Improved radioisotopic assay for cytidine 5′-triphosphate synthetase (EC 6.3.4.2)

ANALYTICAL

91, 46-59 (1978)

BIOCHEMISTRY

Improved Radioisotopic Assay for Cytidine 5’-Triphosphate Synthetase (EC 6.3.4.2)’ JIM C. WILLIAMS,* “‘Dc~purtment

HARUTOSHI GEORGE

KIZAKI,? WEBER~

EMILIO

WEISS,”

AND

of Microhiok~gy.

20014. and tluboratory

Naval McJdicul Rcseurch Institutr. Bethesda. Maryland E?rprrimcntul Oncology. Indiana Uniwrsity School of Medic,inc,. Indiunupolis. Indiunu 46202

for

Received June 2. 1978 An improved radioassay for cytidine 5-triphosphate synthetase is reported which employs thin-layer chromatographic methods and provides a number of advantages over previously available techniques. (i) The method resolves the nucleotides and the degradation products generated during the time course of the enzymatic reaction by ascending chromatography employing polyethyleneimine cellulose plastic-backed sheets. (ii) Determinations of CTP formed and all nucleotide pairs generated during kinetic analysis of CTP synthetase are greatly simplified, further facilitating the detection of extraneous enzymatic activities. (iii) The sensitivity of the assay is enhanced and as little as 50 pmol of product formed was readily detected in supernatant fluids. This was made possible. in part. by the addition of NaF and phosphoenolpyruvate which together maintain the nucleotide triphosphates in the reaction mixture. (iv) A large number of samples can be handled at one time with highly reproducible results. The synthesis of CTP from UTP by enzyme preparations from rat liver, hepatomas. and Sulmonrlla fyphimurium LT2 was quantitated with this method.

Cytidine 5’-triphosphate synthetase* (CTP Sase) is the last enzyme in the de now biosynthesis of pyrimidine nucleotides (11). The bacterial CTP Sase [UTP:ammonia ligase (ADP), EC 6.3.4.21 catalyzes the irreversible transfer of the amide group of glutamine (Gln) or NH, to the C,-position of the uracil moiety of uridine 5’-triphosphate (UTP) in the presence of Mg2+ and adenosine 5’-triphosphate (ATP), whereas the eukaryotic enzyme transfers only the amide group of glutamine (1 I). The ’ The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. s Abbreviations used: CTP Sase, Cytidine 5’-triphosphate synthetase: PCA, perchloric acid; PEP. phosphoenol pyruvate; ME, mercaptoethanol; PEI. polyethyleneimine; GMTS. buffered solution containing Gln. ME. tris (hydroxymethyl) aminomethane. and sucrose (more detailed description under Experimental Procedures). OOO3-2697/78/091I-0046$02.00/O Copyright All rights

Q 1978 by Academic Press. Inc. of reproduction in any form reserved.

46

CTP SYNTHETASE

47

ASSAY

glutaminase activity of the enzyme is enhanced by guanosine 5’-triphosphate (GTP) and the reaction is driven by the cleavage of ATP to ADP and Pi (Reaction [l]). UTP + Gln + ATP -

Mg2+

GTP

CTP +

GIu

+ ADP + Pi.

The enzyme appears to be ubiquitous in nature in both eukaryotes and prokaryotes (11). Assays for CTP Sase are of two general types: (i) a spectrophotometric method (13) based on the increase in absorbance at 290 nm (at acid pH) resulting in the formation of cytidine nucleotides, and (ii) an isotopic technique which depends on HCI or perchloric acid (PCA) hydrolysis of CTP and UTP to CMP and UMP followed by the differential retention of CMP, but not UMP, by the cation exchange resin Dowex 50-H+ (5,20) or separation by paper chromatography (2). Although both methods can be used with equal sensitivity with partially purified preparations (20). they are unsuitable for crude enzyme preparations (5,8.13) because of the presence of interfering enzyme activities. Since CTP Sase specific activity was quite low (7) when compared to other enzymes of nucleotide biosynthesis (22,23), care must be exercised to ensure that the reactants and products are maintained during the time course of the reaction. The inclusion of phosphoenolpyruvate (PEP) in the reaction mixture has aided in the recovery of cytidine nucleotides (7). This paper describes a simple and highly sensitive radiotracer assay system for CTP Sase which measures the formation of CTP. The assay depends upon separation of unreacted UTP from newly synthesized product, CTP, which migrates to a lesser extent during thin-layer chromatography on polyethyleneimine cellulose. Furthermore, the addition of PEP (7) and NaF, which does not interfere with the synthesis of CTP. causes the reactants and products to be maintained during the time course of the reaction. We present evidence that this system is adequate for the assay of rat liver, tumor tissues, and bacterial samples. The highly reproducible chromatographic system for the separation of CTP from UTP made possible the estimation of CTP in crude bacterial and rat liver extracts at concentrations in the picomole range. This assay method was employed in our recent investigation into the behavior of CTP synthetase activity in neopiasia (23). EXPERIMENTAL

PROCEDURES

Preparntion of cell-free extracts. Fifteen percent homogenates (w/v) were prepared at 4°C from rat liver (ACIIN) and tumor (hepatomas 3683F and 3924A) tissues in 0.15 M KCI, pH 7.4. The homogenates were clarified by centrifugation at 100,OOOg for 60 min at 4°C. Salmonella typhimurium LT2 was grown in medium A with added FeCl,.6H,O (0.8 pgiml) and

48

WILLIAMS

ET AL.

ZnSO,.6H,O (5 Fg/ml) and 0.2% glucose as the carbon source (9). The cells were harvested in mid-log phase by centrifugation at 16,300g for 10 min at 4°C. The pellets were resuspended at 4°C in fresh growth medium and concentrated by centrifugation, and the final pellet was resuspended in ice cold 40 mM potassium phosphate buffer, pH 7.4. The suspension was sonicated (Sonifier cell disruptor, Branson Sonic Power Co.) for 5 min at 4°C and clarified by centrifugation, first at 16,300g for 10 min, and then at 100,OOOg for 1 h. The supernatant was divided into two parts. Fraction I was used directly for CTP Sase activity, whereas Fraction II was treated as follows: Streptomycin sulfate was added to a final concentration of 0.5%. and the preparation was left standing in ice for 30 min. The precipitate was removed by centrifugation at 16,300g for 30 min and the supernatant was dialyzed against 100 vol of a solution containing 50 mM glycylglycine, 10 mM @mercaptoethanol, and 10 mM L-Gln for 12 h. This preparation was then assayed for CTP Sase activity. The protein concentration was measured by the technique of Lowry rt al. (15) using bovine serum albumin as the standard. Cyridine 5’-triphosphatr synthrtusr assay. The assay mixture (pH 7.4) for the rat liver and hepatoma samples contained, in a total of 0.2 ml, the following mM quantities: glycylglycine, 70; MgCl*, 18; P-mercaptoethanol (ME), 10; L-Gln, 10; ATP (Na salt), 8; GTP (Na salt), 1; phosphoenolpyruvate (PEP), 8; UTP (Na salt), 2.0; [4-r4C]UTP (ammonium salt, 50 mCilmmo1, 95% uridine nucleotides), 0.026 with a final specific activity of 0.61 &iIpmol; and an appropriate dilution of supernatant fluid. The assay mixture for the S. typhinzurium samples differed only in that the UTP concentration was 0.9 mM. NaF (10 mM>, when added, did not change the concentration of the other components. The reaction was initiated by adding the supernatant fluid (preincubated at 37°C for 3 min) to the reaction mixture which had been warmed to 37°C. Reaction was terminated at appropriate time intervals by adding 10 ~1 of 60% perchloric acid (PCA) and quickly placing the reaction tube in an ice bath. The preparations were processed as follows: (i) The solution was neutralized (pH 6.0-6.5 by adding 15 ~1 of 7.5 N KOH which contained 50 mM ethylenediaminetetraacetic acid (EDTA) and was clarified by centrifugation (4°C) at 1000 t-pm for 20 min (DamoniIEC Division, IEC PR-J centrifuge). (ii) When hydrolysis of the samples was carried out, the reaction tubes, after receiving 10 ~1 of 60% PCA, were placed in boiling water for 0, 30, 60, and 90 min. The samples were then placed in an ice bath and neutralized. Polyethyleneimine (PEI)-cellulose plates were activated by immersion in 10% NaCl for 10 min, dried with cool air, then soaked in distilled deionized water for 10 min, and finally dried with cool air. They were next marked 2.0 cm from the bottom with a soft lead pencil and 10 ~1 of the neutralized supernatant fluids was applied at 2.0-cm intervals. The pro-

CTP SYNTHETASE

ASSAY

49

cedure allowed nine applications per plate. and all samples were applied simultaneously with a carrier system containing 20 nmol each of UTP, UDP, UMP, CTP, CDP, and CMP. Sample application was carried out with great care so that the PEI-cellulose layer was not scratched. After application of the samples the plates were dried with warm air, soaked for 10 min in 500 ml of methanol, and dried with cool air. Before proceeding with ascending chromatography, the plates were predeveloped 5 cm in methanol; this procedure was absolutely essential to prevent trailing of the nucleotides and for maximum separation of UTP from CTP. Ascending chromatography was accomplished in either of two ways: (i) at 4°C in rectangular glass tanks containing 100 ml of 0.8 M (NH&SO4 (Solvent I); or (ii) by predeveloping the plates 3 cm at room temperature in rectangular glass tanks containing 100 ml of 1 M acetic acid, and then placing them into another tank containing 100 ml of 1 M acetic acid: 3 M LiCl(9: 1) (Solvent II), and developing the plates to the top. Upon completion of chromatography (4.0 h) the plates were dried with hot air, marked with radioactive black ink, and exposed to 8 x 10 in. medical X-ray film (Kodak, X-Omat R film, XR-2). The exposure time depended on the need for analysis of all breakdown products of UTP and CTP. When UTP was to be visualized, the film was exposed for 12 h, whereas a 48-h exposure was required to detect low counts per minute in CTP and other nucleotides. After developing the X-ray film, the PEIcellulose plates and X-ray film were matched by the previously applied radioactive black ink, the nucleotides were identified by ultraviolet light, and the appropriate areas were excised and placed into counting vials to which was added 7.5 ml of scintillation fluid (LSC complete, 5.0 g of 2,5diphenyloxazole and 0.2 g ofp-bis-[2-(5-phenyloxazoyl)] benzene per liter of toluene, Yorktown Research). The radioactivity due to 14C was determined with a liquid scintillation system (LS-335, Beckman or Packard TriCarb, Model 3390). The efficiency of counting on PEI-cellulose was 71% in the scintillation fluid. RESULTS Separation

of Nucleotides

by Thin-Layer

Chromatography

Migration rates decreased in the order: uridine (thymidine) > cytidine > adenosine > guanosine nucleotides with diphosphates preceding the corresponding triphosphates (2,18,19). The compounds were resolved in the following ascending order: GTP, ATP, CTP, UTP, CDP, UDP (CMP), UMP, and nucleosides. UDP and CMP were always located in the same spot (Fig. 1). Darkened areas at the top of the X-ray chromatograph did not contain the cytidine bases or their nucleosides. When known 14C-labeled bases and nucleosides were applied, uracil, cytosine, thymine, uridine, cytidine, deoxyuridine, deoxycytidine, and deoxythymidine were not detected at the leading front of the chromatogram, but only uridine.

50

WILLIAMS

ET AL.

FIG. 1. Chromatog~ph~c patterns of the carrier substances and reaction products in 0.8 M (NH&SO, at 4°C. Ten microliters of sample and a carrier solution containing 20 nmol of each compound were applied at the origin (2.0 cm from the bottom of the 20 x 20 cm PEI-cellulose plate), and ascending chromatography was performed. The markers were visualized under a short-wave uv lamp and compared to the darkened areas after exposing the chromatogram to X-ray film for 3 Days. From the origin upwards. the spots are GTP, ATP, unknown (UNK), CTP. UTP, CDP, UDP (CMP), UMP. and (deoxy) nucleosides (N) of uridine and/or deoxyuridine. Column A. products generated during a 60-min incubation of [‘%]CTP and [14C]UTP with liver extracts. Column B. standard separation of [14C]CTP from [Y]UTP. The UNK spot was a contaminant of [‘%]CTP.

CTP SYNTHETASE

ASSAY

51

deoxyuridine, and thymidine. These results were verified by two-dimensional chromatography in which the monophosphates, diphosphates, and triphosphates were resolved ( 18). The resolution of nucleotides (ATP-ADP-AMP, GTP-GDP-GMP, UTP-UDP-UMP, and CTP-CDP-CMP) by 0.8 M (NH&SO, as solvent was quite easily achieved in one dimension (Fig. 2). Drvrlopment of on Isotopic. Method for thr Assuy for Rat Liver arld Hepatoma Prrparations

of CTP Sase Activity

Previous methods (7,20) did not lend themselves to easy manipulation of a large number of samples because they required large sample sizes and since results were of questionable reliability with less-than-purified preparations. The isotopic technique of Genchev (7) was successful in demonstrating CTP Sase activity in lOS.OOOg (for 80 min) supernatant fluids of 25 to 30% homogenates (w/v) of rat liver and other soluble cell fractions by including PEP in the reaction mixture. However, we observed that this method still required a large sample which was replete with nucleotide phosphatase and, more importantly, adenylate kinase activities (1). The interfering enzyme activities in the lOO,OOOg supernatant fluid prepared in a buffered solution (pH 7.4) containing Gin. ME, Tris(hydroxymethyl)aminomethane, and sucrose (GMTS. 7) degraded 50% of the UTP in 30 min (not shown). However, 10 mM NaF in the presence of PEP markedly reduced the degradation of UTP, so that only 3% was lost (not shown). The inclusion of PEP, a substrate for pyruvate kinase (EC 2.7.1.40). by itself failed to reduce the degradation of UTP. It was conceivable that NaF inhibited pyruvate kinase activity (3), but this was shown not to be the case in liver, hepatoma 8999, or S. typhimurium. In fact, there was a moderate increase (1.6-fold) in hepatic pyruvate kinase activity. The activity of a purified rabbit muscle preparation (Calbiochem) showed a 20% inhibition with 10 mM NaF. The effect of NaF in the presence of PEP was to protect the CTP and UTP concentrations in the supernatant fluids of both liver and hepatoma samples (Fig. 3). Seventy percent inhibition of adenylate kinase (1) activity by 10 mM NaF also achieved maintenance of the ATP pool (data not shown). Indeed, other investigators showed that fluoride inhibited ATPase and adenylate kinase activities (3,9,20); these enzymes may reduce the pool of ATP, GTP, CTP, and UTP. Mixing of the supernatant fluids of liver and hepatoma was carried out to test the application of this method (Fig. 4). When liver and hepatoma supernatant fluids were mixed without NaF. but in the presence of PEP, the apparent CTP Sase activity for hepatoma was lowered; however, NaF restored the activity to the characteristic level for hepatoma (Fig. 4). In the presence of 10 mM NaF and PEP, liver CTP Sase activity was markedly increased, whereas only a small increase in hepatoma activity was observed. When tissue preparation was carried out in 0.15 M KCI, the addi-

WILLIAMS

ET AL.

ATP

c FIG. 2. An analysis of nucleotide pairs by chromatography with 0.8 M (NH&SO, at 4°C. The markers were visualized under a short-wave uv lamp and compared to the darkened areas after exposing the chromatogram to X-ray film for 3 days. From left to right and the origin upwards, the spots in the columns are: A. UTP, UDP: B, CTP, CDP; C. ATP. ADP; and D. GTP. GDP. The monophosphate always preceded the diphosphates (not shown)

tion of NaF reduced the degradation of UTP and CTP and 10% and that by hepatoma extracts to 15 and 8%, A comparison of CTP Sase activities in the GMTS buffers showed that KC1 extracted more CTP Sase

by liver extracts to 7 respectively (Fig. 4). and KC1 extraction activity (Table 1).

CTP SYNTHETASE

The Hydrolysis

Method

53

ASSAY

(Table 2)

The hydrolysis of CTP to CMP in the presence of HCl has been the method of choice with the radioassay (7,20), and the products were separated by Dowex 50-H+ resin or paper chromatography (2), which resolved only CMP and UMP. We chose the PEI-cellulose system (Solvent I and II) in order to determine which hydrolysis products were generated. When 5 ~1 of [14C]CTP (478 mCiimmo1, 92% cytidine nucleotides) or [14C]CMP (516 mCi/mmol, 95% cytidine nucleotides) was added to the standard reaction mixture these compounds were recovered by 96 and 97% as [14C]CMP after a 90-min hydrolysis. However, after a 30-min reaction time in the presence of liver and hepatoma extracts followed by hydrolysis, only 87 and 86% of the [14C]CMP were recovered, whereas 100% of the [‘“CJCTP was recovered as CMP with liver extract, and 89% was recovered with hepatoma extracts. Hence, the resolution of nucleotides with the (NH,),SO, system without prior hydrolysis gave consistently higher specific activities (Tables 1 and 2).

The sensitivity of the assay method was enhanced by blanks (i.e., reaction mixture which received PCA before the sample) of 90 to 130 cpm in

LOSS

OF ‘+Z-UTP

LOSS

OF ‘%-CTP I

NaF NaF NaF t

NaF

t t

5z

40

n,0 l ,O

H, 0 - HEPATOMA (39248) ,O - LIVER (ACI/N)

l F 2

- HEPATOMA - LIVER

(3924A)

(ACI/N)

20-

0-

' 0

I IO

I 20

I 30 REACTION

FIG. 3. Comparison of effects of NaF addition [l”C]CTP (Panel B) in 15% 100,OOOg supernatants in 0.15 M KCI.

/ 0

I IO

I 20

I 30

TIME (MIN) on the loss of [“CIUTP of liver and hepatoma

(Panel A) and 3924A prepared

1.2 0

No NaF

5 LA?

0 LlYER tIEPATOMA

35

i

0.90 0.25

- 0.60 0.50

-

0.30 0.75

UVER

0.90 0.25

I T-r 8.2 0

IO mM NaF

PROTEIN CONTENT (mg)

0 1.0

f

L

T #

0.30 0.75

1

0 1.0

:----% # 0.60 0.50

LIVER

I

FIG. 4. Effect of varying the protein concentration and mixing of liver and hepatoma 3683F in the presence and absence of 10 mM NaF. The specific activity of [VIUTP was 6137 cpminmol. Fifteen percent homogenates were prepared in GMTS buffer. and the assays were carried out in the lOO.000~ supernatants. Comparable results were obtained with the 0.15 M KC1 preparations.

v -0

40

45

CTP SYNTHETASE TABLE COMPARISON

OF EXTRACTION

MEDIUM

ON

55

ASSAY I

CTP SYNTHETASE SPECIFIC ACTIVITY

CTP synthetase specific activity” Tissue Liver (ACIIN) Hepatoma 3924A

KW

GMTS”

4.2 ( 100)

(81)

3.4

24.9 (100)

22.4 (90)

KU (Hydrolysis) 3.1 (74) 21.4

(86)

” In the standard assay the activities are expressed as nmol of CTP formed per hour per mg of protein. Percentages of the corresponding controls are in parentheses. b Tissue homogenates were prepared in KCI and GMTS buffered medium (see Experimental Procedures). r Hydrolysis method was carried out on these samples (see Experimental Procedures).

the cytidine nucleotides with the standard method and 30 to 40 cpm with the hydrolysis method. Activities as low as 50 pmol of product formed were readily detected in the standard assay with a specific activity of 1100 cpm per nmol of [‘“CIUTP. In the standard assay the enzyme activity was linear up to 10% conversion of UTP to CTP (i.e., 2000 cpm per CTP spot). The applicability of this technique to samples of liver and hepatoma tissues and S. r~phimuriurn LT2 supernatant fluids were tested. The radioactivity associated with CTP was found to be linear with incubation time up to 30 min and with protein content of liver and hepatoma 3924A up to 1.62 and 1.37 mg, respectively (Fig. 5). When a partially purified (48-fold) preparation of liver enzyme (Kizaki, H., Williams, J. C.. Morris, H. P., and Weber, G., in preparation) was compared with the supernatant fluids, the reaction was also shown to be linear with time, but because of high activity, it was measured for a shorter time period. Extracts of S. typhimurium also exhibited a linear response with time and protein up to 1.09 mg (Fig. 5). CTP Sase from bacterial sources is highly sensitive to nucleotides (20). whereas the mammalian enzyme exhibits moderate responsiveness (16,17). The effect was tested of removing nucleic acids by streptomycin sulfate precipitation with subsequent dialysis to eliminate the soluble factors. The treatment had no effect on the liver or hepatoma 3924A enzyme specific activity, whereas the bacterial enzyme specific activity was increased from 36.0 to 72.0 nmol h-l mg protein-‘. Hence, the crude supernatant fluids derived from liver and hepatoma samples may be used without further treatment, whereas the bacterial samples require the removal of nucleic acids and soluble factors (14).

WILLIAMS

56

ET At.

DISCUSSION

The thin-layer chromatographic radioassay for CTP Sase reported in this paper offers a number of advantages over previously described methods (7,20). CTP Sase activity was readily detected in 15% (w/v) 100,OOOg supe~atant fluids from rat liver and hepatomas without any subsequent treatment (23). The method was also convenient for the assay of bacterial CTP Sase in 100,OOOg supernatant fluids, which had been treated with streptomycin sulfate and dialyzed to remove nucleic acids and soluble factors. This method is more direct because it does not require a hydrolysis step followed by chromatographic separation of CMP from UMP on Dowex 50-H+ resins or paper chromatography. This thin-layer chromatographic technique also allows a large number of samples to be handled at one time and separates CTP and UTP satisfactorily. Most impo~antIy, the (NH&SO, solvent system (Solvent I) resolves GTP, ATP, CTP, UTP, CDP, UDP(CMP), UMP, and other breakdown products, whereas Solvent TABLE EFFECT

2

OF HYDROLYSIS OF CTP AND CMP WITH PERCHLORK ABSENCE OF ENZYME EXTRACTS OF LIVER AND

Isotope in reaction mixturefz No extract [‘V]CTP I%Z]CMP Extract addition (30-min incubation) Liver [Y]CTP [‘V]CMP Hepatoma [‘QCTP [‘“CICMP

ACID IN THE PRESENCE HEPATOMA 3924A

AND

Hydrolytic activity (CPdlO /.LI)

Hydrolysis time” (min)

CTP

CDP

CMP

0 90 0 90

22.759 84 135 229

2063 1599 1206 I26

125 23,641 23,513 24,725

94 96 96 97

90 90

84 494

2078 711

23,057 20,493

IO0 87

90 90

84 I41

1554 605

20,98 1 20,858

89 86

Recovery’ (%x)

n No [“TJlJTP was added to the standard reaction mixtures. CTP and CMP in the reaction mixture was 2.41 pM (0.44 ~C~nmoi) and 2.29 ,uM (0.49 ~C~nmoI~~ respectively. The specific activity of CTP and CMP applied to each lane at time zero was 1100 and II33 cpmpmol. respectively. Incubation with enzyme extracts was carried out at 37°C for 30 min before adding the PCA. b Hydrolysis was performed in a boiling water bath. The final concentration of PCA per reaction tube was 3%. (’ Recovery is expressed as cpmin cytidine nucleotides and is a measure of the stability of the nucleotides in the presence of tissue extracts.

A

I

LIVER

1

CRUDE EXTRACT

I 0

1

TIME

CRUDE EXTRACT

HEPATOMA

I

(MIN)

0

I

H3924A 0.96 m g

C

I

IO

1

I

5

CRUDE EXTRACT

S. typhimurium -

LT2

15

I

I .06 m g I

FIG. 5. Time course of CTP Sase reaction with liver, hepatoma 3924A. and Salnronrlla t~plzimuriu~ LT2 employing 100.000~ supernatants. The specific activity of [“C]UTP was 1165 cpminmol in Panel A and B and 1826 cpminmol in Panel C. Panel A. comparison of partially purified and crude supernatants of liver. Panel B. comparison of crude supernatants of hepatoma 3924A. Panel C, enzyme activity of S. t~phi~nrrriun~ LT2 from a crude supernatant.

1

r

Y

58

WILLIAMS

ET AL.

II resolves CMP and UMP in one dimension. Although the location of the bases, nucleosides, and nucleotides was verified by X-ray film in the preliminary studies, it was not necessary to continue this step in the standard assay system, because the uv-absorbing nucleotide carriers were clearly visualized. Hence, the method can be applied to the analysis of CMP and UMP after hydrolysis and to other nucleotides which are required for the reaction (6). The identification of interfering enzyme activities through the resolution of the breakdown products is important during the purification of CTP Sase. This method should therefore greatly simplify the analysis of extraneous enzymatic activities involved in nucleotide metabolism during purification (16,17) and subsequent kinetic analysis of CTP Sase in which the resolution of nucleotide pairs (GTP-GDP, and ATP-ADP) is required. During these investigations we discovered that NaF provided protection for the CTP produced by preventing degradation of the nucleotides, and, importantly, that NaF did not significantly inhibit pyruvate kinase activity. This enzyme is required for the phosphorylation of the diphosphates to triphophates in the presence of PEP. Hence, the combination of PEP and NaF in the reaction mixture provides protection for the triphosphate pools during the time course of the reaction. This effect is probably due to an inhibition of adenylate kinase by NaF (and possibly nucleotide phosphatases) coupled with the lack of interference with pyruvate kinase activity in the presence of PEP. Specific activities for CTP Sase reported here for rat liver are IO-fold higher than previously observed (7). Hence, this assay is suited for the detection of CTP Sase in crude extracts and for examining the kinetics of the enzymatic reaction. Subsequently, enzyme assays can be confined to the measurement of initial velocities with greater sensitivity in order to minimize possible product inhibitory effects. ACKNOWLEDGMENTS This investigation was supported by the Research and Development Command, Department of the Navy, Research Task No. MR041.05.01.0028 and from the U. S. Public Health, National Cancer Institute, CA-13526 and CA-05034.

REFERENCES 1. Adelman. R. C., Lo, C. H.. and Weinhouse, S. (1968) J. Biol. Chem. 243, 2358. 2. Brockman. R. W.. Shaddix. S. C.. Williams, M., Nelson, J. A., Rose, L. M., and Schabel, F. M. Jr. (1975) Ann. N. Y. Acad. Sci. 255, 501. 3. Bucher. T.. and Pfleiderer. G. (1955) In Methods in Enzymology (Colowick. S. P.. and Kaplan, N. 0.. eds.). Vol. I, p. 435. Academic Press, New York. 4. Chakraborty. K. P.. and Hurlburt, R. B. (1961) Biochim. Biophys. Acfa 47, 607. 5. Cohn, W. E. (1949) Science 109, 377. 6. Genchev. D. D.. and Hadjiolov, A. A. (1969) FEBS Let?. 3, 147.

CTP SYNTHETASE

ASSAY

59

7. Genchev, D. D. (1973) Expcrienria 29, 789. 8. Hurlbert. R. B., and Kammen, H. 0. (1960) J. Biol. C/rem. 235, 443. 9. Kellen, R. A., Kinahan, J. .I., Foltermann. K. F.. and O’Donovan. G. A. (1975) J. Bac,teriol. 124, 764. IO. Kielley, W. W.. and Kielley, R. K. (1951) J. Bid. Chum. 191, 485. Il. Koshland, D. E.. Jr.. and Levitzki, A. (1974) in The Enzymes (Bayer. P. D., ed.). p. 539. Academic Press. New York. I?. Lederer. M. (1964) J. Chron~u~ogr. 13, 232. 13. Lieberman. I. (1956) J. Bit>/. Chew. 222, 765. 14. Long, C. W., and Pardee, A. B. ( 1967) J. Bid. Chrm. 242, 47 15. 15. Lowry. 0. H., Rosebrough. N. J.. Farr. A. L., and Randall, R. J. (1951) J. Bid. Cham. 193, 265. 16. McPartland, R. P.. and Weinfeld, H. (1975) Fed. Proc. 34, 549. 17. McPartland, R. P., and Weinfeld. H. (1976) J. Bid. Chrm. 251, 4372. 18. Neuhard. J.. Randerath. E.. and Randerath, K. (1965) And. Biochcm. 13, ?I I. 19. Randarath. K. (1964) Experirnriu 7, 406. 20. Savage. C. R.. and Weinfeld. H. (1970) J. Bid. Chem. 245, 2429. 21. Siekevitz. P.. and Potter. V. R. (1953) J. Bid. Chem. 200, 187. 22. Williams. J. C.. Weber. G., and Morris. H. P. (1975) Nature (London) 253, 567. 23. Williams. J. C.. Kizaki. H.. Weber. G.. and Morris. H. P. (1978) Nnfurr (London) 21, 71.