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Biochimica et Biophysica Acta, 395 (1975) 221--228 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98346

DETERMINATION OF UTP AND ATP POOL SIZES IN HUMAN TONSILLAR LYMPHOCYTES BY USING ESCHERICHIA COLI RNA POLYMERASE

MARIA SASV.~RI-SZI~KELYa, M,~RTA VITI~Zb, M.~RIA STAUB a and F. ANTONI a alst Institute of Biochemistry, Semrnelweis University of Medicine, 1088 Budapest and bInstitute of Biophysics, Medical School of Debrecen, 4028 Debrecen (Hungary)

(Received December 10th, 1974)

Summary The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts. The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template. A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions. The calculation takes into account the isotope dilution effect caused by the intracellular ATP. Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test. According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per #g RNA.

Introduction The assay of [ 3H] uridine incorporation is generally used for the measurement of the rate of RNA synthesis in whole cells. Recently; however, the question has frequently been raised, how could this method serve as a true index of the rate of RNA synthesis, if the uptake of this tracer is the rate limiting step [1]. These findings made it necessary to correct the data of the incorporation f o r the specific radioactivity of the UTP pool. But the direct measurement of the UTP pool has not been solved, therefore, a few indirect methods were developed for the correction [2,3]. The use of [14C] adenine instead of uridine has also been proposed, because the pool of ATP could easily be determined by using luciferase [4]. Another way was to measure the total amount of uridine nucleotides in the form of UMP [ 5]. The fundamental principle of our method is the same as that of Lindberg

222

and Skoog [6] used for the determination of the deoxyribonucleoside triphosphate pool sizes. RNA polymerase catalyzes the formation of a ribonucleotide polymer from ribonucleoside triphosphates. Using poly(dA-dT) as template, the synthesized polymer consists of AMP and UMP. If one of the ribonucleoside triphosphates present in excess is radioactive, and the other is the only limiting factor of the rate of the reaction, the rate of the incorporation into a macromolecular fraction depends directly on the concentration of the limiting nucleotide. Materials and Methods Chemicals [14C]ATP (53.0 Ci/mol) and [3H]UTP (1.0 C i / m m o l ) w e r e purchased from the Radiochemical Centre, Amersham; poly(dA-dT) was obtained from Miles Laboratories. The [ 3HI UTP was diluted 20-fold with non-labelled UTP, the [14C] ATP was undiluted. ATP and UTP were products of Reanal, Budapest. Preparation o f the cell extract Cells were prepared from freshly removed tonsils of children, 3 - 6 years of age, by the m e t h o d of Piffk5 et al. [7]. The suspension culture of 10 ~ cells per ml was washed twice with ice-cold 0.15 M NaC1 at 0°C. After the collection of the cells by centrifugation (400 X g, 3 rain, 0°C), 0.5 M ice-cold perchloric acid (0.2 ml per 107 cell) was added to the samples at 0°C. The perchloric acid soluble fraction of cells was used for the determination of its nucleotide content after neutralization with KOH. The preparation was carried out strictly at 0° C. The cell extract was stored at --20 ° C or used immediately. Standard conditions for the assay o f ribonucleotides The preparation and the assay of RNA polymerase were carried out according to Burgess [8]. For the experiments the core enzyme (spec. act., 400 units per mg) was used. The standard reaction mixture contained the following components in a final volume of 0.05 ml: radioactive nucleotide, 800--2500 pmol; limiting nucleotide, 25--200 pmol; RNA polymerase, 20 pg; poly(dAdT), 4 pmol; Tris" HC1 buffer, pH 7.9, 1.6 #mol; MgC12, 0.4 pmol; EDTA, 0.004 pmol; dithiothreitol, 0.004 pmol; KC1, 6 pmol. The reaction mixture was incubated at 37°C for 10 min, then chilled in an ice-water bath. An aliquot of 40 pl was placed on a strip of Whatman 3 paper and a descending chromatogram was developed in butanol/acetic acid (3 : 1, v/v) to remove the low molecular weight sub~txates. The newly synthesized RNA remained at the starting point. After drying, the paper was measured in a toluene-based mixture. A standard curve was made with known amounts of ribonucleoside triphosphate whenever the assay was used. Results

The time dependence o f the RNA polymerase reaction Fig. 1 shows the time dependence of the RNA polymerase reaction in the

223 Dmol of UTP

/

~C

/

z~/

x

180 z~

120

m / m

/ /'/ A.// ,~~.

E

~ ? _ _° _ ~ 0 .,..,~v~.~f,.,,~"~/E' _ _"_ _ _ _ ~ 0el

extract

10 20 M[n of incubQtion Fig. 1. T h e t i m e curve o f the R N A p o l y m e r a s e r e a c t i o n . R e a c t i o n s w e r e run w i t h U T P in l i m i t i n g a m o u n t s o f 0 p m o l ( o ) , 4 0 p m o l ( v ) , 1 2 0 p m o l (m), and 1 8 0 p m o l (~). T h e a m o u n t o f [ 1 4 C ] A T P w a s 8 0 0 p m o l .

presence of different amounts of the limiting nucleotide. In this experiment [ 1 4 C] ATP was present in excess and UTP was the limiting nucleotide. On the basis of these results, we decided to use an incubation time of 10 min for further experiments, in which the initial rate of the RNA polymerase reaction could be measured.

Standard curves for the RNA polymerase test (direct method) The initial rate of the RNA polymerase as a function of the concentration of the limiting nucleotide gives a straight line as indicated in Fig. 2. The standard curves of the RNA polymerase test are suitable for the determination of an unknown amount of the limiting nucleotide. Investigation of possible inhibitors present in the cell extracts In the experiments indicated in Table IA, the effect of the addition of a neutralized cell extract on the recovery of ATP has been tested. According to A 4

o f o-

oJ

E G. u2

J

/oJ

o/° ~6o

~6o

pmol of UTP

~o

2~o

pmo{ of

ATP

Fig. 2. T h e initial rate o f R N A p o l y m e r a s e as the f u n c t i o n o f the c o n c e n t r a t i o n o f l i m i t i n g n u c l e o t i d e . ( A ) D i f f e r e n t a m o u n t s o f A T P w e r e a d d e d t o t h e standard r e a c t i o n m i x t u r e in t h e p r e s e n c e o f 2 4 0 0 p m o l [ 3 H I UTP. (]8) D i f f e r e n t a m o u n t s o f U T P w e r e a d d e d t o the standard r e a c t i o n m i x t u r e in t h e p r e s e n c e of 8 0 0 p m o l [ 1 4 C ] ATP. A f t e r an i n c u b a t i o n p e r i o d o f 1 0 m l n , t h e i n c o r p o r a t e d n u c l e o t i d e s w e r e m e a s u r e d as d e s c r i b e d u n d e r M e t h o d s .

224

TABLE I INVESTIGATION

O F P O S S I B L E I N H I B I T O R S P R E S E N T IN T H E C E L L E X T R A C T

In t h e p r e s e n c e o f t h e cell e x t r a c t , the i s o t o p e d i l u t i o n e f f e c t o f A T P , d e r i v e d f r o m cells, was t a k e n into account. A

A d d i t i o n to t h e R N A p o l y m e r a s e r e a c t i o n m i x t u r e

[3H] UTP incorporation (cpm)

ATP (pmol)

No 60 10 20 60

addition pmol ATP pl cell e x t r a c t ~tl cell e x t r a c t p m o l A T P + 1 0 ~tl cell e x t r a c t

50 1250 1200 2420 2410

-(60) 60 120 120

B

A d d i t i o n to the R N A p o l y m e r a s e r e a c t i o n m i x t u r e

[14C] ATP incorporation (cpm)

13TP ( p m o l )

No 60 60 10 60

addition pmol UTP p m o l U T P + 10/.ll s a t d K C I O 4 #l cell e x t r a c t p m o l U T P + 1 0 #l cell e x t r a c t

40 1300 1340 1320" 2620*

-(60) 60 60 120

the results 10 pl of a 4-fold diluted cell extract contained 60 pmol of ATP measured with our assay. When this amount o f the extract was added to 60 pmol of ATP, a total of 120 pmol of ATP was recovered. No inhibitors interfering with our assay could be determined in the cell extract. The same problem has been investigated in the presence of UTP as the limiting nucleotide (Table IB}. If the amount of the [~4C] ATP present in excess was low, the [ ~ 4C] ATP was diluted by the high ATP content of the cell extract, and the dilution effect had to be taken into account when determining the UTP content. This dilution effect was lower, if the amount of [ ' 4 C] ATP was higher (results not presented}, therefore it became possible to eliminate this effect by an appropriately high [14C] ATP concentration. However, the oI

£ t)

100

pmol

of UTP 200

Fig. 3. D e t e r m i n a t i o n o f the A T P and UTP c o n t e n t o f the cell e x t r a c t u n d e r the s a m e e x p e r i m e n t a l c o n d i t i o n s . T h e initial rate o f R N A p o l y m e r a s e as a f u n c t i o n o f t h e c o n c e n t r a t i o ~ o f UTP w a s m e a s u r e d in t h e p r e s e n c e ( L i n e II) o r in the a b s e n c e ( L i n e I) o f 1 0 #1 e x t r a c t . T h e a m o u n t o f [ 1 4 C ] A T P w a s 4 0 0 p m o L A f t e r an i n c u b a t i o n p e r i o d o f 1 0 r a i n , t h e i n c o r p o r a t e d n u e l e o t i d e s w e r e m e a s u r e d as d e s c r i b e d u n d e r M e t h o d s . T h e i n c o r p o r a t i o n m e a s u r e d in t h e p r e s e n c e o f t h e cell e x t r a c t w i t h o u t a d d e d U T P (see a r r o w ) w a s s u b t r a c t e d f r o m e a c h value m e a s u r e d in the p r e s e n c e o f a d d e d U T P p l u s 10/~1 cell e x t r a c t ( L i n e II). A d e t a i l e d e x p l a n a t i o n and c a l c u l a t i o n s are given in the t e x t .

225

T A B L E II ATP AND UTP CONTENT OF HUMAN TONSILLAR

CELLS

Experiment No.

pmol UTP/~g RNA

pmol ATP/pg RNA

1 2 3 4

70 89 80 78

350 326 308

Average

80

340

dilution effect could also be used for the determination of both ATP and UTP pool sizes in the cells under the same experimental conditions (see below). Determination o f A T P and UTP pool sizes under the same experimental conditions The m e t h o d for the determination of both UTP and ATP pool sizes described above (direct method) requires two different experimental conditions and two corresponding standard curves. However, the determination of both UTP and ATP content of cell extracts is also possible in the same experiment taking into account the isotope dilution effect (indirect method). The standard curve for UTP determination indicates the limiting UTP concentration as a function of [~4C] ATP incorporation (Fig. 3, Line I). The arrow on the ordinate in Fig. 3 indicates the incorporation measured in the presence of 10 pl extract and no added UTP. In order to control the influence of the extract on the incorporation of [~4C] ATP, the standard curve has been determined using various amounts of UTP in the presence of constant a m o u n t (10 pl) of extract. Subtracting the value of incorporation obtained with the extract only (see arrow in Fig. 3), from values measured in the presence of 10/~1 extract plus known amounts of UTP, Line II (Fig. 3) was obtained instead of the theoretical Line I expected. The lower slope of Line II could only be a consequence of [14 C] ATP dilution by ATP present in the extract. The same effect was not observed when [ 3 H ] U T P incorporation was measured in the presence of the extract (Table IA) indicating a m u c h lower concentration of UTP in the cells. Line II, calculated in the same way, coincided with Line I (see Table IB). The degree of dilution of [ ~4 C] ATP can be easily calculated by dividing the slope of Line I by the slope of Line II. In this experiment, the dilution was 1.8-fold (Fig. 3). 400 pmol of [ ~4C] ATP were present, consequently the 10 pl of the extract contained 320 pmol o f ATP. Line II can be used for the determination of UTP content in the extracts as indicated by the arrow in Fig. 3. The advantage of this m e t h o d is that UTP and ATP c o n t e n t of the extracts could be determined in the same experiment and lower amounts of [ 14 C] ATP were sufficient.

Discussion A rapid, specific and sensitive m e t h o d has been presented for the determination of ATP and UTP content in biological materials. The neutralized acid

226

soluble fraction of l y m p h o c y t e s was suitable for the RNA polymerase test. The pool sizes of t w o ribonucleotides in human tonsillar cells are shown in Table II. The indicated data are results of four separate experiments using cells from different individuals. (The ATP content of tonsillar lymphocytes was calculated both by the 'direct' and 'indirect' methods and was found to be the same.) These data demonstrate that the method is well reproducible, if the conditions of the preparation of the cell extract and of the RNA polymerase test were standardized. Ribonucleotide pool sizes of different cell types, determined by different methods, were only available for comparison. The ATP pool of Chinese hamster V79 cells determined b y luciferase was 1 - 2 . 5 nmol per 106 cells [4]. The ATP and UTP pools of mouse l y m p h o m a L 51784 cells measured by high-pressure liquid chromatography were 2.4--3.6 and 1.6 nmol per 106 cells, respectively [ 9 ] . The UTP pool of superfused rat liver slices calculated from published data were found to be below 1 pmol per pg RNA [5]. The Novikoff hepatoma cells contain approximately 3 nmol of uracil nucleotides and 8 nmol o f adenine nucleotides per 106 cells, that is 150 pmol and 400 pmol per pg RNA, respectively [10]. Tonsillar l y m p h o c y t e s contain 80 and 340 pmol of UTP and ATP per #g RNA, respectively, that is 160 pmol of UTP and 680 pmol of ATP per 10 6 cells. These cells, consisting mainly of small l y m p h o c y t e s ( 7 0 - 8 0 % ) [11], contain much less RNA (2 #g per 106 cell) than other cell types, for instance, Novikoff hepatoma cells (20 pg per 106 cell). The differences in the data might be due to the different cell types or to the different methods which were used. The calculation of the specific radioactivity of the UTP pool is necessary for correction of [ 3H]uridine incorporation into RNA of cells [1--5,12]. According to our experiments the RNA polymerase test is suitable for the direct measurement of the UTP pool making the determination of the specific radioactivity of the UTP pool possible. Appendix

Mathematical interpretation of calculations (indirect method) The equation of Line I in Fig. 3 is: V = K- c (1) where V = the measured rate of incorporation, c = the concentration of added UTP and K = a constant (the slope of the Line I in Fig. 3). No inhibitors or activators were f o u n d to be present in the cell extract (see Table I), so there was reason to presume that the data of the incorporation measured in the presence of the cell extract and in the presence of added UTP were the sum of the corresponding data of the incorporations measured separately. In this case the equation of Line II will be: V' -- Vcell = K' " (x -- Ccell )

(2)

where V ' = the rate of incorporation measured in the presence of the cell extract plus added UTP; Vcen = the rate of incorporation measured in the

227

presence of the cell extract alone; x = the total a m o u n t of UTP; c~en = the a m o u n t of UTP derived from the cell extract; K' = another constant and x = c + Ceell.

Thus Eqn 2 can be changed into the next form: V'

V¢~u_ - K

--

s

"c

(3)

where c = the a m o u n t of added UTP. If V' = 2V~en then x = 2 C c e n , and Eqn 3 can be changed into the form:

2V¢~n - V¢~n = K' • (2c~ H-cc~ n)

(4)

or simplified: Y c e l l -~ g '

• Ccell

(5)

It means, t h a t substituting the value of Veell into Eqn (5), the value of the UTP c o n t e n t of the cell extract can be calculated from the slope of the Line II, which is equal to K'. The calculation of the ATP c o n t e n t of the cell extract is based on the meaning of the constants: Constant K from Eqn 1 depends on the specific radioactivity of [,4 C] ATP and on the rate constant of the RNA polymerase reaction: K = k~ m

(6)

where k = the rate constant of the RNA-polymerase reaction, A = the radioactivit y of [' 4 C] ATP and m = the a m o u n t of [' 4 C] ATP. According to our experiments (see Table I), the cell extract contained no inhibitors or activators of the RNA polymerase reaction. Therefore the value of k will n o t be changed in the presence of the cell extract, and K' from Eqn 2 will be: K'=k

A m+--'---y

(7)

where y = the a m o u n t of ATP derived from the cell extract. From Eqns 6 and 7, the degree of dilution o f [ ' 4 C ] ATP can be determined f r o m a simple ratio of the slopes: K K'

rn+y m

which means t h a t by substituting the values of K (the slope of the Line I), K' (the slope o f the Line II) and m (the a m o u n t of [ ' 4C] ATP), the ATP c o n t e n t of the cell extract (y) can be calculated.

228

Acknowledgements The authors wish to thank Dr Lambert Skoog for his valuable comments and suggestions during the preparation of this manuscript, as well as Professor S~mdor Damjanovich for his helpful collaboration and advice in the preparation and measurement of RNA polymerase. References 1 2 3 4 5 6 7 8 9 10 11 12

Plagemaun, P.G.W. and Roth, M.F. (1969) Biochemistry 8, 4782--4789 Cooper, H.L. (1973) Anal. Biochem. 53, 49--63 Forsdyke, D.R. (1971) Biochern. J. 125, 721--732 Stambrook, O.J. and Slsken, J.E. (1972) Biochim. Biophys. Acta 281, 45--54 Tseng, J.K. and Gurpide, E. (1974) Biochim. Biophys. Acta 353, 399--406 Lindberg, U. and Skoog, L. (1970) Anal. Biochem. 34, 152--160 Piffk6, P., K6teles, G.J. and Antoni, F. (1970) Prac. Oto-rhino-laryng 32, 350--355 Burgess, R.R. (1972) in Procedures in Nucleic Acid Research (Cantoni, G.L. and Davies, D.R., eds), Vol. II0 Academic Press, New York Snyder, F.F., Henderson, J.F., Kim, S.C. and Paterson, A.R.F. (1973) Cancer Res. 33, 2425--2430 Plagamann, P.G.W. (1971) J. Cell. Physiol. 77, 213--240 Zucker~FrankUn, D. and Barney, S. (1972) J. Exp. Mad. 135, 533--548 Sasv~i-Sz~kely, M. and Staub, M. (1974) Abstr. 9th FEBS Meet. Budapest, p. 152