and dUTP, each at a concentration of 1 mru, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.

Nutritional studies have shown that an exogenous supply of either uracil, uridine, or @dine is required by Tetrahymenapyriformis for growth, suggesting that this organism is unable to synthesize pyrimidine nucleotides via de novo pathways (1, 2). Presumably, these compounds are converted to the pyrimidine nucleotide precursors required for nucleic acid synthesis by “salvage enzymes” which may also play a role in the regulation of the cellular concentration of pyrimidine nucleotides (3,4). One such enzyme from Tetrahymena, uridine-cytidine kinase (EC 2.7.1.48), has been partially purified and shown to be sensitive to feedback regulation by UTP and CTP (5). The activity of a second pyrimidine salvage enzyme, UMP pyrophosi This work was supported in part by National Institutes of Health Predoctoral Fellowship 5 FOI GM 37986-03 and by National Science Foundation Grant GB 6233. ‘Present address: Department of Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77030.

ooO3-9861/78/1871-0153~.00/0 0 1978 by Academic

All rights of reproduction

Press,

1,~.

in any form reserved.

MATERIALS

AND

METHODS

Materials. [S-“C]UraciI was purchased from New England Nuclear Corp. Autoradiography of chromatograms revealed the presence of at least three separate unidentified radioactive compounds that produced unacceptably high background levels in enzyme assays. All impurities were removed by paper chromatography in isobutyric acid:H&lzNI-LOH (66~331, v/v/v). After this purification step, specific activities of the labeled uracil were determined to be 31.0 and 33.5 Ci/mmol for two separate samples. Streptomycin sulfate and the sodium salt of all nucleotides were purchased from Sigma Chemical Co. The magnesium salt of 5-phosphoribosyl 1-pyrophosphate (PRPPY was 3 Abbreviation rophosphate.

264

Copyright

phorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase), which catalyzes the reaction, uracil + Sphosphoribosyl 1-pyrophosphate + UMP + PPi, has been detected in extracts of Tetrahymena (6-8). This report describes the partial purification of UMP pyrophosphorylase and the characterization of some of the properties of this enzyme.

used: PRPP,

5-phosphoribosyl

l-py-

UMP

PYROPHOSPHORYLASE

purchased from P-L Biochemicals. Diethylaminoethyl paper (DE-81) and No. 1 Whatman paper were obtained from H. Reeve-Angel and Co. Triton X-100 was a gift from Rohm and Haas Co. Sephadex G-200 and blue dextran 2000 were purchased from Pharmacia Fine Chemicals. Enzymes were obtained from the following sources: cytochrome c and yeast hexokinase from Nutritional Biochemicals, Inc., bovine pancreas chymotrypsinogen A from P-L Biochemicsls, and bovine hemoglobin from Per&x, Inc. B-Fluorouracil and B-axauracil were a gift from Dr. R. E. Handschumacher, Yale University. Cell growth. Cultures of Tetrahymena pyrifomis GL-7 were grown at 28’C in organic medium containing 2% (w/v) proteose peptone and 0.4% (w/v) liver fraction “L” (Nutritional Biochemicals). Salts prescribed for medium A of Kidder and Dewey (9) were added, omitting the phosphates. Aeration was achieved by using either a reciprocal or a gyratory shaker at 75-90 cycles/n& Cell number was determined using an electronic particle counter (Coulter Electronics, Inc., Model A). For those experiments in which the cells were grown in a chemically defined medium, the medium of Holx et al. (lo), modified to include all ~-amino acids, pyridoxine instead of pyridoxamine, and to exclude pyridoxal HCl, was used. Stock cultures were maintained on this medium for 18 months prior to their use in the experiments described. In such experiments the cells were grown at 28’C in the dark in unshaken cultures. Enzyme assay. The standard assay mixture contained 8.56 gmol of Tris-HCI (pH 7.5), 0.39 pmol of MgC12, 22.3 mnol of PRPP, 0.2 run01 of [2-‘%&racil, and approximately 0.03 mU of enzyme in a final volume of 112 pl. The reaction was started by the addition of enzyme to the preequilibrated (30°C) assay mixture. After 3-10 min, the conversion of uracil to UMP was determined either chromatographically or by the filter disk method of Breitmsn (11). The chromatographic method was used primarily to identify the reaction products when crude extracts were the enzyme source, whereas the filter disk method was usually employed with the purified enzyme. In the chromatographic method, the reaction was stopped by incubation in a boiling water bath for 3 min. Following chilling in ice and centrifugation, a IOgl portion of the supematant was spotted with uv marker compounds on No. 1 Whatman paper and chromatographed in isobutyric acid:HxO:NHIOH (66331, v/v/v). The compounds were detected under ultraviolet light, cut out, and placed in 10 ml of a mixture of Omnitluor (New England Nuclear) and toluene, and radioactivity was counted in a NuclearChicago Series 720 liquid scintillation spectrometer. In the filter disk assay, a 50-d portion of the reaction mixture was pipetted onto a 17-mm DE-81 paper disk and dropped into a beaker containing 20 ml of 10 mru ammonium formate per disk. After three washes in ammonium formate and two rinses in water, the

OF Tetrahvna

pyrifomis

265

disks were dehydrated by successive washes in ethanol and ether prior to determining radioactivity by scintillation counting. Control experiments indicated that less than 0.25% of the substrate was retained on the disk after this procedure, while retention of UMP was 100%. A unit of UMP pyrophosphorylase activity is defined as the amount of enzyme necessary to catalyze the conversion of 1 )unol of uracil and PRPP to 1 pmol of UMP and PPi per minute under the conditions of the standard assay. Specific activity is defined as units of enzyme activity per milligram of protein. Protein levels were determined by the method of Lowry et al. (12) using bovine serum albumin as a standard. Curve fitting and the determination of kinetic constants (Figs. 6 and 7) were carried out with the aid of linear regression analysis. RESULTS

Purification

of UMP Pyrophosphorylase Two liters of Tetrahymena pyriformis GL-7, in the late exponential phase of growth ( lo6 cells/ml), were harvested by centrifugation at 450g for 1 min. Cells were washed twice in phosphate buffer (12.5 mu KzHPOd, 50 mu NaCl, 2 mu KCl, adjusted to pH 7.4) and resuspended in three times the pellet volume of the same buffer. The temperature was maintained at 0-5°C throughout the rest of the purification procedure. Cells were disrupted in 15-ml batches by a tissue homogenizer equipped with a motor-driven Teflon pestle. The pooled homogenates were centrifuged for 20 min at 3O,OOOg,the pellet was discarded, and the cloudy, amber supernatant (Fraction I) was mixed with 0.3 vol of 5% (w/v) streptomycin sulfate for 30 min. Following clarification by centrifugation, the supernatant (Fraction II) was brought to 65% saturation with ammonium sulfate according to the nomograph of diJeso (13). After stirring for 2 h, the sediment obtained by centrifugation was discarded. The supernatant was brought to 80% saturation with ammonium sulfate, then stirred for 2 h, and the precipitate was collected by centrifugation and dissolved in a minimal volume of 0.05 M Tris-HCl, pH 7.5. Following dialysis against 1 liter of the same buffer for 8 h, a small precipitate was removed by centrifugation and the supernatant fluid (Fraction III) was pipetted onto a column (47.8 cm X 6.2 cm2) of Sephadex G-200 which had been preequilibrated with the

266

PLUNKETT

AND MONER

dialysis buffer. The same buffer was used to elute protein from the Sephadex column at a rate of 15 ml/h. Three-milliliter fractions were collected and protein concentration was estimated by determining the absorbance of each fraction at 280 nm. All fractions were assayed for UMP pyrophosphorylase activity by the filter disk method. Those with the highest specific activities were pooled (Fraction IV) and brought to 85% saturation with ammonium sulfate, and the resulting precipitate was collected by centrifugation and redissolved in a minimal volume of 0.05 M Tris-HCl, pH 7.5. Following dialysis for 8 h against 1 liter of the same buffer, Fraction IV was again pipetted onto the Sephadex G-200 column, and eluted and assayed as before. Fractions with the highest specific activities were again pooled to form Fraction V. A summary of the purification procedure is given in Table 1. Fraction V appeared free of contaminating enzyme activities and possible altemative substrates. Autoradiograms of paper chromatographic separations of standard reaction mixtures from which PRPP was omitted showed that all the radioactivity remained with the uracil spot. After incubation of [2-‘4C]uridine 5-phosphate with Fraction V, autoradiography gave no indication of the presence of uridine which might have been produced by contaminating phosphatases (14, 15). Neither was uracil detected in this reaction mixture, ruling out the possibility that uridine, produced by phosphatases, had been further metabolized to uracil by uridine phosphorylase (16,17). The sole radioactive product of the standard assay was chromatographically

identical to UMP and possessed similar spectral characteristics. Incubation of the radioactive reaction product with Escherichia coli alkaline phosphatase produced a compound chromatographically identical to uridine. UMP production, using the Fraction V enzyme, was linear for more than 20 min, although most assays were run for 3-10 min. The standard assay mixture contained 1.0 pg or less of Fraction V protein, which was within the linear range of product formation. Properties of UMP Pyrophosphorylase The molecular weight of UMP pyrophosphorylase was estimated by the gel filtration procedure of Andrews (18) to be 36,060 (Fig. 1). A molecular weight in this range is considerably smaller than that of murine leukemia cell enzymes (19,20), and suggests that the enzyme is likely to exist as a single polypeptide chain or a dimer under the conditions of the assay. UMP pyrophosphorylase is active over a broad pH range, with half-maximal activity at pH 5.6 and 8.4 (Fig. 2). The optimum pH, 7.5, was used in the standard assay mixture. UMP production was linear with increasing temperature between 25 and 40°C the latter representing a well-defined temperature optimum. At temperatures above 4O”C, the rate of UMP formation was markedly decreased. Separate experiments, in which the enzyme was preincubated at a given temperature, cooled, and then assayed in the standard reaction mixture at 30°C suggested that the lower activity observed at temperatures greater than 40°C

TABLE I PURIFICATION OF UMP

Fraction I. Crude extract II. Streptomycin so4 III. (NH&SO+ 65-60’S IV. Sephadex G-

Volume (ml) 180 227

Total protein (mid 639 506

PYROPHOSPHORYLASE

Tot? a$ivity m 227 415

27

38.2

169

37

13.5

143

6

0.49

Specific activity WJ/mg) 0.35 0.82 4.42

Purification factor 1

2 13

10.6

30

29.6

85

200 (1st)

V. Sephadex G200 (2nd)

14.6

UMP

PYROPHOSPHORYLASE

OF Tetrahymena PYrifomk

267

FIG. 1. Estimation of UMP pyrophosphorylase molecular weight by gel filtration. Twenty-five milligrams of Fraction III protein was pipetted onto a column of Sephadex G-296 (46.7 cm x 6.15 cm*) which had been equilibrated with 10 mhr ‘Iris-HCl (pH 8.5). The protein was eluted with the equilibrating buffer at a flow rate of 12 ml/h and collected in 3-ml fractions, and the specific activity was determined by the filter disk method. The column was calibrated with the following proteins of the indicated molecular weights, listed in the order of elution: hexokinase (96,ooO), hemoglobin (69,ooO), chymotrypsinogen (23,ooO), and cytochrome c (13,390). The void volume was determined using blue dextran 2WO (M, - 2 x 106). The ratio of elution volume, V., divided by the void volume, V,, is plotted against the log of the molecular weight.

of 7.6 PM, and their efficiency in competing with uracil for the enzyme at various uracil concentrations was determined. Those bases capable of serving as substrates for the enzyme should show a pattern of competitive inhibition of UMP formation in Lineweaver-Burk plots (Fig. 4). Of the bases tested, only 5-fluorouracil showed such competition with uracil. The ability of UMP pyrophosphorylase to utilize 5-fluorouracil has also been described for bacteria (23) and murine leukemia cells (19, 20). Neither cytosine nor erotic acid (not shown) competed with uracil, confirming the results of nutritional studies indicating both compounds to be nutritionally inert in Tetrahymena. In a separate experiment, 6azauracil also did not compete with uracil for the pyrophosphorylase. This result is compatible with the findings of Holz et al. (24) with Tetrahymena, where this antimetabolite neither affected growth nor inhibited heat-induced synchronous division. It is also in agreement with a preliminary report on pyrophosphorylase activity in cell-free extracts of Tetrahymena (7). UMP pyrophosphorylase activity is, in general, only slightly depressed by the addition of most nucleoside triphosphates to the standard assay mixture (Table II).

was due to irreversible enzyme inactivation. A temperature of 30°C was chosen for the standard assay, since it is close to the optimal growth temperature of the organism, gave adequate enzyme activity, and is the temperature at which the international unit of enzyme activity is defined. The Lineweaver-Burk plots for both PRPP (Fig. 3) and uracil (Fig. 4) yielded straight lines when the other substrate was saturating. Under these conditions, the apparent K,,, values of the enzyme were 6.9 j.rM for PRPP and 0.4 j.&M for uracil. These values indicate a high afXnity of the enzyme for its substrates, and are somewhat lower than those for the UMP pyrophosphorylase from E. coli (21), murine leukemia cells (19, 20), and yeast (22). In addition, Fig. 4 also provides data regarding the specificity of the enzyme for different pyrimidine bases. Nonradioactive bases were added to the standard assay mixture at a concentration

FIG. 2. Effect of temperature on UMP pyrophosphorylase activity. The standard reaction mixture was preincubated at the given temperature for 5 min before adding 10 4 of Fraction V enzyme. After 3 min, UMP production was determined by the filter disk assay. Enzyme activity is expressed as nanomoles of UMP produced per minute per milligram of Fraction V p&&l.

I

10

1.5

2.0

V. x

PH

268

PLUNKE’M’

AND MONER

1.2.

0.8.

-1 ”

1

I

I

I

10

20

30

40

[ 1I PRPP

-1

LIM

FIG. 3. Effect of PRPP concentration of UMP pyrophosphorylase activity. The standard assay mixture was employed, except that the PRPP concentration was varied. UMP production was determined by the filter disk method. The reaction velocity, v, is expressed as nanomoles of UMP produced per minute per milligram of Fraction V protein.

However, the addition of UTP or dUTP, at a concentration of 1 mu, to the reaction mixture results in 86 and 59% inhibition of the reaction, respectively. Since CTP, dCTP, and TTP each produces less than 15% inhibition when added at the same concentration, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. This view is consistent with a previous preliminary report on the Tetruhymena enzyme (7) and the observed properties of E. coli pyrophosphorylase (21). The possibility that UMP pyrophosphorylase activity in Tetrahymena might be increased in response to the presence of specific pyrimidines was investigated using cells grown in chemically defined medium. When uracil, uridine, and UMP were used

separately as exclusive pyrimidine sources, no differences in population doubling time or final cell density were detected. To determine whether these pyrimidine sources affected cellular enzyme activities, homogenates of cells grown with each of the different pyrimidine sources were prepared and the specific activity of UMP pyrophosphorylase was determined. As shown in Table III, the enzyme activity in cells grown in a chemically defined medium containing uracil as the sole pyrimidine source is threefold higher than the activity in extracts of cells grown on either uridine or UMP. Separate assays utilizing these same extracts failed to detect any significant differences in the uridine kinase activity of Tetrahymena grown on these same pyrimidine sources (25).

UMP

PYROPHOSPHORYLASE

OF Tetiahymena

pyriforrnk

269

0.30

0.20

-1 ”

-2.0

- 1.0

0

[

Uracil

1.0 -1

1

2.0

3.0

-1

pM

FIG. 4. Effect of added pyrimidine bases on UMP pyrophosphorylase activity. The standard assay mixture was employed, except that the uracil concentration was varied. The following pyrimidine bases were included at a final concentration of 7.56 p: 5fluorouracil (open triangles), cytosine (open circles), none (solid circles). Reaction velocity, u, is expressed as nanomoles of UMP produced per minute per milligram of Fraction V protein x lo*. DISCUSSION

Like other protozoan ciliates, Tetrahymenu pyriformis possesses no de novo pathways for pyrimidine nucleotide synthesis, and is dependent upon an exogenous supply of pyrimidines for growth (1). Uracil, uridine, or cytidine, but not erotic acid or cytosine, will fulfill the pyrimidine requirement (2). The enzymatic machinery necessary for the conversion of these compounds to nucleotides includes uridine-cytidine kinase, a single enzyme that phosphorylates each of these nucleosides to the corresponding 5’-monophosphate (5). In addition, uridine phosphorylase reversibly converts uracil and ribose-l-phosphate to uridine and inorganic phosphate (16, 17). This enzyme will not, however, degrade cytidine to the

free base. The finding that cytosine is nutritionally inert in Tetrahymena suggests the absence of the corresponding anabolic reaction. Finally, UMP pyrophosphorylase can convert uracil and PRPP directly to UMP and PPi in a single step (6, 7). In agreement with nutritional studies (2), this report has indicated that UMP pyrophosphorylase will not utilize either erotic acid or cytosine as a substrate in this reaction. Bacteria (28) and yeast (19, 26, 27) have separate enzymes for the phosphoribosylation of uracil and erotic acid, whereas both functions appear to be carried out by a single enzyme in various murine tumor systems (19, 20). Since the evidence indicates that Tetrahymena is unable to convert erotic acid to UMP (2), it would be interesting, from an evolutionary stand-

270

PLUNEETT TABLE

II

EFFECT OF NUCLEOSIDE TRIPHOSPHATES ON UMP PYROPHOSPHORYLASE ACTIVITYD

Nucleoside triphosphate

Activity (nmol of UMP/min/mg of protein)

None ATP dATP GTP dGTP CTP dCTP UTP dUTP TTP

23.9 24.9

21.1 17.9 20.8 22.4 20.5 3.3 9.7 20.4

109 104 843 75 86 94 86 14 41 85

a Nucleoside triphosphates were added, to a final concentration of 1.0 mM, to assay mixtures containing 8.56 pm01 of Tri5HCl (pH 7.5), 0.39 ~01 of MgCL, 4.46 ~01 of PRPP, and 0.21 pmol of [2-14C]uracil in a final volume of 0.112 ml. The reaction was started by the addition of 3.3 pg of enzyme Fraction III and incubated for 3 min at 30% before determining UMP production by the filter disk method. All results are the averages of duplicate determinations. TABLE III UMP PYROPHOSPHOR~LASE ACTIVITY IN EXTRACTS OF Tetrahymena GROWN IN CHEMICALLY DEFINED MEDIA WITH DIFFERENT PYRIMIDINE SOURCEZ?

Pyrimidine source

UMP pyrophosphorylase activitv”

Uracil Uridine UMP

218 zt 21 63 + 2 62 f 3

DCells were carried through three serial inoculations in a defined medium utilizing either uracil, uridine, or UMP as the sole pyrimidine source. Ten milliliters of cells grown with each pyrimidine source was then diluted to 325 ml in the homologous medium and grown for an additional 48 h in the dark at 28°C. Cells were harvested and homogenates prepared as described under Materials and Methods. UMP pyrophosphorylase activity was determined in triplicate using the standard assay mixture and the filter disk method. b Picomoles of UMP formed per minute per milligram of protein f SE.

point (30), to determine whether a separate enzyme for orotate phosphoribosylation has been deleted or inactivated in Tetruhymena. Another possibility is that the affinity for orotate of a single pyrophosphorylase enzyme that once utilized both uracil and orotate may have been selectively di-

AND MONER

minished. The pyrophosphorylase enzyme that utilizes orotate and fluorouracil from P388 cells, that are either sensitive or resistant to the analog, have similar affinities for orotate, but the enzyme from the resistant cells has a K, for fluorouracil that is fivefold greater than that of the enzyme from the sensitive cells (20). The data presented in Fig. 4 suggest that UMP pyrophosphorylase from Tetruhymenu is capable of utilizing Sfluorouracil as an alternative substrate. This represents what is probably the first step in the activation of this pyrimidine antimetabolite, which inhibits multiplication in exponentially growing Tetrahymena (31) and can also block oral morphogenesis (32) and cell division (24, 33) in heat-synchronized cultures of this organism. Furthermore, the reversal of the biological effects of fluorinated pyrimidines by uridine may be attributable to the competitive interaction of uracil and 5fluorouracil for UMP pyrophosphorylase. The initial enzymes of de novo pyrimidine biosynthesis are known to be targets for feedback regulatory mechanisms in microorganisms (34). However, since Tetruhymena has no capacity for de novo pyrimidine formation, a possible regulatory role for the salvage enzymes has been investigated. One such enzyme, uridine-cytidine kinase, is active in Tetrahymena, and studies on the partially purified enzyme (5) have demonstrated its sensitivity to feedback inhibition by physiologically attainable concentrations of UTP and CTP (35). The present study suggests that a second salvage enzyme, UMP pyrophosphorylase, may also be sensitive to feedback inhibition. The data in Table II, indicating specific inhibition of enzymatic activity by UTP and dUTP, are in agreement with a preliminary report by Chambers and Brockman (7). The cellular UTP concentration in exponentially growing Tetruhymena has been calculated to be 0.2 mu (35), and the extensive inhibition by UTP reported here (86%) was achieved at a concentration of 1.0 mu. Thus, further studies of the concentration dependence of this inhibition and correlations of the uptake and utilization of uracil with the cellular UTP concentration may provide insight

UMP

PYROPHOSPHORYLASE

into the role of this inhibition in the regulation of pyrimidine nucleotide synthesis in

Tetrahymena. The adaption of mammalian cells to existing nutritional conditions is often associated with changes in the levels of enzyme activities. This appears to be the case with UMP pyrophosphorylase in Tetrahymena. When uracil was the sole pyrimidine source, the enzyme’s specific activity was more than threefold greater than that of cells grown exclusively on either uridine or UMP (Table III). While the value of this response to the organism may be apparent, the mechanism by which it is achieved remains obscure. The finding that the activity of rat hepatoma OMP pyrophosphorylase specifically decreased by 70% when these cells were exposed to uridine as a pyrimidine source may be a related phenomenon (36). REFERENCES 1. KIDDER, G. W. (1965) in Chemical Zoology (Florkin, M., and Scheer, B. T., eds.), Vol. 1, pp. 93-159, Academig Press, New York. 2. KIDDER, G. W., DEWEY, V. C., PARKS, R. E., AND HEINRICH, M. R. (1950) Proc. Nat. Acad. Sci.

USA 36,431-439. 3. KORNBERG, A. (1957) in The Chemical Basis of Heredity (McElroy, W. D., and Glass, B., ede.), pp. 579-608, The Johns Hopkins Press, Baltimore. 4. BROCKMAN, R. W., AND ANDERSON, E. P. (1963) in Metabolic Inhibitors (Ho&&r, R. M., and Quartel, J. H., eds.), Vol. 1, pp. 239-285, Academic Press, New York. 5. PLUNKETT, W., AND MONER, J. G. (1971)

B&him.

Biophys. Acta 260,92-102.

6. HEINRICKSON, R. L., AND G~LDWASSER, E. (1964) J. Biol. Chem. 239,1177-1187. 7. CHAMBERS, P., AND BROCKMAN, R. W. (1965)

Bacterial. Proc. 94. 8. PLUNKED,

W., AND MONER, J. G. (1970) J. Cell

Bid. 47,159A. 9. KIDDER, G. W., AND DEWEY, V. C. (1951) in Biochemistry and Physiology of Protozoa (A. Lwoff, ed.), Vol. 1, pp. 324-400, Academic Press, New York. 10. HOLZ, G. G., ERWIN, J., ROSENBAUM, N., AND

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265-275. 13. DIJESO, F. (1968) J. Biol. Chem. 243,2022-2023. 14. CONNER, R. L., AND MCDONALD, L. A. (1964) J.

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J. Bid. Chem. 243,53&i-5395. 16. EICHEL, H. J. (1957) J. Protozool. (Suppl.) 4, 16. 17. HILL, D. L., AND CHAMBERS, P. (1967) J. Cell.

Physiol. 69,321-330. 18. ANDREWS, P. (1965) B&hem. J. 96,595-605. 19. REYES, P., AND GUGANIG, M. E. (1975) J. Bid.

Chem. 250,5097-5108. 20. -EL, D., DEACON, J., COFFEY, B., AND BAKAMJIAN, A. (1972) Mol. Pharmacol. 8.731-739. 21. MOLLOY, A., AND FINCH, L. R. (1969) FEBS Lett. 5,211-213. 22. NATALINI, P., FIORETTI, E., RUGGIERI, S., VITA, A., AND MAGNI, G. (1975) Experientia 31, 1008-1010. 23. BROCKMAN, R. W., DAVIS, J. M., AND STUI-I~, P. (1960) B&him. Biophys. Acta 40,22-32. 24. HOLZ, G. G., RASMUSSEN, L., AND ZEUTHEN, E. (1962) C. R. Trav. Lab. Carl&erg 33,289-360. 25. PLUNKED, W. (1970) Salvage Enzymes and the Regulation of Pyrimidine Ribonucleotide Synthesis in Tetrahymena pyriformis, Ph.D. thesis, University of Massachusetts, Amherst. 26. LIEBERMAN, E., KORNBERG, A., AND SIMM, E. S. (1955) J. Bid. Chem. 215.403-415. 27. DAHL, J. L., WAY, J. L., AND PARKS, R. E., JR. (1959) J. Biol. Chem. 234,2998-3002. 28. CRAWFORD, I., KORNBERG, A., AND SIMMS, E. S. (1957). J. Bid. Chem. 226,1093-1101. 29. LINDSAY, R. H., TILLERY, C. R., AND Yu, M-Y W. (1972) Arch. Biochem. Biophys. 148, 466-474. 30. HOLZ, G. G. (1966) J. Protozool. 13,2-i. 31. HILL, D. L., STRAIGHT, S., AND ALLAN, P. W. (1970) J. Protozool. 17,619. 32. FRANKEL, J. (1965) J. Erp. 2001.169,113-148. 33. CERRONI, R. W., AND ZEUTHEN, E. (1962) C. R.

Trav. Lab. Carl&erg 32,499-511. 34. G’DONOVAN, G., AND NEUHARD, J. (1970) Bacte-

rid. Rev. 34,278~343. 35. NEXO, B. A. (1975) Biochim. Biophys. Acta 378, 12-17. 36. HOOGENRAAD, N. J., AND LEE, D. C. (1974) J.

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