Properties of deoxythymidine kinase partially purified from noninfected and virus-infected mouse fibroblast cells

WaOLOGY26, 16--27 (1965)

Properties of Deoxythymidine Kinase Partially Purified from Noninfected and Virus-Infected Mouse Fibroblast Cells S A U L K I T A~D D. R. D U B B S

Division of Biochemical Virology~ Baylor University College of Medicine, Houston, Texas Accepted January 20, I965 Deoxythymidine kinase, partially purified from noninfeeted LM mouse fibroblast cells, was rapidly inactivated when incubated in the absence of substrates at 38°C. The half-life of the enzyme was 29-43 minutes whether prepared from LM cells in the logarithmic phase of growth, the phase of negative growth acceleration, the stationary phase, or from LM cells which had been incubated for 24 hours with 50 t~g deoxythymidine per milliliter prior to harvest. The deoxythymidine kinase induced in LM (TK-) cells by herpes simplex virus was also very unstable. In contrast, deoxythymidine kinase prepared from vaecinia-infected LM or LM (TK-) cells was considerably more stable with a half-life greater than 300 minutes. The addition of ATP (15 mM), dTTP (0.025 raM), or deoxythymidine (0.05 to 0.1 raM) significantly protected the LM cell enzyme against thermal inactivation, ttowever, charcoal treatment of the vaccinia-indueed enzyme did not decrease the stability of that enzyme. Immunological differences and differences in apparent Michaelis constants for deoxyuridine were also observed between the vaccinia-induced deoxythymidine kinase and the enzyme from noninfected LM cells. However, the temperature characteristics of the reactions, calculated from the V,~ values at 38 ° and 30 °, were similar, and these enzymes were nearly equal in susceptibility to inhibition by dTTP. With either enzyme, the phosphorylation of deoxyuridine was competitively inhibited by fluorodeoxyuridine, bromodeoxyuridine, iododeoxyuridine, or deoxythymidine. The phosphorylation of deoxythymidine was inhibited by low concentrations of bromodeoxyuridine or iododeoxyuridine, and by high concentrations of deoxyuridine or fluorodeoxyuridine. INTRODUCTION

be presented d e m o n s t r a t e t h a t the vaceiniainduced e n z y m e and the e n z y m e f r o m noninfected L M cells differ immunologically, in kinetic properties, and in t h e r m a l stability. P r e l i m i n a r y reports of these findings have been presented (Kit and Dubbs, 1964a, b).

Following infection b y vaceinia or herpes simplex viruses, d e o x y t h y m i d i n e kinase act i v i t y is induced in mouse fibroblast cells (strain L M ) and in a m u t a n t subline [strain L M ( T K - ) ] , which is defieient in e n z y m e a c t i v i t y (Kit, a n d D u b b s , 19a3, 1964b; Kit et al., 1963a, b, c). D e o x y t h y m i d i n e kinase has now been partially purified from noninfected and fl'om virus-infected cells, and the partially purified e n z y m e s have been c o m p a r e d with respect to: (1) substrate specificity and kinetic properties; (2) feedback inhibition; (3) t h e r m a l stability; (4) metabolite and substrate stabilization; a n d (5) immunological properties. T h e d a t a to

MATERIALS AND METHODS

Growth of cells in culture. L M mouse fibroblast cells and a m u t a n t subline, L ~ I ( T K - ) , were p r o p a g a t e d in suspension cultures as previously deseribed (Dubbs and Kit, 1964d; K i t a n d D u b b s , 1962). L M ( T K - ) cells are resistant to g r o w t h inhibition b y high concentrations of bromo16

PARTIALLY

PURIFIED

DEOXYTHYMIDINE

deoxyuridine (BUdR),~ iododeoxyuridine (IUdR), and deoxythymidine (TdR), and are deficient in deoxythymidine kinase activity (Dubbs and Kit, 1964d; Kit et al., 1963a).2 Virus strains. The I H D strain of vaccinia and the O'Connell strain of herpes shnplex were used. The procedures for the preparation of virus stocks, for the plaque assays, and for the viral induction of deoxythymidine kinase, have been described (Dubbs and Kit, 1964a, b). Enzyme assays. Either deoxyuridine-II3 (UdR-H 3) or TdR-HH3 was employed as substrate in the deoxythymidine kinase assay (Kit et al., 1963b, c). Enzyme preparations catalyzed the phosphorylation of UdR-tI ~ to dUMP-tI 3, but did not catalyze the further phosphorylation of dUMP-H 3 to to dUDP-It 3 and dUTP-HL Thus, with UdR-I.I 8 as substrate, a one-step reaction was studied. Because of the presence of deoxythymidylate kinase and deoxythymidine diphosphate kinase, variable amounts of d T D P - H 3 and dTTP-I-I~ were formed when TdR-H 3 was the substrate. For most of the experiments involving TdR-H 3, dTMP, dTDP, dTTP, and TdR were separated by chromatography on Whatman DE-81 diethylaminoethylcellulose paper (0.4 meq/g) with 4 M formic acid, 0.1 M ammonium formate as the solvent, and the radioactivity of each of the products was determined. For the initial experiments in which UdR-H ~ was the substrate, the

~Abbreviations: TdR, deoxythymidine; UdR, deoxyuridine; BUdR, 5-bromodeoxyuridine; IUdR, 5-iododeoxyuridine; FUdR, 5-fluorodeoxyuridine; dUMP, deoxyuridylate; dUDP, deoxyuridine diphosphate; dUTP, deoxyuridine triphosphate; dTMP, deoxythymidylate; dTDP~ deoxythymidine diphosphate; dTTP, deoxythymidine triphosphate; dCDP, deoxycytidine diphosphate; dCTP, deoxycytidine triphosphate; dATP, deoxyadenosine triphosphate. Recent studies using very high specific activity deoxythymidine-tt~ as substrate have shown that a low level of deoxythymidine kinase activity persists in LM (TK-) cells (E. G. Hampton, personal communication). These experiments have been confirmed in Houston, and it has been estimated that LM (TK-) cells exhibit less than 0.5% the deoxythymidine kinase activity of exponentially growing LM cells.

KINASE

17

products of the kinase assay were also separated on Whatman DE-81 diethylaminoethylcellulose paper. However, after it was determined that dUDP-H 3 and dUTP-H 3 were not formed, dUMP-H 3 and UdR-tt 3 were resolved by chromatography on Whatman no. 4 filter paper. The solvent for chromatography was made by mixing 770 ml of n-butanol, 150 ml of water, and 9.2 ml of 15 N (NI'I-I4)OI'I. After shaking, an additional 10 ml of n-butanol was added to yield a single phase solution. The standard assay mixture contained, in a total volume of 125 ul, 20, 30, or 50 td of enzyme and the following components: TdR-H ~ or UdR-H 3, 0.126 mM (22-26 million counts per minute per micromole); ATP, 9.6 raM; 5{gC12, 12.8 raM; 3-phosphoglycerate, 11.5 mM; and Tris-tICi buffer, pH 8.0 (25°), 96 mM. After an incubation period of 10 minutes at 38 °, the reaction was terminated by the addition of 25 ~1 of 40 % triehloroaeetic acid. Under the conditions of the assay, the amount, of pyrimidine deoxyribonueleoside phosphorylated increased linearly with time (for at least 20 minutes), and with the amount of enzyme added per tube (40-300 #g protein of the $3 fraction or 10-80 t~g protein of the Sephadex fraction). Protein was determined by the method of Bonting and Jones (1957). Partial purification of deoxythymidine lcinase. Modifications of the procedures of Weissman et al. (1960) and Ives et aL (1963) were employed. All steps were carried out at 4-7 °.

Approximately 2-3 g (wet weight) of cells suspended in 5 volumes of 0.15 2F/ KCI, 0.003 23//2-mercaptoethanol, and 0.01 M Tris buffer, pH 8.0 (25 °) were sonicated for 25-40 seconds (Raytheon sonic oscillator, model DFI01, 10kc), and the sonic extracts were centrifuged for i hour at 35,000 rpm (model L Spinco centrifuge, size 40 rotor). The supernatant solution contained 4-5 mg protein (fraction $3) per milliliter (Table I). The pH of fraction $3 was adjusted to g.0 (4 °) by the addition of 0.2 M acetic acid, and, after it had been stirred for i0 minutes, the suspension was centrifuged and the precipitate, consisting of inactive pro-

KIT AND DUBBS

18

TABLE 1 "PARTIAL

PURIFICATION

OF D]]OXYTHYMIDINE

KINASE

Enzyme fraction

Specific activity: #t~raoles dUMP formed per tLg protein in 10 rain. at 3~°

Total protein (mg)

Total enzyme activity: t~raoles dUMP Iorraed in 10 rain. at 38°

LNI cells

$3 HAt-5 AS-50 Sephadex

23.9 56.3 73.6 88.9

53.8 22.0 9.3 8.2

1,286,000 1,239,000 685,000 729,000

Vaceinia-infected LS/I (TK-) cells

$3 ttAc-5 Sephadex

25.0 35.3 95.1

42.3 21.7 7.2

1,058,000 816,000 685,000

Source of enzyme

teins, was discarded. The supernatant fluid, brought to p H 7.0 (4 °) with 0.4 M NaHCO3, generally contained 2.0-2.5 mg protein per milliliter and about 75 % of the enzyme activity of fraction $3 (HAt-5 fraction). An equal volume of saturated (NH4)2S04 solution (pH 7.0) was slowly added, and the suspension was stirred for 30 minutes. The precipitate was collected by centrifugation and redissolved in 3 ml of 0.15 M KC1, 0.003 M 2-mercaptoethanol, 0.01 M Tris buffer, p H 7.0 (25 °) (As-50 fraction). Tubes containing 0.5 or 1.0 g Sephadex G25 or Sephadex G50 were prepared. The Sephadex was equilibrated with: (1) 0.15 M KC1, 0.003 M 2-mercaptoethanol, 0.01 M Tris buffer, p H 7.0 (25 °) or p H 8.0 (25°); or (2) 0.003 M 2-mercaptoethanol, 0.2 M Tris buffer, p H 8.0 (25°). The enzyme solutions were applied to the tubes followed by two void volumes of the buffer solution with which the Sephadex had been equilibrated. The combined eluates were either used immediately or 2-nil aliquots were frozen for 1-4 days at --20 ° in screw-cap test tubes. The frozen enzyme retained 70 % or more of the original activity for at least 6 days (Sephadex enzyme fraction). The Sephadex eluate consisted of about one-fifth to one-seventh of the protein, usually more than half of the initial enzyme activity of the $3 fraction, and 3-5 times the specific activity of the $3 fraction (Table 1).

Kinetics of thermal inactivation of deoxythymidine tcinase. Sephadex enzyme fractions containing 0.38-2.93 mg protein per

milliliter, or tIAc-5 enzyme fractions containing 1.1-4.1 mg protein per milliliter in 0.15 M KC1, 0.003 M 2-mercaptoethanol and 0.01 M Tris-HC1 buffer, p H 7.0 (25°), were incubated with shaking in a water bath at either 38 ° or 65 °. After indicated time intervals, suitable aliquots were pipetted into prewarmed tubes containing the components of the enzyme reaction mixture and the tubes were incubated for another 10 minutes at 38 ° or 30 °. The enzyme reaction was terminated by the addition of trichloroacetic acid and the amount of pyrimidine deoxyribonucleotide formed was determined.

Inhibition of enzyme activity by rabbit antisera. Young New Zealand rabbits were inoculated once a week for 11 weeks with 1.0-ml aliquots of the HAe-5 enzyme fractions prepared from either vaccinia-infected L M ( T K - ) cells or from noninfected LFI cells. The enzyme fractions employed contained 3.4-3.7 mg protein per milliliter and displayed specific activities ranging from 26.6 to 57.9. "Control" samples of blood were collected by cardiac puncture from each of the rabbits prior to immunization. Samples (0.25 ml) of sera obtained from control or immunized rabbits were mixed with 0.25 ml of enzyme and incubated at 38 ° for 30 minutes or at 30 ° for 60 minutes in water-bath shaker, model 2516, Research Specialties Co., Richmond, California. Suitable aliquots were then pipetted into prewarmed tubes containing the components of the reaction mixture and assayed for enzyme activity. Materials. U d R - H 3 and T d R - H 3 were

PARTIALLY PURIFIED DEOXYTHYMIDINE KINASE purchased from New England Nuclear Corporation, Boston, Massachusetts; Sephadex G25 and G50 (coarse grade) from Pharmacia Fine Chemicals, Inc., New York; and d T T P , d C T P , d C D P , dATP, and B U d R from Calbiochem., Los Angeles, California; I U d R was obtained from Sehwarz Bioresearch, Inc., M o u n t Vernon, New York; and F U d R from H o f f m a n n - L a Roche, Inc., Nutley 10, New Jersey. RESULTS

Nucleoside Substrates and Kinetic Properties of Deoxythymidine Kinase Okazaki and Kornberg (1964a, b) have purified deoxythymidine kinase from Escherichia coIi and have demonstrated t h a t the substrate can be a uracil deoxyribonueleoside in which the substituent on carbon 5 of the pyrimidine is hydrogen, a methyl group, or any of several halogens. The specificity for the vaccinia-induced and the L M cell deoxythymidine kinase are similar for the following reasons: 1. The partially purified enzymes from noainfected L M cells and vaccinia-infected L M ( T K - ) cells catalyzed the phosphorylation of T d R - H 3 and U d R - H 3. The ratios of T d R - H 3 to U d R - H 3 phosphorylating activities were 1.09, 1.17, and 1.15, respectively, for the $3, HAt-5, and Sephadex fractions prepared from L M cells. 2. When incubated in the absence of substrate, the T d R - H 3 and the U d R - H 3

19

phosphorylating activities were inactivated at about the same rate. 3. B U d R , I U d R , F U d R , and T d R competitively inhibited the phosphorylation of U d R - H a. With the L~.I cell enzyme, the apparent K~ values were 2.3 X 10 -5 M, 2.3 X 10 - a M , 1.6 X 10 - ~ M , and 9.3 X 10-6M, respectively, for B U d R , I U d R , F U d R , and T d R . The corresponding K~ values for the vaccinia-induced enzyme were 8.3 X 10-c M, 8.3 X 10-° M, 2.9 X 10-5 M, and 7.4 × 10 -6 M. 4. B U d R and I U d R were also potent inhibitors of the phosphorylation of T d R - H a. U d R and F U d R were less effective inhibitors. With the partially purified L M cell enzyme, concentrations of 4.1 X 10-4M F U d R , and 4.4 X 10- 4 M U d R were required to inhibit the phosphorylation of 1.2 X 10- ~ M T d R - H a b y 39% and 59%, respectively. F r o m Lfl~eweaver-Burk plots, the Michaelis constants (K~) and the maximal velocities (V~) have been calculated for the enzyme reactions with U d R - H 3 as substrate (Lineweaver and Burk, 1934; Dixon and Webb, 1958). For the L M cell enzyme, the K~ values at 38 ° and 30 °, respectively, were 3.5 X 1 0 - 5 M and 2.7 X 1 0 - ~ M (Table 2). The corresponding K~ values at 38 ° and 30 ° for deoxythymidine kinase induced b y vaccinia in L M ( T K - ) cells were 7.2 X 1 0 - S M and 4.0 X 10-SM. The differences, although small, were statistically significant at the 5 % level.

TABLE 2 MICHAELIS CONSTANTS (Kin), MAXIMAL VELOCITIES (Vm) AND TEMPERATURE CHARACTERISTICS O F DEOXYTHYMIDINEKINASES FROM LM CELLS AND VACCINIA-INFECTEDLM (TK-) CELLS Source of enzymes

Substrate

LM cells

UdR-H 3

Vaccinia-infeeted LM (TK-) cells

UdR-H ~

Number of assays

Temperature of assay (°C)

14 4 12 6

38 30 38 30

K,? (X 10.5 M) 3.5 2.7 7.2 4.0

4444-

0.4 0.4 0.5 0.3

V~ Temperature (X10-5 M) characteristic (cal°ries)c 2.5 1.7 2.6 1.6

9,200 -11,200 --

Sephadex enzyme fraction. b Mean 4- standard error of the mean. 2.303R (T1T2) (log V~ -- log Vt) c Temperature characteristic = (T2 -- TO , where R equals the gas constant and V2 and V1 are the enzyme velocities at two temperatures, T2 and T1 (Webb, 1963).

20

K I T AND DUBBS 100

were nearly equal in susceptibility to inhibition by dTTP under all the experimental conditions studied. Figure 1 illustrates an >80 experiment in which relatively high levels of UdR-H 3 (0.24 raM) and ATP (12.8 raM) were employed. Fifty per cent inhibition ¢9 6 0 occurred with 0.031 mM to 0.038 mM -O O tlJ dTTP. Okazaki and Kornberg (1964b) have >- 4 0 N shown that dCTP and dCDP activate Z t.~ highly purified deoxythymidine kinase preparations from E. coll. However, Bresnick °~9 20 LM ( T K-)CELLS " ~ : ~ ~ ~ ~ et al. (1964) observed that dCTP inhibited deoxythymidine kinase preparations from 0 I I I I I I adult liver and certain "minimal-deviation" 0.OI 0.03 0.05 0.10 mMdTTP hepatomas. Therefore, the effects of dCDP and dCTP on the enzyme preparations from FIG. 1. Effect of increasing concentrations of d T T P on the p h o s p h o r y l a t i o n of U d R - H 3 b y . noninfected LM cells and vaceinia-infeeted Sephadex enzyme fractions from L M and vaeeiniaLM (TK-) cells were tested. However, infected LM (TK-) cells. neither activation nor inhibition was found in the presence of dCTP or dCDP concenThe Vm values of the LM cell enzyme trations varying from 0 to 0.31 raM. and the vaceinia-induced enzyme were approximately equal at 38 ° and at 30 °. Rates of Therrnat Inactivation of Deoxythymidine Kinase Preparations The temperature characteristics of the reactions, calculated from the V~ values Partially purified deoxythymidine kinase between 30 ° and 38 ° (Webb, 1963), were preparations from noninfected LM ceils 9,200 calories and 11,200 calories, respec- rapidly lost TdR-H 3 and UdR-H s phostively, for the LM and tile vaceinia-induced phorylating activities when preincubated enzymes (Table 2). at 38 ° in 0.01 M Tris-HC1 buffer at pH's ranging from 6.7 to 8.5 (25 °) or 0.2 M TrisFeedbaclc Inhibition of Deozythymidine Ki- HC1 buffer, pH 8.0 (25°). The half-life of nase the HAt-5 enzyme fraction was 29-43 Deoxythymidine triphosphate (dTTP) minutes whether prepared from l-day-old exerts a feedback inhibition on crude or LM cells in the logarithmic growth phase, purified deoxythymidine kinase prepara- 2-day-old cells in the phase of negative tions from bacteria and animal tissues growth acceleration, or 3-day-old, sta(Breitman, 1963; Bresnick et al, 1964; tionary phase cells (Table 3, Fig. 2). HowMaley and Maley, 1962; Okazaki and ever, the HAc-5 enzyme fraction prepared Kornberg, 1964b). The degree of inhibition from vaccinia-infected LM (TK-) cells was is strongly modulated by the concentrations considerably more stable; the half-life of of the substrates, TdR and ATP (Ires et the vaccinia-induced enzyme exceeded 300 al., 1963). To learn whether partially purified minutes (Table 3, Fig. 2). deoxythymidine kinase preparations from The possibility was considered that the noninfected LM cells and vaccinia-infected HAt-5 fraction from vaccinia-infected cells LM (TK-) cells were inhibited by dTTP, contained stabilizing metabolites. In order the enzyme preparations were incubated in to remove soluble metabolites and loosely the presence of varying concentrations of bound substrates, the deoxythymidine kinase dTTP (0 to 0.10 raM). The ATP concen- from vaccinia-infected LM (TK-) cells and tration was varied from 2.4 to 16.8 raM, that from uninfected L M cells were further and the phosphorylation of both TdR-II 3 purified by ammonium sulfate precipitation, and UdR-H 3 were studied. The enzymes redissolved in Tris-HC1 buffer solution, and

PARTIALLY

PURIFIED

DEOXYTHYMIDINE TABLE

21

KINASE

3

]-%KTES OF THERMAL INACTIVATION AT 38 ° OF DEOXYTHYMIDINE ~ I N A S E PREP.kRATIONS

FROM

Enzyme fraction HAt-5

UNINFECTED

AND

~IRUS-INFECTED

Substrate

Virus

Cells

Days after subculture

(rain -1)

(rain.)

UdR-H 3

--

LM LM LM L M (TK-) LM (TK-)

1 2 3 2 2

0.024 (1) 0.023 (4) 0.016 (l) 0.0019 (2) 0.016 (3)

29 30 43 364 45

LM LM (TK-)

2 2

0.021 (2) 0.0017 (1)

33 400

LM L M (TK-)

2 2

0.016 (16) 0.0019 (4)

43 364

-

-

-Vaccini~ Herpes simplex TdR-H 3

-

-

Vaecinia Sephadex

CELLS

UdR-H~

-

-

Vaceinia

ki=~ot ~

tl/2

The velocity c o n s t a n t for t h e r m a l i n a c t i v a t i o n ( / ~ o 0 was calculated from the equation, ki~o~ = (ln v0 - In v ) / t , where v0 and v are the enzyme activities at zero time and time, t, respectively. The half-life, t~/2 was t h e n calculated from the formula, 6/~ = In 2 / k ~ . Values in p a r e n t h e s e s signify t h e n u m b e r of d e t e r m i n a t i o n s .

VACClNIA INFECTED LM(TK-) CELLS

1.2

[]

1.0

t

0.6

~

HERPES S I M P L E X INFECTED

\~LM(TK')CELLS N

ta ID 0

0.4

"~

o.a

LMCELLSXo _

0

i 20

~ 40

MINUTES

! 60 ENZYME

i 80

"~

I I00

PREINCUBATED

I ]20

I ]40

l 160

I 180

AT 3 8 ° C

FIG. 2. Kinetics of thermal inactivation of I-IAc-5 fractions prepared from 2-day-old LM cells and virus-infected LM (TK-) cells. The enzymes were preincubated at pH 7.0 and 38 ° for the indicated times and then were assayed at pI-I 8.0 and 38 °.

filtered through Sephadex G25 or Sephadex G50 gels. Despite this further purification, the half-life of the Sephadex enzyme fraction from vaccinia-infected LSi (TI{-) cells still exceeded 300 minutes whereas, that from uninfected L M cells was 43 minutes.

The rates of inactivation of ~he Sephadex enzyme fractions from noninfected LM cells and vaccinia-infected L M ( T K - ) cells were also studied at 65 °. Figure 3 demonstrates that at 65 ° considerable inactivation of the vaccinia-induced enzyme occurred.

22

K I T AND DUBBS 1.8

1.6

1.4

1.2 VACCINIA,L.M I T K - )

>,.

II--u <[

1.0

la.I

=~

\

"""13 .....

0.8

>.. I%1 z

"'

IN F E C T E D CELLS

El

0.6

0 _.1

0.4

0.21-

I 0 MINUTES

LM C E L L S

I 15 ENZYME

I I 30 4.5 PREINCUBATED

I 60 AT 6 5 " C

FIG. 3. Effect of p r e i n c u b a t i n g Sephadex e n zyme fractions from L M and vaccinia-infected L M (TK-) cells at 65 ° in 0.2 M Tris-HC1 buffer, p H 8.0 (25 °) on the p h o s p h o r y l a t i o n of U d R - H 3 at p H 8.0 and 30 °. TABLE 4 THE EFFECT OF PREINCUBATION WITH DEOXYTHYMIDINE OR D E O X Y U R I D I N E ON TItE K I N E T I C S OF INACTIVATION OF DEOXYTHYMIDINE-]:I3 ]:)HOSPHORYLA_TING ACTIVITY

Enzyme preincubated in solution containing Minutes enzyme preincubated at 38 ° prior to assay

No addition

Deoxy- Deoxythyuridine midine (0.1 mM) (0.1 mM)

ttt~moles dTMP formed per pg protein in 10 min. at 38 ° 0 30 60 120

37.2 18.8 13.1 11.2

43.2 25.6 18.1 13.3

50.4 44.6 41.3 34.7

Nevertheless, the rate of inactivation was slower than that of the enzyme from noninfected LM cells. Partially purified deoxythymidine kinase preparations from vaccinia-infected LM

cells were more stable than the enzyme from noninfected LM cells but less stable than the enzyme from vaccinia-infected LM (TK-) cells. After incubation periods of 60 minutes and 120 minutes at 38 °, respectively, 20 % and 40 % of the activity of the enzyme preparations from vaccinia-infected LM cells was inactivated. However, the deoxythymidine kinase induced in LM (TK-) cells following infection with another DNAcontaining vh~us, herpes simplex, was relatively unstable, exhibiting a half-life of approximately 45 minutes (Table 3, Fig. 2).

Effect of Selected Metabolites on the Thermal Inactivation of L M Cell Deoxythymidine Kinase To learn whether tile LM cell deoxythymidine kinase could be protected against thermal inactivation, the enzyme was preincubated at 38 ° in buffer solutions conraining either substrates of the enzyme, the feedback inhibitor, dTTP, or with metabolites which have been shown to activate the E. cell deoxythymidine kinase (Okazaki and Kornberg, 1964b). After designated intervals, samples were pipetted into prewarmed tubes containing the reaction mixture for the enzyme assay. Preincubation with ATP (15 raM) or with dTTP (0.01 raM), protected the UdR-H 3 phosphorylating activity of the LM cell enzyme against thermal inactivation (Fig. 4). TdR in the preincubation solution also preserved both the UdR-H 3 (Fig. 4), and the TdR-H 3 (Table 4) phosphorylating activities of the LM cell enzyme. However, UdR at concentrations ranging from 0.05 to 0.35 mM, did not protect either the UdR-H 3 or the TdR-H 3 phosphorylating activities. Moreover, the stability of the LM cell, deoxythymidine kinase, was not increased by preincubating the enzyme in the presence of 0.78 mM dCTP, 0.78 mM dCDP, or 0.5 mM dATP (Kit and Dubbs. unpublished experiments). Properties of Deoxythymidine Kinase from L M Cell Cultures Grown in the Presence of TdR As shown in Fig. 4 and Table 4, dTTP or its precursor, TdR, increased the sta-

PARTIALLY PURIFIED

DEOXYTItYMIDINE

KINASE

23

2.0 ~ ~ ~, 1.8

~

1

~

~',.

,>-

-

--o { PLUS 15 mM ,~.TPJ

~ ~

~

"

"

~ V ( PLUSO.OI rnM dTTP) ( PLUS 0.05 mM TdR )

~

>_ }L) tuJ =~ >" N Z ua

1.6

1.4

~ ~

(

P

L

~ .

U

S

~ " " o CONT ROL (B UF F E R SOLUTION} 0.05 mM UdR }

o _1 I.O I

I

L

I

0 50 60 90 120 MINUTES ENZYME PREINCUBATED hT 58°C

F~G. 4. P r e i n c u b a t i o n of L M cell d e o x y t h y m i d i n e kin~se p r e p a r a t i o n s ~t 38 ° in the presence of A T P , d T T P , TdR, or UdR. T h e values shown in t h e figure sre the c o n c e n t r a t i o n s of these m e t a b o l i t e s during the p r e i n e u b a t i o n period. A t the indiea.ted times, aliquots were w i t h d r a w n a n d assayed for U d R - H a p h o s p h o r y l a t i n g activity. I n the enzyme assay tubes, the c o n c e n t r a t i o n s of U d R - t t 3 a n d A T P were 0.26 mM and 15 raM, respectively. I n the experiments involving T d R or d T T P , the c o n c e n t r a t i o n in the enzyme assay tubes of T d R was 0.026 m M and t h a t of d T T P was 0.004 rm~/. E n z y m e assay t e m p e r a -

'mre was 38°C. bility of deoxythymidine kinase in vitro. To learn whether a more stable enzyme could be obtained from LM cells if the intracellular pools of TdR and dTTP were increased, enzyme fractions were prepared from l-day-old and 2-day-old LM cells which had been grown for 24 hours in medium containing 50 ~g/ml deoxythymidine. Activities of these enzyme preparaLions indicated that the addition of deoxythymidine to the culture medium profoundly influenced the deoxythymidine kinase activity of the cells. Normally, the deoxythynfidine kinase activity of LM cells declines to low levels between 55 and 72 hours after subculture. At 48 hours after subculture, the specific activity of the $3 Fraction is about 20-25 enzyme units; at 72 hours, this value generally declines to 5-15 enzyme units. However, $3 Fractions prepared from 3-day-old cells which had been grown in the presence of 50 fxg/mg Tdl~ for 24 hours prior to harvest had a specific activity of 26 enzyme units. In contrast, the addillon of 50 ttg/ml UdR to the culture

medium of 2-day-old cells did not prevent the intracellular decline of deoxythymidine kinase activity observed at 3 days after subculture (Kit and Dubbs, unpublished experiments). The specific activities of the Sephadex fractions, prepared from LM cells which had been grown in medium containing 50 ug/ml TdR, were 106 and 133 enzyme units (Table 5). These values are appreciably higher' than the specific activities of 60-90 usually obtained with the Sephadex fractions of LM cells. The Sephadex fractions from LM cells grown in the presence of TdR were studied for thermal stability by preincubating in buffer solutions at 38 °. The enzymes exhibited the usual marked instability, and approximately 75-82 % of the initial activity was lost after a preincubation period of 2 hours (Table 5). The results indicate that either the partiM purification procedure was effective in removing loosely bound deoxythymidine compounds from tl~e enzyme

24

KIT AND DUBBS TABLE 5

THERMAL INACTIVATION OF SEPHADEX ENZYME FRACTIONS PREPARED FROM L~/~ CELLS GROWN

IN 50 ug/ml TdR Fo~ 24 HOURS PmoR wO ~IARVEST Enzyme partially purified from LM cells: Minutes enzyme preincubated at 38 ° prior to assay

2 days old at 3 days old at time of harvest ~ time of harvest ~ re,moles d U M P formed per ug protein in 10 rain. at 38 °

0 30 60 90 120

106 5~ 40 23 19

133 106 63 -36

TdR (50 ~g/ml) was added to the culture medium 24 hours before the cells were harvested. or t h a t the amounts of metabolites bound were too low to stabilize the purified enzyme.

Charcoal Treatment of Sephadex Enzyme Fraction Prepared from VacciniaInfected LM (TK-) Cells To test the possibility t h a t the stability of the vaccinia-induced deoxythymidine kinase was attributable to enzyme-bound nucleosides or nucleotides, Sephadex enzyme fractions were treated with charcoal. Norite A was thoroughly washed with 5 % triehloroacetie a'Cid, followed b y glassdistilled water and 0.01 M Tris-HC1 buffer, p H 7.0 (25°), containing 0.15 M KCI, 0.003 M 2-mercaptoethanol, and 0.005 M MgC]2. After centrifuging to remove the buffer solution, the Norite A was dried at 100 °. T e n milligrams of the washed Norite A was then added to the vaccinia-induced enzyme solution and stirred for 5 minutes at 4 ° . The suspension was centrifuged to remove the charcoal and the stability of the charcoal-treated enzyme was compared with an untreated enzyme. Charcoal t r e a t m e n t did not cause an inhibition of the vaccinia-induced deoxythymidine kinase activity. Moreover, the charcoal-treated enzyme was as stable as the untreated enzyme. After 2 hours of preincubation a t 38 ° in the absence of substrate, the untreated enzyme exhibited a n 18 % decrease in enzyme activity and the

charcoal-treated enzyme displayed a 12% loss of activity.

Neutralization of Vaccinia-Induced Deoxythymidine Kinase by Rabbit Antisera Rabbits ) were immunized with HAc-5 • enzyme fractions prepared either from uninfected L M cells or from L M ( T K - ) cells infected with vaccinia vh~us. The effects of the antisera obtained from the immunized rabbits on the activity of the L M cell and virus-induced enzymes were studied. T h e enzymes were also preincubated with sera obtained from the rabbits prior to immunization. The activity of the vaccinia-induced deoxythymidine kinase was inhibited when preincubated with antisera obtained from rabbits immunized with the vaccinia-induced enzyme (Table 6). The inhibition decreased with dilution of the antiserum. Antisera which had been heated for 5 minutes at 95 ° prior to mixing with the enzyme failed to inhibit the activity of the vaccinia-induced enzyme. The vaccinia-enzyme a,ntisera did not inhibit the L M cell enzyme. Sera from 7 rabbits which had been inoculated with the L ~ I cell deoxythymidine kinase failed to inhibit either the L M cell enzyme or the vaccinia-induced enzyme. Presumably, these rabbits failed to make sufficient antibodies to the enzyme proteins. I n other control experiments, it was observed t h a t antisera obtained from rabbits immunized with infectious-vaccinia virus did not inhibit the vaccinia-induced deoxythymidine kinase. DISCUSSION Following infection of either Ll~[ cells or L M ( T K - ) cells b y vaccinia virus, a deoxythymidine kinase is induced which is considerably more stable t h a n the enzyme from noninfected L M cells. I t was observed t h a t the t h e r m a l stability of the enzyme from noninfected L M cells could be increased b y the addition of A T P , d T T P , or T d R to the enzyme extracts. The possibility was therefore considered t h a t the relative stability of the vaccinia-induced deoxythymidine kinase was due to stabilizing metabolites rather t h a n to intrinsic differences in protein conformation. Perhaps,

PARTIALLY PURIFIED DEOXYTHYMIDINE KINASE

25

TABLE 6 EFFECT OF RABBITANTISERAON DEOXYTItYMIDINEKINASE PREPARATIONSFROM NONINFECTED LIV[ CELLS AND VACCINIA-INFECTED LM (TK-) CELLS ~ Preincubation of enzyme ~- serum~ Expt. Temp. Time (°C) (min.) a

b

e

38

30

30

30

60

60

Source of enzyme

Vaccinia-infected LM (TK-) cells

Vaecinia-infected LM (TK-) cells

LM cell enzyme

Enzyme incubated w i t h serum from rabbits Prior to immunization (control) Immunized with LM cell enzyme Immunized with vaceiniainduced enzyme

Antiserum Per cent dilution enzyme factor activity 2

100

2

104

2 4 8 16 64 2¢

21 65 89 94 104 112~

Prior to immunization (control) Immunized with LM cell enzyme Immunized with vacciniainduced enzyme

2

100

2

91

2

17

Prior to immunization (control) Immunized with LM eeli enzyme Immunized with vacciniainduced enzyme

2

100

2

500

2

105

-,

In each enzyme assay, Sephadex enzyme fractions were employed. Equal volumes of enzyme and serum were mixed and preineubated. Antiserum was heated for 5 minutes at 95°, cooled, and then preineubated with the enzyme. during vh'al replication, TdR, d T T P , or other nucleotides accumulated in the intracellular space and were bound by the vaccinia-induced deoxythymidine kinase, thereby stabilizing the enzyme against thermal inactivation. This hypothesis is considered unlikely for the following reasons: 1. Partially purified deoxythymidine kinase preparations from vaceinia-infected cells have been studied under various experimental conditions, and in every case these enzyme preparations were markedly more stable than the deoxythymidine kinase from noninfeeted LSi cells. E v e n after the vaccinia-induced deoxythymidine kinase was @tered through Sephadex gels and treated with charcoal to remove possible stabilizing metabolites, the vaceinia-induced enzyme retained its relative stability.

2. On the other hand, deoxythymidine kinase was unstable when purified from LS/I cells in the logarithmic phase of growth, from cultures of L M cells which had been grown in 50 ~g/ml TdR, or from L M ( T K - ) cells infected with herpes simplex vh'us. The enzyme was also unstable when purified from stationary phase LS/I cells. I t would therefore be necessary to assume that stabilizing metabolites accumulated only after vaceinia infection, but not under any of the other physiological conditions. An alternative explanation that stabilizing metabolites are very tightly bound b y the vaeeiniainduced enzyme leads back to the hypothesis that the vaceiniaCinduced enzyme is a new enzyme with unique properties. The hypothesis that vaccinia induces the synthesis of a new enzyme is further sup-

26

KIT AND DUBBS

ported by the observations that the K,, values and the immunological properties of the vaccinia-induced enzyme differ from those of the enzyme from noninfected cells and by experiments involving actinomycin D. In the latter experiments, it has been shown that antecedent treatment of cells for 8 hours, 24 hours prior to vaccinia infection, inhibits more than 95% of the DNAdependent RNA synthesis of LM (TK-) cells and leads to a considerable loss of me cellular RNA. Yet, appreciable deoxythymidine kinase induction occurs after virus infection, suggesting that viral DNA-dependent RNA synthesis, not cellular DNAdependent RNA synthesis, is required for the vaccinia-induced enzyme synthesis (Kit et al., 1963e). These observations are not readily explicable in terms of a derepression of host-cell deoxythymidine kinase in virusinfected cells. The deoxythymidine kinase induced in L M cells b y vaccinia virus and those induced in H e L a cells b y poxviruses (MeAuslan, 1963a, b) are also more stable to t h e r m a l inactivation than the deoxythymidine kinases of the uninfected host cells. Partially purified enzyme extracts from vaccinia-infected L g l cells were more stable t h a n the L M cell enzyme b u t less stable t h a n the enzyme fi'om vaccinia-infected L M ( T K - ) cells. Presumably the extracts from vaccinia-infected L M cells contain a mixture of the L M cell deoxythymidine kinase and the " n e w " vaccinia-induced enzyme. On the other hand, the deoxythylnidine kinase induced in L M ( T K - ) cells following infection with another kind of DNA-eontaining virus, herpes simplex, is much less stable t h a n the vaccinia-induced enzyme. These results support the concept t h a t the capacity to induce a thermally stable deoxythymidine kinase is controlled b y the genome of the virus rather t h a n the host cell. Additional studies of the properties of more highly purified deoxythymidine kinase preparations are planned. A number of m u t a n t strains of vaecinia and herpes simplex viruses, which have altered deoxythymidine kinase-inducing capacities, have been isolated (Dubbs and Kit, 1964a-c). Of particular interest are several

: m u t a n t strains of herpes simplex which induce v e r y little deoxythymidine kinase at 37 °, but induce approximately one-tenth the level obtained with parental virus strains when replication proceeds at 31 ° (Dubbs and Kit, 1964b, c, 1965). I n addition, a m u t a n t H e L a cell line, deficient in deoxythymidine kinase activity, is now available. Comparisons of properties of deoxythymidine kinases induced in the deoxythymidine kinase-defieient cell lines b y parental and m u t a n t virus strains with those of enzymes of uninfected L M and t t e L a cells m a y serve to distinguish conclusively between the "new e n z y m e " hypothesis and the "derepression hypothesis." ACKNOWLEDGMENTS This investigation was supported by grants from the American Cancer Society (E-291A) and the National Science Foundation (GB-620), and by Public Health Service Research Grants (CA 06656-02, and I-KS-AI-2352-02). The experiments were carried out with the able technical assistance of Milton Anken and Corine Higbee. REFERENCES BONTING,S. L., and JONES, M. (1957). Determination of microgram quantities of deoxyribonucleie acid and protein in tissues grown in vitro. Arch. Biochem. Biophysics 65,340-353. BR~ITM~N, T. R. (1963). The feedback inhibition of thymidine kinase. Biochim. Biophys. Acta 67, 153-155. BRESNICK, E., THOMPSON, U. B., MORRIS, H. P., and LIE~ELT, A. G. (1964). Inhibition of thymidine kinase activity in liver and hepatomas by TTP and d-CTP. Biochem. Biophys. Res. Commun.

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DIXON, M., and WEBB., E. C. (1958). "Enzymes." Academic Press, New York. DUBBS, D. R., and KI~', S. (1964a). Isolation and properties of vaccinia mutants deficient in thymidine kinase-inducing activity. Virology 22, 214-225. DUBBS, D. R., and KIT, S. (1964b). Mutant strains of herpes simplex deficient in thymidine kinaseinducing activity. Virology 22,493-502. Du~es, D. R., and KIT, S. (1964e). Herpes simplex mutants with an altered thymidine kinase cistron. Bacteriol. Proc. p. 120. Abstr. 64th Ann. Meeting Am. Soc. Microbiol., Washington, D. C., May 3-7, 1964.

Du~Bs, D. R., and KIT, S. (1964d). Effect of halogenated pyrimidines and thymidine on growth of L-cells and a subline lacking thymidine kinase. Exptl. Cell Res. 33, 19-28.

PAI~TIALLY PUP~IFIED DEOXYTHYMIDINE KINASE D~XBBS, D. R., and KIT, S. (1965). The effect of temperature on induction of deoxythymidine kinase activity by herpes simplex mutants. Virology 25,256-270. IRES, D. H., MORSE, P. A., JR., and POTTER, V. R. (1963). Feedback inhibition of thymidine kinase by thymidine triphosphate. J. Biol. Chem. 238, 1467-1474. KIT, S., and DVBBS, D. R. (1962). Biochemistry of vaccinia-infected mouse fibroblasts (strain LM). I. Effect on nucleic acid and protein synthesis. Virology 18,274-285. K,T, S., and DUB]3S, D. R. (1963). Acquisition of thymidine kinase activity by herpes simplex infected mouse fibroblast cells. Biochem. Biophys. Res. Commun. 11, 55-59. 14IT, S., and DuB]3s, D. R. (1964a). Acquisition of thermostable thymidine-deoxyuridine kinase by vaccinia-infected cells. Federation Proc. 23,382. KIT, S., and DuB~s, D. R. (1964b). The thymidine kinase cistron of vaccinia and herpes simplex viruses. Abstr. 6th Intern. Congr. Biochem., New York, 1964. p. 233. Secretariat, 6th Intern. Contr. Biochem., 9650 Wisconsin Ave., Washington, 14, D. C. KIT, S., DeBts, D. R., PIEKARSKI, L. J., and Hsu, T. C. (1963a). Deletion of thymidine kinase activity from L-ceils resistant to bromodeoxyuridine. Exptl. Cell Res. 31,297-312. KIT, S., PIEKARSKI, L. J., and DURBS, D. i% (1963b). Induction of thymidine kinase activity by vaceinia-infected mouse fibroblasts. J. Mol. Biol. 6, 22-33. KIT, S., PIEK~aSKI, L. J., and DUBBS, D. 1~.

27

(1963c). Effects of 5-fluorouracil, aetinomycin D, and mitomycin C oll the induction of thymidine kinas6 by vaecinia-infeeted L-cells. J. Mol. Biol. 7,497-510. LINEWEAVER,H., and B u ~ : , D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56,658-666. MAI~Y, F., and M~LEY, G. F. (1962). On the nature of a sparing effect by thymidine on the utilization of deoxycytidine. Biochemistry 1, 847-851. McA~JsL~N, B. R. (1963a). Control of induced thymidine kinase activity in the poxvirusinfected cell. Virology 20,162-168. McA~JsLaN, B. R. (1963b). The induction and repression of thymidine kinase in the poxvirusinfected HeLa cell. Virology 2I, 383-389. OXAZA~I, R., and KO~NBER~, A. (1964a). Deoxythymidine kinase of Escherichia coll. I. Purification and some properties of the enzyme. J. Biol. Chem. 239,269-274. O~:Az~i, R., and KORNBERG, A. (1964b). Deoxythymidinc kinase of Escherichia coll. II. Kinetics and feedback eontroI. J. Biol. Chem. 239, 275284. WEeE, J. L. (1963). "Enzyme and Metabolic Inhibitors," Vol. I, p. 792. Academic Press, New York. WEISSM~N, S. M., SMELLIE, R. M. S., and P.~uL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. The phosphorylation of thymidine. Biochim. Biophys. Acta 45, 101-110.