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Activation of thymidine kinase of adult rat liver by the culture filtrate of Clostridium perfringens
352
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96439
ACTIVATION OF T H Y M I D I N E KINASE OF ADULT RAT L I V E R BY T H E CULTURE F I L T R A T E OF C L O S T R I D I U M P E R F R I N G E N S T A K A H I K O S H I O S A K A , Y O S U K E O M U R A , H I R O M I C H I O K U D A AND S E T S U R O F U J I I
Department o/ Enzyme Physiology, Institute /or Enzyme Research, School o/ Medicine, Tokushima University, Tokushima (Japan) (Received N o v e m b e r I9th, 1969)
SUMMARY
I. When adult rat-liver extract was treated with the culture filtrate of Clostridium per/ringens, marked stimulation of the thymidine kinase activity was observed. The extract did not stimulate thymidine kinase of either regenerating liver or Yoshida sarcoma. 2. The activity of the culture filtrate depended on the age of the culture, being maximal after 82 h growth. 3. The activator in the culture filtrate was heat-labile and nondialyzable and was inactivated by pronase. 4- This activator was purified approx. 5oo-fold by a procedure involving (NH4)2S Q fractionation, acetone treatment and refractionation with (NH4)2SO 4.
INTRODUCTION
The first step in the incorporation of thymidine into DNA is known to be phosphorylation of this nucleoside catalyzed by thymidine kinase (ATP:thymidine 2'phosphotransferase, EC 2.7.1.21 ). This enzyme possesses many of the attributes of a rate-limiting enzyme in DNA biosynthesis 1. Thymidine kinase activity is very low in adult rat liver and increases markedly in regenerating liver 2-6. MALEY et al. 7 reported that the increase in thymidine kinase activity of rat liver following partial hepatectomy was greatly impaired by intraperitoneal injection of p-fluorophenylalanine, puromycin or actinomycin D. Accordingly, they suggested that the observed increase in the enzyme activity was associated with synthesis of enzyme de novo and not with activation of pre-existing enzyme. However, we recently obtained evidence suggesting the existence of an inactive form of thynlidine kinase in adult liver which is readily activated by the culture filtrate of Clostridium per/ringens. MATERIALS AND METHODS
Materials
[2-14C]Thymidine was purchased from the Japan Radioisotope Association. ATP, ~-glycerophosphate and calf-mucosa alkaline phosphatase were obtained from Biochim. Biophys. Acta, 204 (197 ° ) 352 358
ACTIVATION OF THYMIDINE KINASE
353
the Sigma Chemical Co. (St. Louis, Mo.). Cl. per]ringens type A SII4-3, was kindly given by Prof. A. Kawada, Department of Food Microbiology, School of Medicine, Tokushima University. Liver was obtained from male albino rats of the WistarKing strain, weighing 15o-2oo g.
Preparation o/adult liver extract 2 g of adult rat liver were homogenized in 25 ml ice-cold Tris-maleate buffer (5 raM, p H 6.5). The homogeuate was centrifuged at 8000 ×g for 15 rain, and the supernatant was used as the liver extract.
Preparation o/ culture /iltrate o[ Cl. per]ringens Bacteria were grown for 82 h at 37 ° in medium consisting of 20 g polypeptone, 5 g yeast extract, 5 g glucose, 0. 5 g sodium thioglyeolate and i . I g Na2CO:, per 1 in distilled water. The culture medium was centrifuged at 12 ooo ×g for 40 rain. The resultant supernatant was used as the culture filtrate.
Thymidine kinase assay Kinase was assayed by the method of EKER8 with the following modification 9. A mixture of Tris-maleate buffer (0.5 M, p H 6.5, 2oo/,1), MgC12 (IOO raM, 25 ~1), ~-glycerophosphate (12o raM, 25/~l), ATP (ioo raM, 25/~1), liver extract (IOO/~1), culture filtrate of Cl. per/ringens (IOO/~l), and E2-14C~thymidine (I raM, 25 ttl, 30 ooo counts/rain) was incubated at 37 ° for 30 rain, and the reaction was stopped by immersing the tubes in boiling water. Aliquots of IOO #1 were withdrawn and placed on DEAE-cellulose paper disks (4 cm × 4 cm). The paper was dried and immersed in about 3 ° m l of I mM ammonium formate for IO rain. The washing liquid was discarded and the paper was washed in distilled water. This procedure was repeated twice. Finally the paper was placed in 95 % ethanol and dried at 80 °. By this procedure, E2-14C]thymidine was effectively removed while E14C]thymidine monophosphate was retained on the paper. The d~ied paper was placed in a counting vial containing 6 ml of the toluene-phosphorus mixture (IOO rag/1 of 1,4-bis-2,2-(5-phenyloxazolyl)benzene and 4 g/1 of 2,5-diphenyloxazole in toluene). Radioactivity was counted in a Packard liquid scintillation counter. Protein concentration was determined by the method of LOWRY et al. TM or by biuret method n,l~ with bovine serum albumin as a standard. In the biuret method, the protein of the culture filtrate was estimated after trichloroacetic acid precipitation. Before trichloroacetic acid precipitation, a much higher value of the protein concentration was obtained. The different result obtained by this method seems to indicate that most of the contaminating materials are of relatively low molecular weight.
RESULTS
Activation o] adult rat-liver thymidine kinase by the culture/iltrate o/ Cl. per]ringens When the extract of adult liver was treated with various amounts of the culture filtrate of Cl. per]ringens described in MATERIALSAND METHODS, the thymidine kinase activity was considerably stimulated (Fig. I). The amount of culture filtrate was expressed as mg protein, which was estimated by the biuret method. The effect was maximal on addition of culture filtrate containing 0.06 mg protein and did not increase on addition of more filtrate (Fig. I).
Biochim. Biophys. Acta, 204 (197o) 352-358
354
T.
SHIOSAKAet al.
The liver extract and the culture filtrate were pre-incubated for various periods before the addition of other constituents and the resulting stimulatory effect of the culture filtrate on adult liver thymidine kinase was examined. As shown in Fig. 2, activation of the liver thymidine kinase did not change on pre-incubation for various periods.
_ Z_2 e m
6.0
C
0
-~ ~ 4 . C F_-O 2.c
2~
/
I
i
0.05 OJO -Culture f[Itrate](mg protein)
E
ol
~
i
i
J
5 10 15 20 25 Preincubation time (rain)
L
50
Fig. I. E f f e c t of t h e c o n c e n t r a t i o n of C1. per/ringens c u l t u r e f i l t r a t e on a d u l t r a t - l i v e r t h y m i d i n e k i n a s e a c t i v i t y . T h y m i d i n e k i n a s e a c t i v i t y was e x p r e s s e d as n m o l e s of T M P forme d per nag prot e i n of t h e liver e x t r a c t . Fig. 2. E f f e c t of p r e - i n c u b a t i o n t i m e on t h e s t i m u l a t o r y effect of C1. per#ingens c u l t u r e fi l t ra t e . The p r o c e d u r e w a s as described in MATERIALS AND METHODS, e x c e p t t h a t t h e buffer, t h e e x t r a c t a n d t h e c u l t u r e f i l t r a t e were p r e - i n c u b a t e d a t 22 ° for t h e t i m e s i n d i c a t e d before a d d i t i o n of o t h e r c o n s t i t u e n t s . T h y m i d i n e k i n a s e a c t i v i t y was e x p r e s s e d as n m o l e s of T M P f o r m e d pe r m g p r o t e i n of t h e l i v e r e x t r a c t .
The result suggests that the liver thymidine kinase is fully activated during the incubation time required for the assay of enzyme activity. The activity of the culture filtrate depended on the age of the culture. Maximal activity was obtained after growth for 82 h (Fig. 3). On the other hand, the cell number reached a m a x i m u m 6 h after inoculation and then gradually decreased, The protein concentration also increased and reached a m a x i m u m at 66 h suggesting that cell lysis partly occurred during culture. The protein concentration was estimated by the biuret method, in which the culture filtrate was treated with 5 % of trichloroacetic acid and the estimation was carried out on the precipitated material.
6.0
~5.c ~'X -
•
4 .C
.4
. . . .
:2 3.C
2.0 ~ ~o ~
o~'
/ " /' /
30 60 90 Age of culture (h)
~o.2.~c ~,,o 4o.2 ~o.~.o~ ~o,i 120
'~°o~-
Fig. 3' S t i m u l a t i o n of a d u l t l i v e r t h y m i d i n e k i n a s e b y t h e c u l t u r e f i l t r a t e of Cl. perfringens a t v a r i o u s c u l t u r e ages. Cl. per/ringens was i n o c u l a t e d i n t o t h e p o l y p e p t o n e m e d i u m as d e s c r i b e d in MATERIALS AND METHODS a n d i n c u b a t e d a t 37 ° for t h e t i m e s i n d i c a t e d . The p r o t e i n c o n c e n t r a t i o n w a s e s t i m a t e d b y t h e b i u r e t m e t h o d n , 12. 0 - 0 , a c t i v i t y of a d u l t r a t l i ve r a d d e d t o C1. per/ringens; O - - - O , cells of p o p u l a t i o n ; x - • - x , protein.
Biochim. Biophys. ,4cta, 204 (197 o) 352-358
ACTIVATION OF THYMIDINE KINASE
355
Analysis o/reaction products The products of the reaction of liver thymidine kinase activated by the culture filtrate were analyzed b y the method of WEISSMAN et al. 2. The supernatant obtained after boiling the reaction mixture was collected, and i ml of this fluid was applied to an ECTEOLA-cellulose column (0.030 mequiv/ml), 8 cm × I cm. The column was eluted successively with 5o-ml portions of water, o.oi M HC1, 0.05 M HC1 and 0.5 M HC1 to remove thymidine, TMP, T D P and TTP, respectively. The effluent was collected in 6-ml fractions with a collector. An aliquot (I ml) of each fraction was introduced into a counting vial containing IO ml scintillation liquid (IO g 2.5-diphenyloxazole, 25 ° mg 1,4-bis-2,2-(5-phenyloxazolyl)benzene, ioo g naphthalene, dioxane to I 1). Radioactivity was determined in a Packard Tris-Carb liquid scintillation counter. As shown in Fig. 4, TMP was separated from the reaction mixture of adult liver thymidine kinase activated by the culture filtrate. The TMP fraction was collected and neutralized with 0.5 M NaOH. An aliquot (2 ml) of this fraction was mixed with I m g of calf-mucosa alkaline phosphatase and incubated for 3 h at 37 °. After incubation, the reaction mixture was applied to an ECTEOLA-cellulose column, as described above. Most of the TMP fraction was recovered in the thymidine fraction, as shown in Fig. 5. The product of the action of the activated adult liver thymidine kinase was confirmed to be TMP by paper chromatography 8. 420 ~ P O . O 1M ~ - O . 0 5 M ' - ' I I - - 0 . 5 M - - - ¢ HCt HCI HC[
2100 C
E
OfT
- - H 2 0 "----4~-- 0.01M ~ HCI
~-0.05 M ~ I ~ HC[
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8
' Thym;d[nc
200
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~
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0
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.9
o
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50 100 Eluate (rnl)
150
200
~
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~00
150
200
Eluate (rnl)
Fig. 4. S e p a r a t i o n of t h e p r o d u c t s f r o m t h e reaction m i x t u r e of a d u l t liver t h y m i d i n e kinase a c t i v a t e d b y t h e c u l t u r e filtrate. T h e p r o c e d u r e w a s as described in t h e t e x t . Fig. 5. E C T E O L A - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y of alkaline p h o s p h a t a s e - t r e a t e d T M P fraction. T h e p r o c e d u r e w a s as described in t h e t e x t .
Examination o/the reaction When A T P was omitted from the reaction mixture, no thymidine kinase activity was observed, as shown in Table I. Heat treatment (IOO°, IO min) of the liver extract or the culture filtrate also reduced the thymidine kinase activity considerably (Table I). The culture filtrate was pre-treated with various enzymes and its stimulatory effect on adult liver thymidine kinase activity was examined. The treatment with pronase decreased the stimulatory effect of the culture filtrate as shown in Table n . Biochim. Biophys. Aaa, 204 (I97 o) 352-358
T. SHIOSAKA et al.
356 TABLE I EFFECT
OF H E A T I N G L I V E R E X T R A C T OR C U L T U R E
FILTRATE AND OF ABSENCE
OF
ATP
ON T H Y M I -
DINE KINASE ACTIVITY
Complete mixture: (1.25 mg) adult liver extract; o.2 mg protein c u l t u r e filtrate of C1. per/ringens; 2. 5/~moles ATP; 2. 5/~moles MgCI2; 3.0/2moles ~-glycerophosphate; 25 m/~moles (about 3 ° ooo counts/miD) [2-14C~thylnidine; ioo #moles Tris-HC1 buffers (pH 6.5) in a final v o l u m e of 0. 5 ml. The reaction was carried o u t for 3 ° mid at 37 ° and t e r m i n a t e d b y heating in a b a t h at ioo c) for 5 miD. The t h y m i d i n e kinase activities were expressed as nmoles of TMP formed per mg protein of the liver extract. The protein concentration was determined b y the m e t h o d of LOWRY et al. TM.
Fraction
Thymidine kinase (nmoles /mg protein)
Inhibition (°/o)
Complete s y s t e m
4.46 o.oo o.2o
-ioo 96
o.19
96
Minus A T P Boiled adult liver extract Boiled culture filtrate of Cl. perfringens
TABLE II EFFECT
OF PRE-TREATMENT
OF T H E C U L T U R E F I L T R A T E OF
Cl. per#ingens
WITH VARIOUS ENZYMES
ON ITS STIMULATORY EFFECT The culture filtrate (1.2 rag) and various enzymes (2oo/xg) were incubated with 0.02 3I Tris-HC1 buffer (pH 8.0) in a final volume of 0. 4 ml at 37 ° for 30 miD. After incubation, the mixture was diluted Io-fold with 5 mM T r i s - m a l e a t e buffer (pH 6.5), and the diluted solution (o.i ml) was used for activation of adult liver t h y m i d i n e kinase, as described in MATERIALS AND METHODS. I n the control, the culture filtrate (1.2 rag) and various enzymes (2oo ttg) were separately incubated with 0.02 M Tris-HC1 buffer (pH 8.0) in a final volume of 0.2 ml at 37 ° for 3 ° mill. After incubation, the solutions of culture filtrate and of enzymes were combined and diluted Io-fold with 5 mM T r i s - m a l e a t e buffer (pH 6.5), and the diluted solution (o.i ml) was used for activation of a d u l t liver t h y m i d i n e kinase, as described in MATERIALS AND METHODS.
Enzyme
None Pronase Trypsin Chymotrypsin Lysozyme
Adult liver thymidine kinase activity activated by the culture filtrate treated with enzyme (nmoles)
1.51 2.90 2.59 3.19
Control (nmoles)
2.72 2.92 2.84 2.7o 2.75
All these results suggest that the active principle in the culture filtrate is of protein nature.
E][ect o] culture filtrate on thymidine kinase of various tissues The effect of the culture filtrate of Cl. per]ringens on the thymidine kinases of various proliferating tissues such as regenerating liver and Yoshida sarcoma was tested. The culture filtrate activated the thymidine kinase activity of an extract of adult rat liver 56-fold but did not affect activities of regenerating liver or Yoshida sarcoma, as shown in Table III. Bioehim. Biophys. ,4cta, 2o 4 (197 o) 352-358
ACTIVATION OF THYMIDINE KINASE
357
TABLE III EFFECT OF CULTURE FILTRATE OF Cl. per/ringens ON THYMIDINE KINASE ACTIVITY OF ADULT RAT LIVER, REGENERATING LIVER AND YOSHIDA SARCOMA R e g e n e r a t i n g l i v e r w a s p r e p a r e d as follows: r a t s were p a r t i a l l y h e p a t e c t o m i z e d b y t h e m e t h o d of HIGGINS AND ANDERSON 13, a n d t h e l i v e r was r e m o v e d 32 h a f t e r t h e o p e r a t i o n . A s c i t e s t u m o r cells of 5 - d a y - o l d Y o s h i d a s a r c o m a were collected b y c e n t r i f u g a t i o n . A f t e r t h e m a t e r i a l s were w a s h e d twice w i t h isotonic saline solution, t h e y were h o n m g e n i z e d in 2 vol. of ice-cold 5 mM T r i s - m a l e a t e buffer (pH 6.5). The h o m o g e n a t e was c e n t r i f u g e d a t 8000 × g for 15 rain a n d t h y m i d i n e k i n a s e in t h e s u p e r n a t a n t was a s s a y e d as d e s c r i b e d in MATERIALS AND METHODS. The t h y m i d i n e k i n a s e a c t i v i t i e s were e x p r e s s e d as nmoles of T M P forme d pe r m g p r o t e i n of t h e extracts.
Source o/ thymidinc kinase
A d u l t r a t liv er Regenerating liver Yoshida sarcoma
Thymidine kinase (nmoles/mg protein) Control
Treated with culture [iltrate
o.0 5 2.22 7.85
2.82 2.28 8.31
Purification All manipulations were carried out in the cold. Cl. per/ringens was grown in culture medium for 82 h, and then the culture filtrate was obtained b y eentrifugation (I2 ooo×g, 15 rain). Solid (NH4)eSO4 was then added slowly with stirring to 95 % saturation. The mixture was left to stand overnight and then filtered, and the liquid was discarded. The precipitate was dissolved in 5 mM Tris-maleate buffer (pH 6.5) and dialyzed overnight against the same buffer. Aliquots of the (NH4)2SO,-treated fraction were chilled on ice, and acetone was added slowly to 86 % saturation at --20 °. The mixture was centrifuged at 12 ooo ×g for 15 min. The resultant precipitate was collected b y centrifugation (12 ooo ×g, 15 rain) and dissolved in 5 mM Tris-maleate buffer (pH 6.5). Solid (NH4)2S04 was added to the solution to 30% saturation. The precipitate was dissolved in 5 mM Tris-maleate buffer and dialyzed overnight against the same buffer. By these procedures, the activator was purified about 5oo-fold and the recovery was 72 %, as shown in Table IV. In this purification, protein concentration was estimated b y the method of LOWRY et al. 1°. Attempts to obtain further purification
T A B L E IV PURIFICATION OF ADULT LIVER ACTIVATOR FROM THE CULTURE FILTRATE OF el. per/ringens The a c t i v i t y w a s e x p r e s s e d as n m o l e s of T M P formed. The p r o t e i n c o n c e n t r a t i o n w a s e s t i m a t e d b y t h e m e t h o d of LOWRY et al. 1°.
Procedure
Vol. (ml)
Total protein (mg)
Total activity (Izmoles)
Speci[ic activity (nmoles/mg)
Yield (%)
Culture filtrate 95 % (NH~)~SOi 86 ~o a c e t o n e f r a c t i o n 3o ~o (NH4)~SO4
99o 27 17 6.0
12 4oo 13o 3 °.0 18.3
58.4 5o.7 42-9 42.3
4.6o 39o.o 143o.o 2311.5
ioo
87 74 72
Biochim. Biophys. Acta, 2o 4 (197 o) 352-358
358
T. SHIOSAKA et al.
by low-temperature ethanol fractionation, treatment with calcium phosphate gel or colmnn chromatography on DEAE-cellulose, AE-cellulose or CM-cellulose proved unsuccessful. DISCUSSION
A direct relation is known between the rate of cell division and the activity of thymidine kinase ~a*-l~. Moreover, thymidine kinase is scarcely detectable in adult rat liver but rapidly appears in regenerating liver~,6,14,1~,TM. Thus there must be some regulatory mechanism for induction and subsequent repression of thymidine kinase in liver tissue. Our results showed that adult rat-liver thymidine kinase activity was greatly stimulated when the liver extract was treated with the culture filtrate of Cl. per-
/ringens. The product of the action of the activated enzyme was confirmed to be TMP by ECTEOLA-cellulose column chromatography, paper chromatography and treatment with calf-mucosa alkaline phosphatase. A shown in Table I, omission of ATP or heat treatment of the liver extract or of tlle culture filtrate reduced the thymidine kinase activity considerably. The active principle in the culture filtrate was heat-labile and nondialyzable and was inactivated by pronase. The results suggest that the active principle is of protein nature. The culture filtrate did not stimulate the thymidine kinase of regenerating liver. Therefore, it seemed that the thymidine kinase in adult liver is present in an inactive form and is activated during liver regeneration. Experiments are in progress on the mode of activation of adult liver thymidine kinase by the culture filtrate. REFE RENCES I E. tARESNICK, V. B. THOMPSON, H. P. MORRIS AND A. G. LIEVELT, Biochem. Biophys. Res. Commun., 16 (1964) 278. 2 S. M. WEISSMAN, R. M. S. SMELLIE AND J. PAUL, Bioehim. Biophys. Acta, 45 (196o) lOl. 3 H. H. HIATT AND T. t3. ~BOJARSKI, Biochem. Biophys. Res. Commun., 2 (196o) 35. 4 N. FAUSTO AND J. L. VAN LANCKER, J. Biol. Chem., 240 (1965) 1247. 5 P. A. BIANCHI, A. R. CRETHORN AND K. V. SHOOTER, Biochem. Biophys. Acta, 61 (1962) 728 6 F. J. BOLLUM AND B. R. POTTER, Cancer Res., I9 (1959) 561. 7 G. F. MALEY, M. G. LORENSON AND F. MALEY, Biochem. Biophys. Res. Commun., 18 (1965) 364 • 8 P. EKER, J. Biol. Chem., 240 (1965) 2607. 9 E. BRESNICK AND R. J. KARJALA, Cancer Res., 24 (1964) 841. IO H. LOWRY, N. S. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. I I J. GOA, Scan& J. Clin. Lab. Invest., 5 (1953) 218. 12 R. PARVIN, S. V. PANDE AND T. A. VENKITASUBRAMANIAN,Anal. Biochem., 12 (1965) 219. 13 G. M. HIGGINS AND R. ~VL ANDERSON, Arch. Pathol., 12 (1931) 186. 14 P. A. BIANCHI, J. A. BUTTER, J, A. CRETHORN AND K. V. SHOOTER, Bioehim, Biophys. Acta, 48 (1961) 218. 15 F. J. BOLLUM AND V. l{. POTTER, J. Biol. Chem., 233 (1958) 478. 16 R. MANTSAVlNOS AND E. S. CANELLAKIS, J. Biol. Chem., 234 (1959) 628. 17 R, E. BELTZ, Arch. Biochem. Biophys., 99 (1962) 304 . i8 P. A. BIANCHI, Biochim. Biophys. Acta, 55 (1962) 547.
Biochim. Biophys. Acta, 204 (197 o) 352-358
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96439
ACTIVATION OF T H Y M I D I N E KINASE OF ADULT RAT L I V E R BY T H E CULTURE F I L T R A T E OF C L O S T R I D I U M P E R F R I N G E N S T A K A H I K O S H I O S A K A , Y O S U K E O M U R A , H I R O M I C H I O K U D A AND S E T S U R O F U J I I
Department o/ Enzyme Physiology, Institute /or Enzyme Research, School o/ Medicine, Tokushima University, Tokushima (Japan) (Received N o v e m b e r I9th, 1969)
SUMMARY
I. When adult rat-liver extract was treated with the culture filtrate of Clostridium per/ringens, marked stimulation of the thymidine kinase activity was observed. The extract did not stimulate thymidine kinase of either regenerating liver or Yoshida sarcoma. 2. The activity of the culture filtrate depended on the age of the culture, being maximal after 82 h growth. 3. The activator in the culture filtrate was heat-labile and nondialyzable and was inactivated by pronase. 4- This activator was purified approx. 5oo-fold by a procedure involving (NH4)2S Q fractionation, acetone treatment and refractionation with (NH4)2SO 4.
INTRODUCTION
The first step in the incorporation of thymidine into DNA is known to be phosphorylation of this nucleoside catalyzed by thymidine kinase (ATP:thymidine 2'phosphotransferase, EC 2.7.1.21 ). This enzyme possesses many of the attributes of a rate-limiting enzyme in DNA biosynthesis 1. Thymidine kinase activity is very low in adult rat liver and increases markedly in regenerating liver 2-6. MALEY et al. 7 reported that the increase in thymidine kinase activity of rat liver following partial hepatectomy was greatly impaired by intraperitoneal injection of p-fluorophenylalanine, puromycin or actinomycin D. Accordingly, they suggested that the observed increase in the enzyme activity was associated with synthesis of enzyme de novo and not with activation of pre-existing enzyme. However, we recently obtained evidence suggesting the existence of an inactive form of thynlidine kinase in adult liver which is readily activated by the culture filtrate of Clostridium per/ringens. MATERIALS AND METHODS
Materials
[2-14C]Thymidine was purchased from the Japan Radioisotope Association. ATP, ~-glycerophosphate and calf-mucosa alkaline phosphatase were obtained from Biochim. Biophys. Acta, 204 (197 ° ) 352 358
ACTIVATION OF THYMIDINE KINASE
353
the Sigma Chemical Co. (St. Louis, Mo.). Cl. per]ringens type A SII4-3, was kindly given by Prof. A. Kawada, Department of Food Microbiology, School of Medicine, Tokushima University. Liver was obtained from male albino rats of the WistarKing strain, weighing 15o-2oo g.
Preparation o/adult liver extract 2 g of adult rat liver were homogenized in 25 ml ice-cold Tris-maleate buffer (5 raM, p H 6.5). The homogeuate was centrifuged at 8000 ×g for 15 rain, and the supernatant was used as the liver extract.
Preparation o/ culture /iltrate o[ Cl. per]ringens Bacteria were grown for 82 h at 37 ° in medium consisting of 20 g polypeptone, 5 g yeast extract, 5 g glucose, 0. 5 g sodium thioglyeolate and i . I g Na2CO:, per 1 in distilled water. The culture medium was centrifuged at 12 ooo ×g for 40 rain. The resultant supernatant was used as the culture filtrate.
Thymidine kinase assay Kinase was assayed by the method of EKER8 with the following modification 9. A mixture of Tris-maleate buffer (0.5 M, p H 6.5, 2oo/,1), MgC12 (IOO raM, 25 ~1), ~-glycerophosphate (12o raM, 25/~l), ATP (ioo raM, 25/~1), liver extract (IOO/~1), culture filtrate of Cl. per/ringens (IOO/~l), and E2-14C~thymidine (I raM, 25 ttl, 30 ooo counts/rain) was incubated at 37 ° for 30 rain, and the reaction was stopped by immersing the tubes in boiling water. Aliquots of IOO #1 were withdrawn and placed on DEAE-cellulose paper disks (4 cm × 4 cm). The paper was dried and immersed in about 3 ° m l of I mM ammonium formate for IO rain. The washing liquid was discarded and the paper was washed in distilled water. This procedure was repeated twice. Finally the paper was placed in 95 % ethanol and dried at 80 °. By this procedure, E2-14C]thymidine was effectively removed while E14C]thymidine monophosphate was retained on the paper. The d~ied paper was placed in a counting vial containing 6 ml of the toluene-phosphorus mixture (IOO rag/1 of 1,4-bis-2,2-(5-phenyloxazolyl)benzene and 4 g/1 of 2,5-diphenyloxazole in toluene). Radioactivity was counted in a Packard liquid scintillation counter. Protein concentration was determined by the method of LOWRY et al. TM or by biuret method n,l~ with bovine serum albumin as a standard. In the biuret method, the protein of the culture filtrate was estimated after trichloroacetic acid precipitation. Before trichloroacetic acid precipitation, a much higher value of the protein concentration was obtained. The different result obtained by this method seems to indicate that most of the contaminating materials are of relatively low molecular weight.
RESULTS
Activation o] adult rat-liver thymidine kinase by the culture/iltrate o/ Cl. per]ringens When the extract of adult liver was treated with various amounts of the culture filtrate of Cl. per]ringens described in MATERIALSAND METHODS, the thymidine kinase activity was considerably stimulated (Fig. I). The amount of culture filtrate was expressed as mg protein, which was estimated by the biuret method. The effect was maximal on addition of culture filtrate containing 0.06 mg protein and did not increase on addition of more filtrate (Fig. I).
Biochim. Biophys. Acta, 204 (197o) 352-358
354
T.
SHIOSAKAet al.
The liver extract and the culture filtrate were pre-incubated for various periods before the addition of other constituents and the resulting stimulatory effect of the culture filtrate on adult liver thymidine kinase was examined. As shown in Fig. 2, activation of the liver thymidine kinase did not change on pre-incubation for various periods.
_ Z_2 e m
6.0
C
0
-~ ~ 4 . C F_-O 2.c
2~
/
I
i
0.05 OJO -Culture f[Itrate](mg protein)
E
ol
~
i
i
J
5 10 15 20 25 Preincubation time (rain)
L
50
Fig. I. E f f e c t of t h e c o n c e n t r a t i o n of C1. per/ringens c u l t u r e f i l t r a t e on a d u l t r a t - l i v e r t h y m i d i n e k i n a s e a c t i v i t y . T h y m i d i n e k i n a s e a c t i v i t y was e x p r e s s e d as n m o l e s of T M P forme d per nag prot e i n of t h e liver e x t r a c t . Fig. 2. E f f e c t of p r e - i n c u b a t i o n t i m e on t h e s t i m u l a t o r y effect of C1. per#ingens c u l t u r e fi l t ra t e . The p r o c e d u r e w a s as described in MATERIALS AND METHODS, e x c e p t t h a t t h e buffer, t h e e x t r a c t a n d t h e c u l t u r e f i l t r a t e were p r e - i n c u b a t e d a t 22 ° for t h e t i m e s i n d i c a t e d before a d d i t i o n of o t h e r c o n s t i t u e n t s . T h y m i d i n e k i n a s e a c t i v i t y was e x p r e s s e d as n m o l e s of T M P f o r m e d pe r m g p r o t e i n of t h e l i v e r e x t r a c t .
The result suggests that the liver thymidine kinase is fully activated during the incubation time required for the assay of enzyme activity. The activity of the culture filtrate depended on the age of the culture. Maximal activity was obtained after growth for 82 h (Fig. 3). On the other hand, the cell number reached a m a x i m u m 6 h after inoculation and then gradually decreased, The protein concentration also increased and reached a m a x i m u m at 66 h suggesting that cell lysis partly occurred during culture. The protein concentration was estimated by the biuret method, in which the culture filtrate was treated with 5 % of trichloroacetic acid and the estimation was carried out on the precipitated material.
6.0
~5.c ~'X -
•
4 .C
.4
. . . .
:2 3.C
2.0 ~ ~o ~
o~'
/ " /' /
30 60 90 Age of culture (h)
~o.2.~c ~,,o 4o.2 ~o.~.o~ ~o,i 120
'~°o~-
Fig. 3' S t i m u l a t i o n of a d u l t l i v e r t h y m i d i n e k i n a s e b y t h e c u l t u r e f i l t r a t e of Cl. perfringens a t v a r i o u s c u l t u r e ages. Cl. per/ringens was i n o c u l a t e d i n t o t h e p o l y p e p t o n e m e d i u m as d e s c r i b e d in MATERIALS AND METHODS a n d i n c u b a t e d a t 37 ° for t h e t i m e s i n d i c a t e d . The p r o t e i n c o n c e n t r a t i o n w a s e s t i m a t e d b y t h e b i u r e t m e t h o d n , 12. 0 - 0 , a c t i v i t y of a d u l t r a t l i ve r a d d e d t o C1. per/ringens; O - - - O , cells of p o p u l a t i o n ; x - • - x , protein.
Biochim. Biophys. ,4cta, 204 (197 o) 352-358
ACTIVATION OF THYMIDINE KINASE
355
Analysis o/reaction products The products of the reaction of liver thymidine kinase activated by the culture filtrate were analyzed b y the method of WEISSMAN et al. 2. The supernatant obtained after boiling the reaction mixture was collected, and i ml of this fluid was applied to an ECTEOLA-cellulose column (0.030 mequiv/ml), 8 cm × I cm. The column was eluted successively with 5o-ml portions of water, o.oi M HC1, 0.05 M HC1 and 0.5 M HC1 to remove thymidine, TMP, T D P and TTP, respectively. The effluent was collected in 6-ml fractions with a collector. An aliquot (I ml) of each fraction was introduced into a counting vial containing IO ml scintillation liquid (IO g 2.5-diphenyloxazole, 25 ° mg 1,4-bis-2,2-(5-phenyloxazolyl)benzene, ioo g naphthalene, dioxane to I 1). Radioactivity was determined in a Packard Tris-Carb liquid scintillation counter. As shown in Fig. 4, TMP was separated from the reaction mixture of adult liver thymidine kinase activated by the culture filtrate. The TMP fraction was collected and neutralized with 0.5 M NaOH. An aliquot (2 ml) of this fraction was mixed with I m g of calf-mucosa alkaline phosphatase and incubated for 3 h at 37 °. After incubation, the reaction mixture was applied to an ECTEOLA-cellulose column, as described above. Most of the TMP fraction was recovered in the thymidine fraction, as shown in Fig. 5. The product of the action of the activated adult liver thymidine kinase was confirmed to be TMP by paper chromatography 8. 420 ~ P O . O 1M ~ - O . 0 5 M ' - ' I I - - 0 . 5 M - - - ¢ HCt HCI HC[
2100 C
E
OfT
- - H 2 0 "----4~-- 0.01M ~ HCI
~-0.05 M ~ I ~ HC[
0 5 M----4 HCL
hyrnld~ne
30C E
8
' Thym;d[nc
200
-~,
~
5oo
~ 100
0
g
.9
o
0
50 100 Eluate (rnl)
150
200
~
o6
50
~00
150
200
Eluate (rnl)
Fig. 4. S e p a r a t i o n of t h e p r o d u c t s f r o m t h e reaction m i x t u r e of a d u l t liver t h y m i d i n e kinase a c t i v a t e d b y t h e c u l t u r e filtrate. T h e p r o c e d u r e w a s as described in t h e t e x t . Fig. 5. E C T E O L A - c e l l u l o s e c o l u m n c h r o m a t o g r a p h y of alkaline p h o s p h a t a s e - t r e a t e d T M P fraction. T h e p r o c e d u r e w a s as described in t h e t e x t .
Examination o/the reaction When A T P was omitted from the reaction mixture, no thymidine kinase activity was observed, as shown in Table I. Heat treatment (IOO°, IO min) of the liver extract or the culture filtrate also reduced the thymidine kinase activity considerably (Table I). The culture filtrate was pre-treated with various enzymes and its stimulatory effect on adult liver thymidine kinase activity was examined. The treatment with pronase decreased the stimulatory effect of the culture filtrate as shown in Table n . Biochim. Biophys. Aaa, 204 (I97 o) 352-358
T. SHIOSAKA et al.
356 TABLE I EFFECT
OF H E A T I N G L I V E R E X T R A C T OR C U L T U R E
FILTRATE AND OF ABSENCE
OF
ATP
ON T H Y M I -
DINE KINASE ACTIVITY
Complete mixture: (1.25 mg) adult liver extract; o.2 mg protein c u l t u r e filtrate of C1. per/ringens; 2. 5/~moles ATP; 2. 5/~moles MgCI2; 3.0/2moles ~-glycerophosphate; 25 m/~moles (about 3 ° ooo counts/miD) [2-14C~thylnidine; ioo #moles Tris-HC1 buffers (pH 6.5) in a final v o l u m e of 0. 5 ml. The reaction was carried o u t for 3 ° mid at 37 ° and t e r m i n a t e d b y heating in a b a t h at ioo c) for 5 miD. The t h y m i d i n e kinase activities were expressed as nmoles of TMP formed per mg protein of the liver extract. The protein concentration was determined b y the m e t h o d of LOWRY et al. TM.
Fraction
Thymidine kinase (nmoles /mg protein)
Inhibition (°/o)
Complete s y s t e m
4.46 o.oo o.2o
-ioo 96
o.19
96
Minus A T P Boiled adult liver extract Boiled culture filtrate of Cl. perfringens
TABLE II EFFECT
OF PRE-TREATMENT
OF T H E C U L T U R E F I L T R A T E OF
Cl. per#ingens
WITH VARIOUS ENZYMES
ON ITS STIMULATORY EFFECT The culture filtrate (1.2 rag) and various enzymes (2oo/xg) were incubated with 0.02 3I Tris-HC1 buffer (pH 8.0) in a final volume of 0. 4 ml at 37 ° for 30 miD. After incubation, the mixture was diluted Io-fold with 5 mM T r i s - m a l e a t e buffer (pH 6.5), and the diluted solution (o.i ml) was used for activation of adult liver t h y m i d i n e kinase, as described in MATERIALS AND METHODS. I n the control, the culture filtrate (1.2 rag) and various enzymes (2oo ttg) were separately incubated with 0.02 M Tris-HC1 buffer (pH 8.0) in a final volume of 0.2 ml at 37 ° for 3 ° mill. After incubation, the solutions of culture filtrate and of enzymes were combined and diluted Io-fold with 5 mM T r i s - m a l e a t e buffer (pH 6.5), and the diluted solution (o.i ml) was used for activation of a d u l t liver t h y m i d i n e kinase, as described in MATERIALS AND METHODS.
Enzyme
None Pronase Trypsin Chymotrypsin Lysozyme
Adult liver thymidine kinase activity activated by the culture filtrate treated with enzyme (nmoles)
1.51 2.90 2.59 3.19
Control (nmoles)
2.72 2.92 2.84 2.7o 2.75
All these results suggest that the active principle in the culture filtrate is of protein nature.
E][ect o] culture filtrate on thymidine kinase of various tissues The effect of the culture filtrate of Cl. per]ringens on the thymidine kinases of various proliferating tissues such as regenerating liver and Yoshida sarcoma was tested. The culture filtrate activated the thymidine kinase activity of an extract of adult rat liver 56-fold but did not affect activities of regenerating liver or Yoshida sarcoma, as shown in Table III. Bioehim. Biophys. ,4cta, 2o 4 (197 o) 352-358
ACTIVATION OF THYMIDINE KINASE
357
TABLE III EFFECT OF CULTURE FILTRATE OF Cl. per/ringens ON THYMIDINE KINASE ACTIVITY OF ADULT RAT LIVER, REGENERATING LIVER AND YOSHIDA SARCOMA R e g e n e r a t i n g l i v e r w a s p r e p a r e d as follows: r a t s were p a r t i a l l y h e p a t e c t o m i z e d b y t h e m e t h o d of HIGGINS AND ANDERSON 13, a n d t h e l i v e r was r e m o v e d 32 h a f t e r t h e o p e r a t i o n . A s c i t e s t u m o r cells of 5 - d a y - o l d Y o s h i d a s a r c o m a were collected b y c e n t r i f u g a t i o n . A f t e r t h e m a t e r i a l s were w a s h e d twice w i t h isotonic saline solution, t h e y were h o n m g e n i z e d in 2 vol. of ice-cold 5 mM T r i s - m a l e a t e buffer (pH 6.5). The h o m o g e n a t e was c e n t r i f u g e d a t 8000 × g for 15 rain a n d t h y m i d i n e k i n a s e in t h e s u p e r n a t a n t was a s s a y e d as d e s c r i b e d in MATERIALS AND METHODS. The t h y m i d i n e k i n a s e a c t i v i t i e s were e x p r e s s e d as nmoles of T M P forme d pe r m g p r o t e i n of t h e extracts.
Source o/ thymidinc kinase
A d u l t r a t liv er Regenerating liver Yoshida sarcoma
Thymidine kinase (nmoles/mg protein) Control
Treated with culture [iltrate
o.0 5 2.22 7.85
2.82 2.28 8.31
Purification All manipulations were carried out in the cold. Cl. per/ringens was grown in culture medium for 82 h, and then the culture filtrate was obtained b y eentrifugation (I2 ooo×g, 15 rain). Solid (NH4)eSO4 was then added slowly with stirring to 95 % saturation. The mixture was left to stand overnight and then filtered, and the liquid was discarded. The precipitate was dissolved in 5 mM Tris-maleate buffer (pH 6.5) and dialyzed overnight against the same buffer. Aliquots of the (NH4)2SO,-treated fraction were chilled on ice, and acetone was added slowly to 86 % saturation at --20 °. The mixture was centrifuged at 12 ooo ×g for 15 min. The resultant precipitate was collected b y centrifugation (12 ooo ×g, 15 rain) and dissolved in 5 mM Tris-maleate buffer (pH 6.5). Solid (NH4)2S04 was added to the solution to 30% saturation. The precipitate was dissolved in 5 mM Tris-maleate buffer and dialyzed overnight against the same buffer. By these procedures, the activator was purified about 5oo-fold and the recovery was 72 %, as shown in Table IV. In this purification, protein concentration was estimated b y the method of LOWRY et al. 1°. Attempts to obtain further purification
T A B L E IV PURIFICATION OF ADULT LIVER ACTIVATOR FROM THE CULTURE FILTRATE OF el. per/ringens The a c t i v i t y w a s e x p r e s s e d as n m o l e s of T M P formed. The p r o t e i n c o n c e n t r a t i o n w a s e s t i m a t e d b y t h e m e t h o d of LOWRY et al. 1°.
Procedure
Vol. (ml)
Total protein (mg)
Total activity (Izmoles)
Speci[ic activity (nmoles/mg)
Yield (%)
Culture filtrate 95 % (NH~)~SOi 86 ~o a c e t o n e f r a c t i o n 3o ~o (NH4)~SO4
99o 27 17 6.0
12 4oo 13o 3 °.0 18.3
58.4 5o.7 42-9 42.3
4.6o 39o.o 143o.o 2311.5
ioo
87 74 72
Biochim. Biophys. Acta, 2o 4 (197 o) 352-358
358
T. SHIOSAKA et al.
by low-temperature ethanol fractionation, treatment with calcium phosphate gel or colmnn chromatography on DEAE-cellulose, AE-cellulose or CM-cellulose proved unsuccessful. DISCUSSION
A direct relation is known between the rate of cell division and the activity of thymidine kinase ~a*-l~. Moreover, thymidine kinase is scarcely detectable in adult rat liver but rapidly appears in regenerating liver~,6,14,1~,TM. Thus there must be some regulatory mechanism for induction and subsequent repression of thymidine kinase in liver tissue. Our results showed that adult rat-liver thymidine kinase activity was greatly stimulated when the liver extract was treated with the culture filtrate of Cl. per-
/ringens. The product of the action of the activated enzyme was confirmed to be TMP by ECTEOLA-cellulose column chromatography, paper chromatography and treatment with calf-mucosa alkaline phosphatase. A shown in Table I, omission of ATP or heat treatment of the liver extract or of tlle culture filtrate reduced the thymidine kinase activity considerably. The active principle in the culture filtrate was heat-labile and nondialyzable and was inactivated by pronase. The results suggest that the active principle is of protein nature. The culture filtrate did not stimulate the thymidine kinase of regenerating liver. Therefore, it seemed that the thymidine kinase in adult liver is present in an inactive form and is activated during liver regeneration. Experiments are in progress on the mode of activation of adult liver thymidine kinase by the culture filtrate. REFE RENCES I E. tARESNICK, V. B. THOMPSON, H. P. MORRIS AND A. G. LIEVELT, Biochem. Biophys. Res. Commun., 16 (1964) 278. 2 S. M. WEISSMAN, R. M. S. SMELLIE AND J. PAUL, Bioehim. Biophys. Acta, 45 (196o) lOl. 3 H. H. HIATT AND T. t3. ~BOJARSKI, Biochem. Biophys. Res. Commun., 2 (196o) 35. 4 N. FAUSTO AND J. L. VAN LANCKER, J. Biol. Chem., 240 (1965) 1247. 5 P. A. BIANCHI, A. R. CRETHORN AND K. V. SHOOTER, Biochem. Biophys. Acta, 61 (1962) 728 6 F. J. BOLLUM AND B. R. POTTER, Cancer Res., I9 (1959) 561. 7 G. F. MALEY, M. G. LORENSON AND F. MALEY, Biochem. Biophys. Res. Commun., 18 (1965) 364 • 8 P. EKER, J. Biol. Chem., 240 (1965) 2607. 9 E. BRESNICK AND R. J. KARJALA, Cancer Res., 24 (1964) 841. IO H. LOWRY, N. S. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. I I J. GOA, Scan& J. Clin. Lab. Invest., 5 (1953) 218. 12 R. PARVIN, S. V. PANDE AND T. A. VENKITASUBRAMANIAN,Anal. Biochem., 12 (1965) 219. 13 G. M. HIGGINS AND R. ~VL ANDERSON, Arch. Pathol., 12 (1931) 186. 14 P. A. BIANCHI, J. A. BUTTER, J, A. CRETHORN AND K. V. SHOOTER, Bioehim, Biophys. Acta, 48 (1961) 218. 15 F. J. BOLLUM AND V. l{. POTTER, J. Biol. Chem., 233 (1958) 478. 16 R. MANTSAVlNOS AND E. S. CANELLAKIS, J. Biol. Chem., 234 (1959) 628. 17 R, E. BELTZ, Arch. Biochem. Biophys., 99 (1962) 304 . i8 P. A. BIANCHI, Biochim. Biophys. Acta, 55 (1962) 547.
Biochim. Biophys. Acta, 204 (197 o) 352-358