J. Cell Sci. 64, 13-25 (1983) 13 Printed in Great Britain © The Company of Biologists Limited 1983

THYMIDYLATE FROM ACETABULARIA I. PROPERTIES OF THE EGON J. DE GROOT AND HANS-GEORG SCHWEIGER Max-Planck-Institut fur Zellbiobgie, Ladenburg bei Heidelberg, West Germany

SUMMARY The occurrence of a dTMP kinase a8 well as a dTDP kinase in Acetabularia mediterranea has been demonstrated. A test system was developed by which it was possible to estimate the enzyme activity in an individual Acetabularia cell or even in a cell fragment. The enzyme catalyses the phosphorylation of dTMP in the presence of ATP. In the test system described, dTTP is formed as well as dTDP. This indicates that there is also a dTDP kinase present in the enzyme preparation. Characteristics of the enzyme such as pH optima at pH 7-0 and 8-75, a 6 temperature optimum at 45 °C, a Km value of 3-3 X 10~ M and a high specificity for ATP were established. In homogenates that were preserved at — 70°C the enzyme activity was retained even after many weeks. Freezing at — 70 °C and then thawing resulted in an increase in enzyme activity. The enzyme was inhibited by low concentrations of dTTP. After centrifugation of homogenates the greater part of the enzyme activity was found in the sediment. From the observation that purified chloroplast preparations contained most of the dTMP kinase activity, and that chlorophyll and enzyme activity cosedimented in a linear sucrose gradient, it was concluded that the enzyme is located in the chloroplasts.

INTRODUCTION The unicellular green alga Acetabularia is a classical object for studies on dif- ferentiation at the cellular level (for references see Schweiger, 1969; Schweiger & Berger, 1979). During its vegetative phase it exhibits a highly species-specific mor- phogenesis (Hammerling, 1931; Schweiger & Berger, 1981). The subsequent generative phase is characterized by a high number of nuclear divisions, and cyst and gamete formation (Schulze, 1939; Schweiger, 1969; Schweiger & Berger, 1979). During the generative phase of the life cycle the number of nuclei is increased from one to more than 106. This, of course, means that the amount of DNA is also substan- tially increased and this leads to the question of how the cell regulates its metabolism so that the production of deoxyribonucleotides is significantly augmented. In previous studies we have shown that two that are involved in the deoxyribonucleotide metabolism are subjected to pronounced regulation at the begin- ning of the generative phase, i.e. when the cell needs large amounts of deoxyribo- nucleotides. The activities of the two enzymes, a and a dCMP deaminase are dramatically increased and it was demonstrated that this increase is due to de novo synthesis of the enzymes. On the basis of inhibitor studies it was concluded that both enzymes are coded for and translated in organelles (Bannwarth & Schweiger, 1975; Bannwarth, Ikehara& Schweiger, \977a,b, 1982). In this paper we will deal with another enzyme, a (EC 2.7.4.9) (de Groot & Schweiger, 1980, 1981). This enzyme, which catalyses the formation of dTDP, 14 E. jf.de Groot and H.-G. Schweiger deserves special interest since its substrate, the dTMP, originates on the one hand in a thymidine kinase catalysed reaction, which probably represents a salvage pathway (Cleaver, 1967), and on the other hand in a reaction that is catalysed by a thymidylate synthetase. So far, the thymidylate kinase is supposed to be the only enzyme involved in the de novo synthesis pathway that catalyses phosphorylation of a deoxyribonucleo- side monophosphate, while the other three deoxyribonucleoside triphosphates are being formed by phosphorylation of diphosphates that are products of a ribonucleo- side diphosphate reduction reaction.

MATERIALS AND METHODS Cells of Acetabularia mediterranea were used throughout (Berger, Sandakhchiev & Schweiger, 1974). In early experiments Erd-Schreiber medium was used (Hammerling, 1963; Schweiger, 1969) but was later replaced by a fully defined artificial medium (Muller, 1962; Berger & Schweiger, 1980). Selected cells were washed twice with medium, carefully brushed, blotted on filter paper and homogenized in 05 ml of 0-1 M-Tris-HCl buffer (pH7-5) containing 10mM-ATP and 20 mM- MgCh, in a glass homogenizer at 4°C. This homogenate was used as the enzyme preparation. The reaction was started by adding S^iCi in 5^1 of the substrate [we

RESULTS Although anion-exchange column chromatography is a laborious method, it was used to separate dTMP, dTDP and dTTP since it had been proved to be a very efficient separation method in contrast to paper and thin-layer chromatography (Fig. 1). There is no cross contamination between the peaks. After incubation with labelled dTMP, the dTDP peak and also the dTTP peak contained radioactivity. This in- dicated that the enzyme preparation also catalysed phosphorylation of dTDP. Therefore, the rates of formation of both dTDP and dTTP were used to give the Properties of thymidylate kinase of Acetabularia 15 activity of dTMP kinase. Usually activity was measured in a homogenate starting with three cells. However, this number by no means represented the lower limit of sensitiv- ity of the method. The amount of material obtained from one cell and even fragments of a cell was sufficient to perform reliable estimations. In order to exclude the possibil- ity that microbial contamination interfered with the determinations, enzyme activity was estimated in axenic and non-axenic cells. No significant difference in enzyme activity was found between axenic and non-axenic cells (results not shown). It is of major importance to monitor the pH in the homogenate and during the test since the content of the central vacuole is extremely acidic and the enzyme activity strongly depends on pH. The enzyme activity exhibited two pronounced maxima, at pH7-0 and 8-75 (Fig. 2). A steep decrease in the enzyme activity was measured below pH 6 and above pH9-5. Experiments were performed in order to find out whether inactivation at suboptimum pH is due to a reversible or an irreversible effect. Controls were in- cubated twice for 30 min at pH 7-5, 5-5 and 10-5, respectively. The enzymic activities measured under these conditions were in agreement with the values expected from the pH curve. Samples that had been incubated at pH 55 or 10-5 for the first half hour and then at pH 7-5 for the second half hour exhibited a total enzyme activity that was

dTMP

4M-HCOOH/ 4M-HCOOH/ 0-4M-NH4COOH 1-2M-NH,COOH

dTTP 3-5 ° x

3-0 1 « TJ 2-0 I

1-6 S

10 £ O 0-5 i O

25 50 Fraction number

Fig. 1. Anion-exchange chromatography of the reaction products of thymidylate phoaphorylation. The homogenate from 10 cells with maximum caps was incubated at pH7-5 in the presence of [me//ry/-3H]dTMP at 45 °C for 1 h. Unlabelled dT, dTMP, dTDP and dTTP were added before chromatography. 16 E. jf. de Groot and H.-G. Schweiger

1-6

o 1-4 x

E 10 c 8 0-8

| 0-6

E I" 0-4

0-2

10 pH

Fig. 2. Effect of pH on phosphorylation of dTMP in vitro. Homogenate was incubated at 45 °C for 1 h in the presence of [me//ry/-3H]dTMP. The buffers used were 0-05 M- Tris/maleate (first estimation (O); second estimation (•)) and 01 M-glycine (A). The pH was adjusted with 0-1 M-NaOH. only a little higher than for those in which incubation was performed at pH 5-5 or 10-5 for 60 min (Table 1). Since the rate of the enzymic reaction remained almost the same over a period of 60 min (Fig. 3), one may conclude that at pH 5-5 as well as at pH 10-5 the inactivation of the enzyme is to a great extent irreversible.

Table 1. Influence of non-optimum pH on enzyme activity pH Incubation Incubation Enzyme activity 0-30 min 30-60 min (cts min"' cell" 'x 10"4)

7-5 7-5 10-6 5-5 5-5 2-8 10-5 10-5 2-7 5-5 7-5 3-3 10-5 7-5 4-5

Homogenate was incubated at 45 °C for 1 hat pH7-5, 5-5 and 10-5 (controls). Another part of the homogenate was incubated either at pH 5-5 for 30 min or at pH 10-5 for 30 min. After rapid change of the pH values to 7-5 both homogenates were incubated for further 30min. Properties of thymidylate kinase o/Acetabularia 17

x 5

I4

I a

20 40 60 80 100 120 Time (min)

Fig. 3. Time course of dTMP phosphorylation in vitro. Homogenates from cells with cyst-bearing caps were incubated for different times in the presence of [methyl-3H]dTMP. Values are given as radioactivity in dTDP and dTTP per cell.

The enzyme exhibited a characteristic temperature dependence, with an optimum of about 45 °C (Fig. 4). Heating of the homogenate to 100 °C for 5 min resulted in complete inactivation. From a Lineweaver-Burk plot of data for enzyme activity at different substrate concentrations Km was estimated to be 3*3 10~6M (Fig. 5). The enzyme was strongly inhibited by low concentrations of dTTP (Fig. 6). At a con- centration of 10/iM the enzyme activity was reduced by 50%. The enzyme reaction was dependent upon ATP. ATP could only be partially replaced by CTP, UTP and GTP (Table 2). The optimum concentration of ATP was 5 mM or lower (Fig. 7). At higher concentrations the enzyme activity was substantially lower. The concentration of Mg2"1" in these experiments was 10 mM. The stability of the enzyme in long-term experiments depended upon temperature (Fig. 8). While there was no decrease in enzyme activity after 55 days at —70 °C, only half of the activity was retained after 10 days at +5°C. At25-5°Call the activity was lost within less than 10 days. Experiments not shown here indicated that in contrast to the dTMP kinase the dTDP kinase was significantly activated during storage at — 70 °C. Storage at 5°C resulted in a rapid

Properties of thymidylate kinase of Acetabularia 19

X 4

i c E | i

CO .2

20 40 60 80 100 120 dTTP

Fig. 6. Effect of dTTP on dTMP kinase activity. Samples were incubated for 1 h at 45 °C in the presence of dTTP, [methyl-^H]dTMP and a homogenate from cyst-bearing caps. Values are given as radioactivity in dTDP and dTTP per cell. decrease in the activity of the dTDP kinase, while the dTMP kinase activity declined at a significantly lower rate. At +5 °C no activation was observed. In order to answer the question of where in the cell the dTMP kinase is located, a number of experiments were performed. Separation of cell wall and cytoplasm was performed by a method of gentle centrifugation (Schweiger, 1966). By far the greater

Fig. 4. Effect of temperature on dTMP phosphorylation in vitro. Homogenates from cells with cyst-bearing caps were incubated for 1 h at the given temperature in the presence of [methyl-3H]dTM?. Values are given as radioactivity in dTDP and dTTP per three cells. Fig. 5. Lineweaver-Burk plot of dTMP phosphorylation in homogenates from cells in the stage of cyst formation. Addition of unlabelled dTMP was necessary to obtain the correct concentrations of substrate ([S]). The concentration of ATP in all incubations was 10 mM. Values are given as radioactivity in dTDP and dTTP per cell. 20 E. jf. de Groot and H.-G. Schweiger

Table 2. Comparison of several different nucleotides as phosphate donors Enzyme activity Addition (ctsmin^ceir1 X 10 None 0 ATP 11-6 CTP 1-9 UTP 1-3 GTP 1-1 AMP 0

Homogenates from cells with half-maximum cap diameter were incubated for 1 h at 45 °C in the presence of different phosphate donors at a concentration of 10 mM, and [methyl-3H]dTMP. Values are given as radioactivity in dTDP and dTTP per cell.

4 -

c E I 2

to

10 15 20 ATP (ITIM) Fig. 7. ATP dependence of the phosphorylation of dTMP. Homogenates from cells with cyst-bearing caps were incubated for 1 h at the given ATP concentrations in the presence of [me

Table 3. dTMP kinase activity in the sediment and supernatant fractions

Enzyme activity (ctsmin-'ceir'x 10"3) Stage of development Supernatant Sediment

Prior cap formation 4 40 Half-maximum diameter caps 11 69 Maximum diameter caps 16 104 Cysts 24 123

Homogenates corresponding to 12 cells of each developmental stage were centrifuged for 20 min at 20 000£ and 4°C. The pellets were rehomogenized in the same volume of buffer as the super- natant; 500 /ul of each fraction were incubated for 1 h at 37 °C in the presence of [methyl-^H]dTMP. Values are given as radioactivity in dTDP and dTTP per cell.

E 3

20 40 60 Time (days)

Fig. 8. Temperature stability of dTMP kinase. Homogenate from 54 cells with cyst- bearing caps in 9 ml incubation buffer was divided into 500-^1 fractions. After storage at different temperatures the fractions were incubated for 1 h at 45 °C in the presence of [methvl-^H]dTMP. Values are given as radioactivity in dTDP and dTTP per cell. (•——•) 21-5°C; (• •) 5°C; (• A) -70°C. 22 E. J. de Groot and H.-G. Schweiger part of the enzyme activity was found in the cytoplasm. In order to find out whether the enzyme is associated with membranes, organelles or other particles, a homogenate of cells of Acetabularia was centrifuged for 20min at 20 000 £ and 4°C. About 75 % of the enzyme activity was located in the pellet (Table 3). On the basis of chlorophyll content, the enzyme activities of homogenates and highly purified chloroplasts

50 i- -|50

4-5

40 40

E x 9 30 30 r a o C 2-5 a i £ 20 20 •

1-5

10 10

0-5

3 4 5 6 Fraction (number) I 50% •+ ->20% I Sucrose Fig. 9. Distribution of the dTMP kinase activity after centrifugation of a homogenate through a linear sucrose density gradient. Cytoplasm obtained by centrifugation of anucleate cells with small caps was washed three times in isotonic buffer (0-4M-sucrose, 005M-Tris, 0-01 M-NaCl, pH7-8) and subjected to a linear sucrose density gradient centrifugation (20% to 50% sucrose, w/w; 6ml vol.; 60min; 28 000 g; 4CC). After fractionation of the gradient, enzyme activity and chlorophyll content were estimated in each fraction (0-7 ml). Values for enzyme activity are given as radioactivity in dTDP and dTTP. Properties of thymidylate kinase of Acetabularia 23 (Shephard & Levin, 1972) were compared. The results (not shown) support the idea that the enzyme activity is associated with the chloroplasts. Further support was obtained from an experiment in which cytoplasm obtained by gentle centrifugation of anucleate cells was washed three times in isotonic buffer (0-4M-sucrose, 0-05M- Tris, 0-OlM-NaCl, pH7-8) and subjected to a linear sucrose density gradient centrifugation (20% to 50% sucrose, 60min, 28 000^, 4°C). Fractionation of the gradient and estimation of the enzyme activity and the chlorophyll content of each fraction showed that enzyme activity coincided with the chlorophyll content (Fig. 9).

DISCUSSION The experiments presented here demonstrate the occurrence of a dTMP kinase and a dTDP kinase in Acetabularia. The method used for determining the enzyme activ- ity is highly specific because of the excellent capacity for separation of the products of the enzymic reaction. In addition, the method is sensitive enough to allow the estimation of enzyme activity in individual Acetabularia cells or even fragments of a cell. Remarkable features of the enzyme are the double-peak pH curve and the inhibit- ory effect of low concentrations of dTTP. A double peak with similar pH optima was described for a dTMP kinase from mouse hepatoma (Kielley, 1979). The pronounced feedback inhibition by dTTP is in good agreement with results obtained from other biological materials (Nelson & Carter, 1969; Cheng & Prusoff, 1973; Smith & Eakin, 1975). The thermal stabilities of the dTMP kinase and the dTDP kinase are different. The dTDP kinase activity exhibits a rapid decrease after storage at 5°C, while the dTMP kinase activity remains relatively stable. Investigations on the green alga Chlamydomonas reinhardti (Swinton & Chiang, 1979) and KB cells (Ooka & Daillie, 1977) have also revealed differences in the temperature dependence of the phos- phorylating enzymes. The dTMP kinase activity per Acetabularia cell has been estimated as 17 X lO'^molmin"1, which is of the same order of magnitude as described for the dCMP deaminase (Bannwarth et al. 1982) and higher than the activity of the thymidine kinase (Bannwarth et al. 1977a). It should be noted that the = - affinity of dTMP for the enzyme from Acetabularia (/Cn 3 3x 10~6M) is sig- nificantly higher than for the enzyme from Ranapipiens (Km = 0-5 X 10~3 M) (Atkin- son & Roth, 1971), Escherichia coli B (Km = 2-4 X 10~4M) (Nelson & Carter, 1969), 4 mouse hepatoma (Km = 1-9 X 10~ M) (Kielley, 1970) and embryonal chicken liver (Kn = 9 X 10~5M) (Smith & Eakin, 1975). This high affinity suggests that the enzyme plays an important role in the cell by providing DNA precursors. The majority of the enzyme activity is easily pelleted by centrifugation. This is in contrast to results for dTMP kinase in other biological materials (Wanka & Poels, 1969; Kielley, 1970; Cheng & Prusoff, 1973; Smith & Eakin, 1975; Cossu, Grippo, Marino & Molinaro, 1979; Kaufman & Davidson, 1979). From the fact that purified chloroplasts contain dTMP kinase activity that compares well with the total activity on the basis of chlorophyll content, and that in linear sucrose density gradient centrifugation chlorophyll and enzyme activity parallel each other, it is suggested that at least the greater part of the enzyme is either closely associated with or is a constituent 24 E. J. de Groot and H.-G. Schweiger of the chloroplasts. The finding that thymidine kinase is located in the plasma mem- brane outside the chloroplasts (Bannwarth et al. 1977a,b), in combination with the generally accepted idea that, except for ATP, nucleotides do not enter the chloroplasts (Heldt, 1976), indicates that the thymidine kinase and the chloroplast dTMP kinase are not functionally linked to each other. This might be related to the fact that the thymidine kinase is a salvage-pathway enzyme while the dTMP kinase under con- sideration catalyses a reaction that is part of a truly de novo synthetic pathway, which obtains its substrate from the thymidylate synthetase reaction.

The authors appreciate fruitful discussions with Professor Horst Bannwarth, the expert technical assistance of Mrs Rita Menze and the careful preparation of the manuscript by Mrs Brigitte Nagel.

REFERENCES ARNON, D. J. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta uu/gam. PI. Physiol. 24, 1-15. ATKINSON, B. G. & ROTH, J. S. (1971). Control of the synthesis of DNA precursors during Rana pipiens development. Biochim. biophys. Ada 240, 12-22. BANNWARTH, H., IKEHARA, N. & SCHWEIGER, H.-G. (1977a). Thymidine phosphorylating enzymes mAcetabularia: evidence for the occurrence of a thymidine kinase. Proc. R. Soc. Land. B, 198, 155-176. BANNWARTH, H., IKEHARA, N. & SCHWEIGER, H.-G. (19776). Nucleocytoplasmic interactions in the regulation of thymidine phosphorylation in Acetabularia. Proc. R. Soc. Land. B, 198, 177-190. BANNWARTH, H., IKEHARA, N. & SCHWEIGER, H.-G. (1982). Deoxycytidine monophosphate deaminase mAcetabularia: properties and regulation in the early generative phase. Eur.jf. Cell Biol. 27, 200-205. BANNWARTH, H. & SCHWEIGER, H.-G. (1975). Regulation of thymidine phosphorylation in nucleate and anucleate cells of Acetabularia. Proc. R. Soc. Land. B, 188, 203-219. BERGER, S., SANDAKHCHIEV, L. & SCHWEIGER, H.-G. (1974). Fine structural and biochemical markers of the Dasycladaceae. J. Microsc. 19, 89-104. BERGER, S. & SCHWEIGER, H.-G. (\980). Acetabularia: techniques for study of nucleo-cytoplasmic interrelationships. In Handbook of Phycological Methods. Developmental and Cytological Methods (ed. E. Gantt), pp. 47-57. Cambridge University Press. CHENG, Y. C. & PRUSOFF, W. H. (1973). Mouse ascites sarkoma 180 thymidylate kinase. General properties, kinetic analysis, and inhibition studies. Biochemistry 12, 2612-2619. CLEAVER, J. E. (1967). Thymidine metabolism and cell kinetics. In Frontiers of Biology (ed. A. Neuberger & E. L. Tatum), vol. 6. Amsterdam: North Holland. Cossu, G., GRIPPO, P., MARINO, M. & MOLINARO, M. (1979). Biosynthesis of dTTP during differentiation of chick embryo myoblasts in culture: changes in thymidine kinase, thymidylate kinase and thymidylate synthetase activities. Cell Differ. 8, 323-329. DE GROOT, E. J. & SCHWEIGER, H.-G. (1980). Thymidylate kinase activity and nuclear divisions in Acetabularia mediterranea during the generative phase. Protoplasma 105, 355. DE GROOT, E. J. & SCHWEIGER, H.-G. (1981). Regulation of the thymidylate kinase in Acetabularia mediterranea. Eur.jf. Cell Biol. 24, 5. GIBOR, A. & IZAWA, M. (1963). The DNA content of the chloroplasts of Acetabularia. Proc. natn Acad. Sri. U.SA. 50, 1164-1169. HAMMERLING, J. (1931). Entwicklung und Formbildungsvermogen von Acetabularia mediter- ranea. I. Die normale Entwicklung. Biol. Zbl. 51, 633-647. HAMMERLING, J. (1963). Nucleo-cytoplasmic interactions in Acetabularia and other cells. A. Rev. PI. Physiol. 14, 65-92. HELDT, H. W. (1976). Metabolite transport in intact spinach chloroplasts. In The Intact Chloroplast (ed. J. Barber), vol. 1, pp. 215-234. Amsterdam, New York, Oxford: Elsevier. Properties of thymidylate kinase of Acetabularia 25 HURLBERT, R. B. (1957). Preparation of nucleoside diphosphates and triphosphates - chromatographic separation. Meth. Enzym. 3, 793-805. KAUFMAN, E. R. & DAVIDSON, R. L. (1979). Altered thymidylate kinase substrate specificity in mammalian cells selected for resistance to iododeoxyuridine. Expl Cell Res. 23, 355-363. KIELLEY, R. K. (1970). Purification and properties of thymidine monophosphate kinase from mouse hepatoma.7. biol. Chem. 245, 4204-4212. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. MOLLER, D. (1962). Uber jahres- und lunarperiodische Erscheinungen bei einigen Braunalgen. Botanica mar. 4, 140-155. NELSON, D. J. & CARTER, C. E. (1969). Purification and characterization of thymidine 5'-mono- phosphate kinase from Escherichia coli B.jf. biol. Chem. 244, 5254-5262. OOKA, T. & DAILLIE, J. (1977). Temperature dependent phosphorylation of (3H) thymidine and its incorporation into DNA by KB cells. Expl Cell Res. 104, 319-324. RANDERATH, K. (1965). Diinnschichtchrvmatographie. Weinheim: Verlag Chemie. SCHULZE, K. L. (1939). Cytologische Untersuchungen an Acetabularia mediterranea and Acetabularia wettsteinii. Arch. Protistenk. 92, 179-225. SCHWEIGER, H.-G. (1966). Ribonuclease-Aktivitat in Acetabularia Planta 68, 247-255. SCHWEIGER, H.-G. (1969). Cell Biology of Acetabularia. Curr. Top. Microbiol. lmrnun. SO, 1-36. SCHWEIGER, H.-G. & BERGER, S. (1979). Nucleocytoplasmic interrelationships in Acetabularia and some other Dasycladaceae. Int. Rev. Cytol. (suppl.) 9, 11-44. SCHWEIGER, H.-G. & BERCER, S. (1981). Pattern formation in Acetabularia. In Cytomorphogenesis in Plants (ed. O. Kiermayer) Cell Biology Monographs, vol. 8, pp. 119-145. New York, Vienna: Springer Verlag. SCHWEIGER, H.-G., BREMER, H. J. & SCHWEIGER, E. (1963). Der Ribonucleinsaure-Stoffwechsel in Erythrocyten. I. Freie Nucleotide und Phosphateinbau in kernhaltigen Erythrocyten. Hoppe- Seyler's Z. Physiol. Chem. 332, 17-26. SHEPHARD, D. C. & LEVIN, W. B. (1972). Biosynthesis in isolated Acetabularia chloroplasts. J. Cell Sri. 54, 279-294. SMITH, L. K. &EAKIN, R. E. (1975). Partial purification and characterization of thymidylate kinase from embryonic chick liver. Archs Biochem. Biophys. 167, 61-71. SWINTON, D. C. & CHIANG, K. S. (1979). Characterization of thymidine kinase and phosphoryla- tion of deoxyribonucleosides in Chlamydomonas reinhardti. Molec. gen. Genet. 176, 399-409. WANKA, F. & POELS, C. L. M. (1969). On the problem of an enzymatic regulation of DNA synthesis in Chlorella. Eur. jf. Biochem. 9, 478-482.

(Received 21 April 1983-Accepted 25 April 1983)