Human Thymidine Kinase

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ance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom ..... 100. 103. 100. 103. 1W. 100. YI. 44. 80. 38. CTP. GTP. UTP. ITP. dATP. dGlP. dCTP.

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 258, No. 11, Issue of June 10. pp. 7000-7004.1983 Printed in U S.A.

Human Thymidine Kinase PURIFICATION AND PROPERTIES OF THE CYTOSOLIC ENZYME OF PLACENTA* (Received for publication, November 8, 1982)

T. Eng Gan, Jane L. Brumley, and Martin B. Van Der WeydenS From the Department of Medicine, Monash University, Alfred Hospital, Prahran, 3181, Victoria, Australia

Cytosolic thymidine kinase (EC 2.7.1.21) has been purified 5200-fold to apparent homogeneity from normal humanplacenta. The purification includes sequential affinity chromatography on blue-Sepharose and a thymidine column. The molecular weight of the enzyme determined by gel filtrationand sucrose density ultracentrifugation is 92,000. The subunit molecular weight is 44,000, suggesting that the enzyme is a dimer in its native state.With isoelectric focusing, placental thymidine kinase demonstrated a single form with an isoelectric point of 9.1. The final purified enzyme preparation exhibits no immunological cross-reactivity with human mitochondrial thymidine kinase.

RESULTS

of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-3014,cite the authors, and include a check or money order for $5.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

The abbreviations used are: dThd, thymidine; dTMP, thymidine monophosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonicacid.

7000

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Subcellular Fractionation and Purification of Thymidine Kinase-Subcellular fractionation studies performed on two separate occasions showed that 70 to 85% of the totalactivity in placental crude homogenate occurred in the 100,000 X g supernatant with the remainder of activity sedimenting with the 15,000 x g pellet. The results of the purification procedure are summarized in Table I. The enzyme preparation obtained after Step 1 when stored at -70 “C in the indicated buffer showed no appreciable loss of activity for up to two weeks. Optimum condition for enzyme binding to blue-Sepharose (Step 3) was found to be within a pH range of 7.5 to 8.9 with a 0.02 M Tris-HC1 buffer. As shown in Fig. 1 for its dissociation from the blue-Sepharose complex, thymidine kinase was eluted as a single peak of activity with 0.5 M Tris-HC1, pH Thymidine kinase (ATP:thymidine 5’-phosphotransferase, 8.0, containing 5 mM ATP and 10 mM MgClz. Enzyme prepEC 2.7.1.21), an enzyme of the pyrimidine salvage pathway, arations obtained after Steps 2 and 3 when stored at -70 “C catalyzes the phosphorylation of thymidine to thymidine 5’- in the respective buffers outlined in thepurification procedure monophosphate (1, 2). In mammalian tissue, two distinct were stable for up to 72 h. The enzyme preparation obtained activities occur with cytosolic or mitochondrial localization after Step4 was labile with loss of more than 70% activity in and it is the cytosolic activity which increases with cellular 24 h when stored at either 4, -20, or -70 “C in 0.2 M TrisDNA synthesis, whereas the mitochondrial activity remains HC1 and 200 PM dThd.2 Effortsto stabilize this activity with relatively constant during various phases of the cell cycle (3- EDTA ( 5 mM), ATP ( 5 mM), M&12 (10 mM), bovine serum 7). Interest in the human enzymes has been recently stimu- albumin (10 mglml), and protease inhibitors including soylated by the finding that the levels of the mitochondrial or bean trypsininhibitor (0.01 mg/ml), sodium heparin (10 cytosolic thymidine kinase activities of involved neoplastic units/ml), and aprotinin (10 units/ml) were unsuccessful. The tissue of patients with a variety of lymphoproliferative disor- final enzyme preparation (Step 4) when subjected to electroders are useful indicators for predicting either tumor behavior phoresis showed a single Coomassie blue staining band with or the clinical course of these patients (8-10). Mitochondrial identical Rf of 0.32 (Table 11) to the single peak of thymidine thymidine kinase has been purified to near homogeneity from kinase activity, and with the outlined purification procedure human liver (11), but our attempts to develop a radioimmu- the overall yield was about 16%. noassay for cytosolic thymidine kinase has necessitated isoPhysical Properties and SubunitMolecular Weight-When lation and definition of its properties. Inthis report, we the purified enzyme was denatured with sodium dodecyl suldescribe a purification procedure for cytosolic thymidine ki- fate, dithiothreitol, and 0-mercaptoethanol and electrophonase from human placenta and some characteristics of this resed on 7.5% polyacrylamide gels, a single Coomassie blue highly purified enzyme not previously reported are described. stained band was observed which corresponded to the single band of the autoradiograph of I’25 radiolabeled purified enEXPERIMENTAL PROCEDURES’ zyme (Step 4) preparation processed in the same fashion (Fig. 2). By comparison of the migration of these bands to proteins *This research was supported in part by grants from the Anti- of known subunit molecular weight, the subunit molecular to be 44,000 k 1,500 Cancer Council of Victoria, Australia. The costs of publication of this weight of thymidinekinaseappears article were defrayed in part by the payment of page charges. This (mean f S.D. of 4 determinations). Because of the relative article must therefore be hereby marked “advertisement” in accord- lability of the final enzyme preparation, some of the physical ance with 18 U.S.C. Section 1734 solely to indicate this fact. properties of thymidine kinase were determined with I’25 To whom reprint requests should be addressed. Portions of this paper (including “Experimental Procedures,” radiolabeled protein of Step 4 enzyme preparations. With Figs. 1 to 4,and Tables I to 111) are presented in miniprintat theend sucrose density ultracentrifugation, the final enzyme prepaof this paper. Miniprint is easily read with the aid of a standard rations yielded a single peak of catalytic activity with an S Z O . ~ magnifying glass. Full size photocopies are available from the Journal value of 5.2 and a molecular weight of 92,000 (Table 11). Gel

Human Placental Thymidine Kinase

DISCUSSION

Mammalian cytosolic thymidine kinase has been partially purified and characterized from a few tissue sources (27-33). We have purified thymidinekinase 5200-fold to apparent homogeneity from human placenta and the critical aspect of our preparation was the sequential elutionof the enzyme from blue-Sepharose and a thymidine-Sepharosecolumn. The final purified enzyme was presumed to be the cytosolic form. That this is indeed the case is suggested by the findings that the bulk of human placentalthymidinekinase occurs in the cytosol fraction; the behavior of the purified enzyme with polyacrylamide electrophoresis, isoelectric focusing, orsucrose density ultracentrifugation is quite distinct from human liver mitochondrial thymidine kinase (11) and these properties compare favorably with those reported for the cytosolic thymidine kinase(s) from a variety of mammalian sources (28-32). In addition, rabbit immune serum elaborated with purified human placenta thymidine kinase preparations does not cross-react withpurified mitochondrial thymidine kinase activity. In concert, these findings are compatible with the suggestion that human mitochondrial and cytosol thymidine kinase are products of distinct gene loci on human chromosome 16 and 17, respectively (34, 35). The final enzyme preparation appears to be homogeneous based on the findings that: it yields a single protein band corresponding to enzyme activity with polyacrylamide electrophoresis at pH8.3; with isoelectric focusing, it migrates as a single activity peakwith a PI of 9.1 corresponding to a single protein band; and the final enzyme preparations yield single protein and IlZ5 enzyme preparationsa single autoradiographic band with sodium dodecyl sulfate electrophoresis. The native molecular weight of placental cytosolic thymidine kinase of 92,000, determined either with sucrose density ultracentrifugation or gel filtration of the radiolabeled enzyme, are in good agreement with that obtained with the enzyme either from HeLa cells (28, 29) or from human mye-

locytic blasts (32), but is distinct to the molecular heterogeneity of highly purified human liver mitochondrial enzyme (11) orthe molecular weight of 177,000 reported for the cytosolic form of the human breast carcinomacell line MCF7 (33). The subunitmolecular weight of 44,000 for the human placental enzyme, determined with sodium dodecyl sulfate electrophoresis, would suggest that this activity in its native state exists as a dimer. The catalytic lability of the final enzyme preparations has precluded extensive characterization of its kinetic characteristic and attempts tostabilize this activity were unsuccessful. Despite this, the pH optimum of 7.4 for human placental thymidine kinase is similar to that reported for the cytosolic enzyme from a numberof mammalian sources (5,28,31). The substrate specificity, including the compounds which could donate a phosphate inthe reaction and potential nucleosides, are in accordance with previous observations (28,29, 31, 36). The presentpurification procedure is adaptable for the isolation of cytosolic thymidinekinase from other tissue sources as with this method the enzyme from human CEM lymphoblasts has been purified to h~mogeneity.~addition, In our previous findings have suggested that human mitochondrial thymidine kinase and deoxycytidine kinase activity reside with the same protein (11) whereas nucleoside specificity reportedhere for the highly purified cytosolic activity is entirely compatible with the suggestion that human cytosol deoxycytidine kinase and thymidine kinase are different enzyme entities (37). Acknowledgments-We would like to thank the labor ward nursing staffs of Margaret Coles HospitalandQueenVictoriaHospital, Melbourne for the supply of fresh human placenta. We would also like to thank Dr. P. H. Ellims for his help and advice, and M. Brown and A. Devlin for the typing of the manuscript. REFERENCES 1. Okazaki, R., and Kornberg, A. (1964) J. Biol. Chem. 239, 269274 2. Okazaki, R., and Kornberg, A. (1964) J. Biol. Chem. 2 3 9 , 275284 3. Brent, T. P. (1971) Cell Tissue Kinet. 4,297-305 4. Kit, S., Dubbs, D. R., and Frearson, P. M. (1968) J. Biol. Chem. 240,2565-2573 5. Adler, R., and McAuslan, B. R. (1974) Cell 2 , 113-117 6. Kit, S. (1976) Mol.Cell. Biochem. 1 1 , 161-182 7. Munch-Petersen, B., and Tyrsted, G. (1977) Biochim. Biophys. Acta 478,364-375 8. Ellims, P. H., Van Der Weyden, M. B., and Medley, G. (1981) Cancer Res. 4 1,691-695 9. Ellims, P. H., Gan, T. E., Medley, T., and Van Der Weyden, M. B. (19811 Blood 58,926-930 10. Ellims, P. H., Gan, T. E., and Van Der Weyden, M. B. (1981) Br. J.Haematol. 49,479-481 11. Ellims, P. H., and Van Der Weyden, M. B. (1980) J. BWZ. Chem. 255, 11290-11295 12. Kowal, E. P., and Markus, G. (1976) Prep. Biochem. 6,369-385 13. Ellims, P.H., Hayman, R. J., and Van der Weyden, M. B. (1979) Biochem. Biophys. Res. Commun. 8 9 , 103-107 14. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Bid. Chem. 1 9 3 , 265-275 15. Bensadoun, A., andWeinstein, D. (1976) Anal. Biochem. 70, 251-257 16. Kit, S., Leung, W.-C., and Kaplan, L. A. (1973) Eur. J . Biochem. 39,43-48 17. Weber, K.,and Osborn, M. (1969) J. Biol. Chern. 244,4406-4412 18. Kit, S., Leung, W.-C., Trkula, D., Dubbs, D. R., and Jorgensen, G. (1973) Intervirology 2 , 137-151 19. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529539 20. Laurent, T. C., and Killander, J. (1964) J. Chrornatogr. 14, 317-

T. E. Gan, J. L. Brumley, and M. B. Van Der Weyden, unpub-

lished data.

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filtration of the radiolabeled Step 4 preparations with BioGel A - 0 . 5 ~ yielded a single peak of radioactivity with a Stokes radius of 41 A. The isoelectric point of the purified enzyme is 9.1 (Fig. 3). Rabbit immune serum was prepared with preparations of human placental thymidine kinase obtained after Step 4 of the purification procedure and absorbed against human liver mitochondrial extracts. This antiserum showed precipitation lines of identity with crude placental extract, partially purified placental thymidine kinase (Step 2 and 3 of the purification procedure) but no immunological reactivity with either crude human liver extract or purified liver thymidine kinase or placental mitochondrial extract (Fig. 4). Properties of the Reaction-The effect of pH range of 4.4 to 10.0 Mes buffers (50 mM) were used for the pH range of 4.4 to 6.5, Hepes buffers (50 mM) for the pHrange 7.0 to 8.0, and Tris-HCl(50 mM) buffers for pH range 7.4 to 10.0. Thymidine kinase exhibited a sharp pH optimum of 7.4. Preliminary substrate specificity studies showed the following. (a) Mg2+ is the preferred divalent cation although Caz+ and Mn2+showed greater than 20% of the activity with M e . ( b ) Compounds that showed ability to donatea phosphategroup include GTP, CTP, dATP, dGTP, and dCTP. No compounds tested had greater activity than ATP, with dATP showing 60% and the other compounds less than 30% of the activity with ATP. (c) Nucleoside substrate specificity studies showed that amongst the compounds tested only 5 iododeoxyuridine, 5 diazodeoxyuridine, and 5 bromodeoxyuridine produced significant degrees of inhibition of activity (Table 111). The apparent K , for thymidine was determined to be 3 PM.

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Human Placental Thymidine Kinase

7002 330

21. Ackers, G. K. (1967) J. Biol. Chem. 242, 3237-3238

22. Smith, M. H. (1968) in Handbook of Biochemistry (Sober, A, ed) pp. 610-617, The Chemical Rubber Co., Cleveland, OH 23. Andrews, P. (1965) Biochem. J. 46,595-606 24. Martin, R. G., and Ames, B. N. (1961) J. B i d . Chem. 236,13721379 25. Naitukatis, J. L. (1981) Methods Enzymol. 73, 2-52 26. Ouchterlony, 0.(1962) in Progress in Allergy (Kallos, P., and Waksman, B. H., eds) Vol. VI, pp. 30-154, Karger, Base1 27. Her, M. O., and Momparler, R. L. (1971) J . Biol. Chem. 246, 6152-6158 28. Kit, S., Leung, W.-C., Trkula, D., and Jorgensen, G. (1974) Int. J. Cancer 13, 203-218 29. Kit, S., Leung, W.-C., Trkula, D. (1973) Arch. Biochem. Biophys. 158,503-513

30. Kizer, D. E., and Holman, L. (1974) Biochin. Biophys. Acta350, 193-200 31. Taylor, A. T., Stafford, M. A., and Jones, 0.W. (1972) J. Biol. Chem. 247, 1930-1935 32. Lee, L.-S., and Cheng, Y. C. (1976) J . Biol. Chem. 2 5 1 , 26002604 33. Brongert, D.A., Monaco, M. E., Pinkus, L., Aitken, S., and Lippman, M. E. (1981) Cancer Res. 41,604-610 34. Elsevier, S. M., Kucherlapati, R. S., Nichols, E. A., Creagan, R. P., Giles, R. E., Ruddle, F. H., Willecke, K., and McDougall. J. K. (1974) Nature (Lond.)251,633-635 35. Willecke, K., Reber, T., Kucherlapati, R. S., and Ruddle, F. H. (1977) Somatic Cell Genet. 3, 237-245 36. Lee, L.-S., and Cheng, Y.-C. (1976) Biochemistry 15, 3686-3690 37. Cheng, Y.-C., Damin, B., and Lee, L . 3 . (1977) Biochim. Bzophys. Acta 481,481-492

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7003

Human Placental Thymidine Kinase H a l e c u l a r w i g h t and Stoker radius detemination

SupplerentalElaterialto Humn ThymidineKinase. P u r i f i c a t i o n and P m p e r t i e so ft h e Cytosolic Enzyme of Placenta. by T . Eng Can. Jane L. Bvumley and M r t i n 8. Van Der Weyden.

4 1 were pooled and 0.2 mg

The p u r i f i e d dThd kinase preparations (Step r a d i o l a b e l l e dw i t h1 l Z 5u r i n gt h e

a &io-Gel A-0.5 M mlum ( 2 . 5 x 90 ml which both equllibrated

EXPERlMNTAL PROCEWRES Elaterials

phosphate buffer. pH 7.4 containlng 0.15 M sodim chloride.

T h y l - l ' C ]

Catalale.imnoglobulin

henerrham.

N, N'rethylene-bilacrylamide,

(100 nCilml)

Nucleosides,nucleotides.

Fractions O f 1.7 m1

N.N.N'.N'-tetramethylethyienedia*

determinedby

t r a n s f e r r i n were purchased frm Siw. M-Sepharore 48,

se~umalbumin andhurnan

ide.bovine

(5 C i l m l ) and

dThd2 (56 m C i l n m l ) . [!ethyl-'H]dThd

were purchased fm t h e RadiochemicalCentre, dlth,othre?taI.acrylamide,

were purchased fmm

Blue-Sephamre CL-60. Sephadex 6-25. markerr f o r gels and blue dextran

ampholyte$ were purchased frm LKB Pmdukter.Bio-Gel

P h a m c i a .M h o l i n ec a r r i e r

was obtalned f m m &io-RadLaboratories.Ultra

A 0.5

p u r e sodium dodecylrulfate and anmniun

persulfate were Obtained fmm A J ~ XChemicals. Sydney.

A l o t h e r reagents were c a m r c i a t

Preearation of Thymidine

Sephamse A f f i n i t y M t r i x

The thymidineSepharore

affinity matrix

Of

KOwaland

2-3 u m l e l of thymidinederivative

was 25 di 1131.

f'qthymidine

Of

One

klnare i s d e f i n e d a s t h e amunt of enzyme which can convert 1 n m l e of

unit of thymidine

Aftercentrlfugationat

The r a t i o o f thymidinekinase

imdiately after delivery

phosphate buffer pH 7.4. containing 0.25 M sucm$e and 5 d4 DTT and centrifuged at subsequent c e n t r i f u g a t i o na t

Mtochondria were preparedby

After

was obtainedby

thawing ~n l i q u i d n i t r o g e n . Enzyme P u r i f i c a t i o n Homgenization.Cctyledonr O f human placenta(100 q)were homgenized i n one volume l u l v l Of 50 d4 sodium phosphate b u f f e r pH 7.1 containing 0.25 M wcmle and 5 d4 OTT ? n a blender for 2 min a t 4'C at vhich tenperatwe a l l subsequent steps were p e r f o m d .

Step I .

15,000 g f a r 30 .in.

Step. 2 . Amanium s u l f a t ef r a c t i o n a t i o n . ammnium s u l f a t e

111

TO thesupernatant

A l i q u o t r 170-1W us1 o f pooledStep

4 enzyme preparations

volume Of Freund's cowlete adjuvant and e m l s i f i e d . A l i q u o t r

injected intradermally into

mle r a b b i t s 1251.

shaved parts

Of

Y ~ R mxed with

an equal

were New Zealand white

(50 pl)oftheemllion

t h e back and p m x i m l l i n b r

Of

Four t o e i g h t weeks afterprimaryimniration.antibodyproduction

was

rere i m n i r e d a g a i n a t t h i s The i m n i z e d Tabbits wre bledweekly and a n t i rerum 1261.

assessed usingtheOuchterlonytechnique

equal vOIume

The r a b b i t s

(*/VIOf 50

d4 sodium phosphate b u f f e r pH 7.4 containing 0.25 4 I lucmse.

The hmagenate was centrifuged a t 1WO g f o r 10 min a t 4'C and theSupernatant IO Obtained The m i t o c h m d r i a l p e l l e t IO obtained was washed f i v e f o l d a t 15.000 g f o r IS min a t 4%. i n 0.154 M sodium c h l o r i d e I o l u t i o n .

The p e l l e t was then lysed by rapidfreezethawing

After centrifugation

a t 15,000 g f o r 30 min. the p r e c i p i t a t e was suspended in appmrimately 45 ml of 20 Mi Trir-

in

One w 1 m o f t h e e x t r a c t was incubated w i t h tw voIum1 O f r a b b r t i m n e sevum a t 25% f o r 60 mi" and then centnfuged a t 100,000 g a t 4 O C for 1 how. P u r i f i c a t i o n o f human IiYer thvmidine Llnare liquidnitrogen.

Nan neoplastic h m n l i v e r was obtained a t autopsyandpmcerred t h e p u r i f i c a t i o n procedure.except

h m n l i v e r thymldine kinare

as o u t l i n e d above i n

was e l u t e d from the

colum w i t h 2W m i l l l l ~ t r e r o f 0.5 M T r i l HCI b u f f e r pH 8.0 containing

thymidine affinity

10% glycerol. 5 nW D T I and 300 pM dThd.

front Step 1, c r y s t a l l i n e

added t o 406 r a t u r a t l o n and s t i r r e d f o v 30 mi".

which *ere determined by absorbance a t 2 8 0 nm.

The finalpreparationreprerented

I

4000 f o l d

p u r i f i c a t j o n w i t h a s p e c l f i c a c t i v i t y o f 31.4 nmlerlm~nlmg p m t e i n r i t h a CTPlATP r a t l o of 0.72.

(20 m l )o f a Sephadex 6-25 c o l u m (2.5 x 27 cm) this preparation were sequentially applied to e q u l l i b r a t e d and elutedwithbuffev A. Ten m l l l l i t r e rarnpler *ere c o l l e c t e d and fractions H C I b u f f e r . pH 8.0 containing 10% g l y c e m l and 5 ti4 DTT ( b u f f e r A).Aliquots

TMLE I PURIFICATION OFHLQMPLACENTALTHYMIDINEKINASE

* P r D r e n f

containing thymidine kinase activity pwled. Step. 3. Blue repmamse a f f i n i t y chmmtagraphy.Allqwts from Step 2 were applied to

CL-68 mlum (2.5 x 6 . 5

I Bluerephamre

A.

e q u i l i b r a t e d and washed w i t h b u f f e r

fractions containing actlvity

E.).

Step 3 were applied to

n

5940

IS

a s i n g l e peak and

2.

Amamurn s u l f a t81.5 e (0-401) & 6-25 gel filtration

1555

the t h y m d i n e a f f i n i t y

I5 "11 Of the enzyme preparation f m m colum (0.5 x 4.5 cm) e q u i l i b r a t e d w i t h

repharose 3. Blue a f f i n i t y Chmmatography

31.W

55.7

1.7 57

The calum

B).

Iris-HCI pH 8.0 containing 106 g l y c e m l and 5 # OTT (buffer

0.167

15.7

93.83

4,

Thymidine a f f i n i t y colum

was then washed sequentially w t h 200 m i l l i l l t r e s o f b u f f e r 8 c o n t a i n l n g 2W UM dlhd, and 50 m l l l i l i t r e r Of 0.5 M Trir-HCI buffer pH 8.0containing 106 g l y c e m l . 5 d4 Om and300 dThd.

Ten m i l l i l i t r e f r a c t i o n s

0.018

Placental supevnatant

were pooled.

The c o l u m was washed t o zem absorbance a t 280 nm w t h 100 ml of buffer A and

20 rnl o f 0.2

%

97.3

1.

100

MgC12. 5 nt4 DTT and 101 g l y c e m l , and ten

Thymidine k i n a s ea c t i v i t ye l u t e d

Step. 4 . Thymidine a f f i n i t yc h r m t o g r a p h y .A l i q u o t r b u f f e r A.

Yfeid

- f o l d mg u n i t s l n gn m l e r t n i n

prev~ou~ly

The colum was then washed w i t h 200 ml O f 0.5 M

Trll-HCl pH 0.0 containing 5 d4 ATP, 10 d4 m i l l i l i t r ef r a c t i o n rc o l l e c t e d .

(10 m1) o f enzym preparation

US n ipt se cRl fci ct l P v iut yr i f i c a t i o n

were collected,arrayedforthymidinekinase

0.052

3

99

5212

and a c t i v i t y i n

t h e peak f r a c t i o n r pooled. Polyacrylamde g e l electropherenr

TABLE I 1

E l e c t r o p h o r e r l r was performed w i t h Canalca apparatusemploying, amide (0.2%). N.N,N'.N'

w i t h minor m d i f i c a t i m p ,

The gels (5 x 90 m) were polymerired frm acrylamide ( 5 % ) , b i r a c r y l -

the method o f Kit (16).

tetramthylethylenedia~ine (0.05%) i n 200 d4

TPiS

HC1. pH 8.9

Sedimentation c o e f f i c i e n t

P r i o r t o e l e c t r a p h o r e r i r the enzyme contalning 50 YM dThd, 2 n*l MgCI2 and 2 . 5 d4 ATP. preparations were concentrated10-fold by u l t r a f i l t r a t i o n on a YX 10 f i l t e r . i n 20 nEl TriS

5 d4 DTT and 101 g l y c e m l . One hundred m i c r o l i t r e rarnples i n 25% glycerol were layered Onto t h e gel, togetherwithbromphenolblue as themarker. HCI b u f f e r pH 8.9 containing

E l e c t r o p h o r e $ I I was perfanned at 4'C

2.5 W g e l .

forapproximately

3 hours w i t h a constantcurrent

The runmng b u f f e r was 0.18 M T n r - g l y c i n e b u f f e r

20 ut4 dThdand

2

npI

OTT.

~ e c t l o n l . l m r r e d i n 100

A t completion yl O f

O f

e l e c t m p h o r e r i r gels were s l i c e d ,"to

thethymldlne

klnare reactlo"mlxture

a t 37'C.

Proteln was stainedwith Coornarrie b n l l i a n t b l u e f o r 1 7.5% acetic acidcontaining 10% m t h a n o l .

Samples and standards forIodivmdodecylsulfate t o 85'C

f o r1

by Yeber and Orborn ( 1 7 ) .

minute.

2.5 m

and assdyed f o r 1

hour

hour and desta7ned i n

(SO81 gel e l e c t m p h o r e r i r were d l a l y r e d

dgdmst 10 nM T r i p HCI b u f f e r pH 6.8 c o n t a i n i n g 1%SDS, 2% 8-mrcaptoethanal. thenheated

at

pH 8.9 containing 1 nt4 MgCll

as descvibed

used includedphosphorylase

b (94.000). bovlne serum albumin (67,000),ovalbumin (43,000). carbomc anhydrase (30,000), m y bean t r y p r l n l n h l b i t o r

(20.1001 and o l a c t a l b u m n (14.400).

l s o e l e c t r i i fDCYIIng IsOelectrlC focusing war p e r f o m d u s i n g t h e melhod o f Kit (18) "7th minor mdiflcdtions. The gel conrlrted of 4% acrylamlde, 0.11% blsacrylamide. 0.05% N,N,N',N' tetramethylethylenedlamlne. 0.005% amniumpersulfate. m7cmlltrer

Of

applied to the (w/v

1

1OX glycerol and 2% arnpholiner (pH 7-11) and s i x t y 1.25 RN MgC12 and 2% anphollner wem

enzyme ppepwation 7n 101: glycerol,

The upper and lower b u f f e r Chdnberr R W 0.4% ("/VI NaOH and 0.2% acld. The s a n v l e l were focused a t 4'C with1 wdlgel up t o 400 v o l t s and a t

gels.

IUlfUriC

2 houri. The gels were processed and arrayedfor dThd k i n a r e a c t l v l t y as F o l l o w i n gf i x a t i o ni n 10% TCA Overnight. gels were stainedforproteln as The pH O f each aliquot was detemlnedwith a radiomete? (Copenhagen) microelectmdeafterlnCubdtion of g e l s l l c e s i n 1000 y l of d l i t l l l e d x a t e v f o r 1 h i a t .C'+

thisvoltagefor

described above. dercrrbed above.

HO1ecul.r

(A')

weight

Condi t i o n r

sucrose density ultracentrifugation

92,000

gel f > l t r a t i o n

92,000

(2)

PAGE

94.000 t 1500

sodium dadecyl s u l f a t e

44.000 i 1500 0.32 i 0.02

(41 (41 (31

9.1

(21

pnGE ( R f ) Isoelectricpoint

5

16W

(4)

0.1% OTT and

The sampler were SubJeCted toelectraphorerls

The subunit mlecular weightstandards

(x 1 0 ~ ~ 1 1

Stakesradius

a

The value given i s t h e

rem

f

1 SO.

The f i g u r e 7n parenthesisindicatesthe

nunber

Of

83

detenmnat7ons

16

Downloaded from www.jbc.org by guest, on July 13, 2011

c e n t r i f u g a t i o n a t 100.000 g fov 60 min. The Various s u b c e l l u l a r f r a c t i o n s were suspended i n 50 nW phosphate b u f f e r pH 7.4 containing 101 g l y c e m l and 5 d4 MT and lysed by rapid freeze

The hormgenate was centrifuged at

(S20,w) yere calculated

The standads used were human t r a n r f e r n n

Human l i v e r was obtained at autopsy and h m g e n i z e d a t t h e r l a r e r t speed i n a blender i n

1000 9.

15.000 g f o r 15 minutes.

t h e m t o c h o n d l i a had been remved, the soluble supernatant fraction

IO

a SY 50L r o t o r

(275 "1) were c o l l e c t e d and assayed for thymidine

SedimentationCoefficients

h r t i n and k n e r 1241. Of

5 n rl

tire if no antibody was detected. pooled. The i m n e r a b b l t sewm was absorbed against human l i v e r mitochondrial e x t n c t .

were hamgeniled

Slowest speed f o r 15 seconds i n one volum lwlv) of 50 d4

blender at the

Of

m

5 d4 DTT.and

i n a Beckmn LZ-6% centrifugewith

1'C,

as described above.

the m t h o d

and bovinealbumin,themigration

activitywitheither

Subcellulm Fractionation Cotyledons o f human placentaobtained

accordingto

41 was detemined

Preparatlon of r a b b i t i m n e serum

CTP ( 5 nt4) or ATP ( 5 RN) as thephosphate donor was used as a convenient qualitative means o f d i f f e r e n t i a t i n g rn7tochondrial and CytoIOliCthymidinekinase w i t h a value o f less than 0.4. i n d i c a t i n g excess c y t o s e l l ca c t i v i t y( 8 ) .P m t e l n was determined by a m d i f i c a t i o n O f thenethod Of towry e t a 1 (14,151.

In a

human thymidinekinase(Step

a t 35,000 r p f o r 18-24 h w r r , a l i q u o t l

as

p4C]dThd t o [14C]dTW

were

for waglobin which

gradrent. 5-20% ( w l w l i n 10 RN T r i r HC1 (pH 7.4)containing

k i n a s ea c t i v i t y

was arrayed by d e t e m i n i n g t h e c o w e m i o n o f

previouslydescribedexceptthefinalconcentration

gently

IUCPOI~

nt4 b!gC12.

Enzyme A l l a y

dThd t o dTWlnin.

Tho e l u t i o n v0Ium.1 O f t h e s t a n d a r d p m t e i n l

detemination

linear

were

bound per ml of wet gel. Thymidinekinase

were collected.

and e l u t e d w i t h 50 nt4 The colum was Caliblatedwith

serum albumin, ovalbumin and myoglobxn.

f o l l o w i n g t h e absorbance a t 280 nm f o r a l l standards except

The sedimentation behaviour of

was preparedusingtheprocedure

Elarkur (12) and with three separate prepavatim

6 , transferrin,bovine

was d e t e m i n e d a t 410 nn and the void volure was determinedby e l u t i o n o f b l u e d e x t r a n . The Kav O f each standard was detemined by the mthod of Laurent and K i l l a n d e r (201. The Stokes radius O f dlhd kinase was calculated by the m t h a d O f Ackerr (21) and theStoker r a d i i u r e d f o r t h e standards wre obtained fmm t h e l i t e m t u r e (221. The mlecular weight O f dThd klnare was determinedby the method o f Andrewr (231 S20,u

prepamtlons of the highest purity available.

p r o t e i n was was appliedto

Of

chloramine-T method (19).Thispreparation

Human Placental Thymidine Kinase

7004 THE EFFECTOFVARIOUSNUCLEOSIOES

T l L E 111 AN0 PHOSPHATE DDNOFS ON THE ACTIVITY OF HUHlW PLACENTU THYUlOlNE .~. KINASE . "

Percent a c t i v i t y

Additlonal nucleoside NOW

LOO

Adenosine Gvamrine cytidine Uridine Deoxyadenorine DLOryguanOsine Oeoxycytidine Dwxyuridine 5 iododeoxyuridine 5 diamdeoxyuridine 5 fluomdeoxyuridine 5 bmdeoxyuridine

100

Phosphate donor ATP CTP GTP

100

103 100

UTP

ITP dATP dGlP dCTP dTlP

103

103

1W 100

Percent a c t i v i t y

-50

LOO 29 24 20

'40

I7 62

36 22

-30

.I

YI 44 80

- 20

38

Fornucleoside r p e c i f l c l t y Studles assays were p e r f o m d i n d u p l i c a t e w i t h 50 YU [I4C] thymidine. 5 N4 ATP. 10 C44 %Cland Z 50 $I o f theindlcated C-und. R e S ~ l t sare expressed as percent T I P perfonned i n the absence o f the additional purlne or p y r i m l d i n c c m o w d . For phosphate donor studies the concentration Of nucleotides used was 5 N4 and IO nn UgCl2. Results a r e expressed as percent T I P p e r f o m d w i t h ATP as the phosphate donor. Each assay contained 2-5 ug of t h e p u r i f i e d enzyne (step 4).

-10

FRACTION

NUMBER

Isoelectricfocusingof h u m p l a c e n t a lc y t o s o l i ct h p i d i n ek l n a r ea c t i v i t y Obtained a f t e r Step 4 o f t h e p u r i f i c a t i o n pmcedure. l r o f o c v r r i n g w i t h 4Z pDlyacrylamldegels were p e r f o m d as described under 'ExperimentalPmcedures'. A c t i v i t y w l t h ATP [e-) and w i t h C l P (M).

Fig. 4 .

Double i m n o - d i f f u r l o np r e c i p i t i nr e a c t i o n of anti-hymnplacentalthymidine kinase r a b b i t r e m wlthvariousantigens. l m n e SeNm (25 "1.1 was used i n the centre well and various other preparations 125-103 ug) i n the perlpheral wells O f themicm-ouchterlonyplate. The w e l l s a r e derlplated 1 a t the top and sequentially nmbered i n t h e clockwise direction. with w e ll 1 containing CN& placenta extracti 2 purified placcntdl thynidine kinase (Step 2 ) ; 3 p u r i f i e d placental thymidine klnare (Step 3 ) ; 4 crude l i v e r e x t r a c t ; 5 p u r i f i e d h w n llver thymidinekinase (Step 4); 6 Dlacental mltochondrial e r t n c t .

FRACTION NUMBER f i g . 1.

E l u t i o n pattern o f h m n p l a c m t a l c y t o s o l i c t h p l d i n e kinasewithBlueSephsmre a f f i n i t y chrmdtography. Pn a l i q w t (IO n l ) o f Step 2 preparation ofthe purificatlon pmcedure was applied to the c o l m h i c hwas e l u t e d w i t h 0.5 M T r i s o f 10 ml were HClpH 8.0 contalning 5 C44 ATP and 10 nn ngclZ.Fractions Collected and a c t i v i t y o c c w r l n g as s h m by fractlons 20-26 were pooled. A c t i v i t y w i t h ATP (a".).

e-

e"

L-

A f i g . 2.

B

C

D

Polyacrylamideelectmphoresis o f h m n placentalcytosolicthymidinekinase obtainedafter Step 4o ft h ep u r i f i c a t l o n pmcedure. Panel A. Enzym preparation applied to 7.5: polyacrylamidegel and pmcerred as described I n the 'Experinmtal Pmcedures' Panel 8. Sane preparationappliedto SDS-polyacryldmide gel as describedunder'Experimental peccdurer'. Panel C s h s Sm polyacrylamide gels o f the radio-labelled Step 4 enzym prepamtion as described under 'Experimmtal Pmcedures'. Panel 0. Standards appliedto SDSpolyacrylamidegels: phosphovylase. nolecular weight (94.030). bovine serum albumin 168.000). Ovalbmin (43.000). Cdrbmic anhydrase (30.000). t r y p s i n i n h i b i t o r 120.100) and .I l a c t a l b m i n (14.400). The R f o f thesingle band i n Panel B i s i d e n t i c a l t o the R f oftheautoradiograph band i n Panel c corresponding t O a m l H u l a r weight O f 4 4 . W . The w r m s i n d i c a t e standards,

-

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Fig. 3.

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