A~c~rv~s OF BIOCHEMISTRY AND BIWHYSICS Vol. 188, No. 1, May, pp. 64-69, 1978
Identification SHIJHEI *Department
of True Thymidine YIJYAMA,*
SONDRA
of Zoology, University of Toronto, Biology, Queen’s Uniuemty, Received
Kinase
October
in Tetrahymena
CORFF,*
AND
PAUL
Pyriformis G. YOTJNGT
Toronto, Ontario, Canada ML5 IAI, and tZ)epartrnent Kingston, Ontorio, Canada K71 3N6 31, 1977; revised
January
ST
of
2, 1978
Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg’+, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Althbugh the mitochondrial fraction also by Mg”+ and not signifiexhibited ATP-dependent phosphorylation, it is not stimulated cantly inhibited by dTTP. Nucleoside phosphotraniferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphot.ransferase activit.ies were separated hy establishing the presence of both enzymes in this polyacrylamide gel electrophoresis, organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.
Thymidine is phosphorylated to 2’-deoxythymidine 5’-monophosphate (dTMP) by either thymidine kinase (TK,’ EC 2.7.1.75) or nucleoside phosphotransferase (NPT, EC 2.7.1.77). Thymidine kinase utilizes nucleoside triphosphates (preferentially ATP) as phosphate donors, is very specific with regard to the substrate phosphorylated (thymidine or deoxyuridine only), requires Mg2+, and is feedback inhibited by dTTP (1, 2). Nucleoside phosphotransferase, on the other hand, utilizes a wide range of nucleoside mono- and diphosphates as phosphate donors and phosphorylates a wide range of nucleosides, is not dependent upon Mg”+, and is generally inhibited by NaF but not by dTTP (3-7). The mechanism of thymidine phosphorylation in Tetrahymena pyriformis has been controversial. An early report indi’ Abbreviations u.sed: TK, thymidine nucleoside phosphotransferase; DEAE, ethyl; ME, mercaptoethanob mt-lysate, lysate; cyto-lysate, cytoplasmic lysate; ethylimino.
kinase; NPT, diethylaminomitochondrial PEI, phospho64
0003.9861/78/1881-0064$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights ot reproduction m any form reserved.
cated that the thymidine kinase of this organism was unusual in that it was not feedback inhibited by dTTP (.%).The above conclusion was later disputed by other workers (9, 10). The ATP-dependent phosphorylation of thymidine by Tetrahymena extract was much less than the AMP-dependent phosphorylation of thymidine and the thymidine kinase-like activity could not be separated (by gel filtration and DEAESephadex chromatography) from ATPase and nucleoside phosphotransferase activities (10). Also, the activities of ATPand AMP-dependent phosphorylation increased at the same rate in synchronous cultures (9). It was therefore claimed that thymidine kinase was absent in Tetrahymena and that thymidine phosphorylation in the ATP-dependent reaction was catalyzed by the combined actions of ATPase and nucleoside phosphotransferase (9, 10). In this paper, we present evidence that demonstrates the presence of a true thymidine kinase (EC 2.7.1.75) in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis ST.
THYMIDINE MATERIALS
AND
KINASE
METHODS
Organism and culture method. Tetrahymena pyriformis ST was grown in Neffs medium (11) to 1 to 3 X 10” cells/ml with vigorous shaking. In this medium, cells at this range of concentration are in early to midlogarithmic growth phase. Thymidine kinase and nucleoside phosphotransferase assays. The TK and NPT assays followed the method of Ives et al. (12) and have been reported previously (7). The complete TK assay contained, in a volume of 200 ~1, final concentrations as follows: 0.01 M ATP, 0.005 M MgCl,, 0.01 M mercaptoethanol (ME), 0.005 M NaF, 0.05 M Tris-HCl, pH 8.0, and 0.02 mM [methyl-,‘H]thymidine (0.25 &i/nmol, AmershamSearle). The complete NPT assay mixture was identical to that for TK except that 0.01 M AMP was substituted for ATI’, and NaF was omitted. The amount of cell extract was adjusted such that all of the data in this paper lie well within the linear range of the reaction. Data are expressed as nanomoles of dTMP formed per milligram of protein per minute. Preparation of cell extract. A crude cell lysate was prepared b,v washing cells once with distilled water and then lysing cells with 1% Triton X-100 in 0.01 M Tris. pH 8.0. A postmitochondrial supernatant and crude mitochondria were prepared as follows. Cells were homogenized in 0.35 M sucrose, 0.005 M MgCL, 0.01 M Tris, pH 7.0 using a hand emulsifier (Fisher). Whole cells, nuclei, and debris were removed by centrifugation at 600~ for 3 min. A postmitochondrial supernatant was then prepared by centrifugation at 77oOg for 10 min. Triton X-100 was added to 1%. The crude mitochondrial lysate was prepared by washing the above pellet once with the sucrose-Tris-MgL’ buffer using the same centrifugation procedure. The pellet was then resuspended in the sucrose-Tris-Mg” buffer and Triton X-100 was added to 1%. The mitochondrial suspension cleared in about 2 min, indicating complete lysis. All the above operations were performed at 0 to 4°C.J Polyacrylamide gel electrophoresrs. Polyacrylamide gel electrophoresis for TK using postmitochondrial supernatant or crude mitochondrial lysate was carried out following the method of Kit et al. (13). Following electrophoresis, the gels were sliced into 2mm sections and each section was incubated with 200 ~1 of the appropriate assay mixture. Incubation was for 2 h at 28°C with shaking. Other assays. Protein was determined by the method of Lowry et al. (14) using bovine serum albumin as standard. RESULTS
The experiments in this study were designed to identify thymidine kinase and to distinguish it from nucleoside phosphon transterase.
IN Tetrahymena
65
pyriformis
dTTP Sensitivity To examine whether or not a true TK is present in Tetrahymena, the sensitivity of the thymidine phosphorylating reaction to dTTP was examined. A crude mitochondrial lysate (mt-lysate) and a postmitochondrial supernatant (cyto-lysate) were prepared from logarithmically growing Tetrahymena cells and assayed for thymidine phosphorylating activity at different concentrations of dTTP under three different sets of conditions, i.e., ATP plus NaF, AMP alone, and AMP plus NaF. NaF is known to inhibit ATPase (15) as well as NPT. The results are shown in Figs. 1A and 1B. A
0
1
0 dTTP
c
“‘a....
._c 0 ‘0 ii
a..
2 mM
bL...~.
. ... . ..
...*. -....o. ~E” , o r ..% /-.-.. -L
/ T 0-
-......,,.
I 1 dTTP
I
.‘. . ... . ..
‘-....O ~
I
1 2
mM
. . . . 0 . . . . . . . .a . . . . . . . . . . . . . . . . . . . . . . . . . .
1
OO dCTP
2
mM
FIG. 1. Sensitivity of thymidine phosphorylating reaction to dTTP and dCTP. (A) dTTP sensitivity of ATP-dependent reaction (in presence of 5 mM NaF). V-V, cyto-lysate; U-U, mt-lysate. (B) dTTP sensitivity of AMP-dependent reaction in the absence or presence of NaF. Q-V, cyto-lysate (no NaF); Cl- --Cl, mt-lysate (no NaF); M, cyto-lysate (plus NaF); 0- - -0, mt-lysate (plus NaF). (C) dCTP sensitivity of ATP-dependent reaction. V-V, cytolysate; Cl- - 4, mt-Iysate. nm-nanomoles.
66
YUYAMA,
CORFF,
When ATP (plus NaF) was the phosphate donor, thymidine phosphorylation by the cyto-lysate was strongly inhibited by dTTP, whereas the reaction by the mt-lysate was not inhibited (Fig. 1A). In the absence of dTTP, the specific activity of the cyto-lysate was lo- to 12-fold greater than the mt-lysate (Fig. 1A). When AMP (without NaF) was the phosphate donor, phosphorylation by the cytolysate and by the mt-lysate were both slightly inhibited by dTTP (Fig. 1B) and the phosphorylation by the mt-lysate was slightly more sensitive to dTTP than that of the cyto-lysate (Fig. 1B). However, neither the degree nor the pattern of inhibition by dTTP was similar to that observed when using ATP as the phosphate donor. In the absence of dTTP, the specific activity of the cyto-lysate was about 60% of that of mt-lysate. When AMP (plus NaF) was the phosphate donor, the effect of dTTP on phosphorylation was similar to that observed when using AMP alone (no NaF) (Fig. 1B). Again, the phosphorylation reaction by the mt-lysate was slightly more sensitive to dTTP than that by the cytolysate (Fig. 1B). Since some TKs are also sensitive to dCTP (16), the effects of dCTP were examined using ATP (plus NaF) as the phosphate donor. With the cyto-lysate, 1 mM dCTP inhibited the phosphorylation by 30% and 2 mM dCTP, by 50%. Phosphorylation by the mt-lysate showed no dCTP sensitivity. The above data suggest that the cytoplasm of Tetrahymena contains typical TK and NPT. They also suggest that ATP-dependent phosphorylation of thymidine by mt-lysate is primarily not due to typical TK. NaF Sensitivity In the second series of experiments, the effects of NaF on the thymidine phosphorylating reaction were examined. The ATPdependent phosphorylation by the cyto-lysate showed little sensitivity to NaF (Fig. 2A), whereas it was markedly inhibited in the mt-lysate (at 5 mM, about 80% inhibition; at 10 mM, about 80%; Fig. 2A). NaF inhibited the AMP-dependent phosphoryl-
AND
YOUNG
lOI--
5p--.-.....y ......p......__. p._.._..._.__. 9 P, 2.5
‘j ii m
E \ D E
5.0
25
10
No F, mM
20 ....“*,....h ‘*., *.
t
1
I
I
I
2.5
5.0
7.5
10
NoF,
mM
FIG. 2. Sensitivity of thymidine phosphorylating reaction to NaF. (A) NaF sensitivity of ATP-dependent reaction. V-V, cyto-lysate; U - Xl, mt-lysate. (B) NaF sensitivity of AMP-dependent reaction. V- - -V, cyto-lysate; Cl- - -El, mt-lysate.
ation by either fraction (at 5 mM, 30-35% inhibition; at 10 mM, 55-60%; Fig. 2B). As mentioned earlier (in the dTTP sensitivity experiment), the specific activity of ATP-dependent phosphorylation by the cyto-lysate was greater than that by the mt-lysate, whereas the reverse was true for the specific activity of AMP-dependent phosphorylation. This observation was confirmed in the NaF sensitivity study. ibIg”+ Dependence The above observations showed that the characteristics of phosphorylation of thymidine by the cyto-lysate were those of either TK or NPT, depending upon the phosphate donor used, ATP or AMP, and that mt-lysate did not contain typical TK. In the third series of experiments, the Mg2+ dependence of the reaction was tested using the crude cell lysate. As seen in Fig. 3, Mg2+
THYMIDINE
3ot
IN Tetrahymena
rl
I
1
addition of AMP (Fig. 4B). This increased amount of phosphorylation was, at least in part, sensitive to dTTP. Although present data suggest a possible inhibitory effect of AMP on TK activity, it is not conclusive since there is no agent which inhibits NPT activity completely. Separation of TK and NPT by Polyacrylamide Gel Electrophoresis
; 5p-----T OO
67
pyriformis
0 _,,_.... ‘.‘.’ ..0,,,,,..,_....
,...,__... ... .; 25 ___,._..... .‘.,_......... 0 \ .cu ‘d ; 20 2 \
KINASE
/ 2.5 MgCI,,
I 5.0
I 7.5
1 10
mM
FIG. 3. Effects of Mg’+ on thymidine phosphorylating reaction using crude cell lysate. V-V, ATPdependent phosphorylation; Cl- - -Cl, AMP-dependent phosphorylation; O--O, ATP plus AMP-dependent phosphorylation.
concentrations of 2.5 to 5 mM stimulated the phosphorylation reaction 4- to 6-fold when ATP was the phosphate donor. When Mg”+ was absent and EDTA (10 mM) was added, there was a further 44% decrease in the phosphorylation of thymidine. No marked stimulation of phosphorylation by Mg2+ was observed when AMP (no NaF) was the phosphate donor (Fig. 3). Similarly, when both AMP and ATP were present as phosphate donors, Mg”+ did not stimulate the phosphorylation markedly (Fig. 3). Moreover, the presence of ATP greatly lowered the level of phosphorylation observed with AMP alone. ATP appeared, therefore, to inhibit the AMP-dependent phosphorylation. In order to verify this assumption, the effect of ATP on the NPT activity was examined in the presence of 1 mM dTTP, which completely inhibits the TK activity. As seen in Fig. 4A, phosphorylation of thymidine by AMP is strongly inhibited by ATP. In the reciprocal experiment, the effect of AMP on the ATP-dependent phosphorylation was tested. The amount of thymidine phosphorylation was increased by the
The results of our initial experiments provided evidence for the presence of TK in the cytoplasm of Tetrahymena. When the cyto-lysate was fractionated on 5% polyacrylamide gels, the TK was separated from the NPT (Fig. 5A). TK ran as a sharp peak and the activity was completely inhibited by 1 mM dTTP (Fig. 5A). NPT appeared to have two components, a sharp peak and a relatively broad peak (Fig. 5A). It is not clear whether the broad peak was an aggre-
ATP
0
(mM)
2
I AMP
3
4
(“‘M)
FIG. 4. Effects of ATP and AMP on the thymidine phosphorylating reaction using cell lysate. (A) Effects of ATP on AMP-dependent phosphorylation. Each reaction mixture contained 1 mM dTTP to inhibit TK activity. (B) Effects of AMP on ATP-dependent phosphorylation. X, the levels of phosphorylation in the presence of 1 mM dTTP.
68
YUYAMA,
CORFF,
AND
YOUNG
as the cytoplasmic enzyme and the broad peak migrated slightly slower than the corresponding peak. The product of the phosphorylation reaction was characterized to ensure that it was 5’-dTMP. Aliquots of reaction mixtures from the peak of TK activity in Fig. 5A and from the sharp peak of NPT in Fig. 5A were chromatographed against standards; PEI cellulose plates (BDH) were developed in one dimension using 0.25 M LiCl (17). In both cases, only thymidine (the substrate) and 5’-dTMP (the product) were radioactively labeled (Fig. 6). 20
IO
30
40
DISCUSSION
SI ice
Number
FIG. 5. Separation of TK and NPT activities of Tetratrymena extract by polyacrylamide gel electrophoresis. (A) TK and NPT activities of cyto-lysate. -, ATP-dependent phosphorylation; - - -, ATP-dependent phosphorylation in the presence of 1 mM dTTP; --, AMP-dependent phosphorylation. (B) TK and NPT activities of mt-lysate. F, ATP-dependent phosphorylation; - - -, ATP-dependent phosphorylation in the presence of 1 mM dTTP; --, AMP-dependent phosphorylation. Note the difference in the scales of TK and NPT. BPB = bromophenol blue marker.
gated form or a second enzyme. The high NPT activity seen on top of the gel (Fig. 5A) suggests the possibility of aggregation. The important point, however, is that TK and NPT are clearly separable on the gel, demonstrating the presence of both enzymes in the cyto-lysate of Tetrahymena pyriformis. When the mt-lysate was fractionated, the patterns of enzyme activities were quite similar to those of the cyto-lysate, although the NPT activity was much higher than that of TK, as expected (Fig. 5B). TK ran as a relatively small and sharp peak which migrated at the same rate as the cytoplasmic TK and was completely inhibited by dTTP (Fig. 5B). NPT had two components; the sharp peak migrated at the same rate
In this study, we have shown that thymidine kinase is present in Tetrahymena pyriformis ST. This is contrary to the previous reports that proposed the absence of TK in Tetrahymena (9, 10). Our conclusion is based on the fact that TK (thymidine phosphorylating enzyme which is ATP dependent and dTTP sensitive) is separated from NPT (AMP dependent) using electrophoresis. Since the ATP-dependent phosphorylation of thymidine, which is stimulated by Mg2+ and is inhibited by dTTP, is clearly demonstrable both with crude cell lysate and postmitochondrial supernatant of Tetrahymena, we are at a loss to explain earlier results (8, 9, 10). TK in Tetrahy-
‘:
‘.., r
dTMP
4r
w
z
3-
0 ; 0 ”
z-
l-
0
5
10
15
20
Fractions
FIG. 6. Identification of products of thymidine phosphorylating reaction using Tetrahymena extract. PEI plates were sliced transversely into 19 strips. The “H radioactivity of each strip was determined. p, ATP-dependent phosphorylation; - - -, AMP-dependent phosphorylation. Arrows with dTMP and dT indicate the center of the migrated position of the marker. 0 = origin.
THYMIDINE
KINASE
menu is a peak enzyme whose activity is highest during the S phase of the cell cycle (18), while NPT activity increases continuously during the cell cycle (9, 18). This observation also supports our conclusion that TK is present in Tetrahymena. The TK of Tetrahymena is similar to that found in the cytosol of other organisms (1, 16), except that it exhibits some degree of dCTP sensitivity which has previously only been reported for some mitochondrial TKs (16). However, TK of Tetrahymena is clearly not mitochondrial, because most of the TK activity is demonstrable in the postmitochondrial supernatant. Although the crude mitochondrial fraction exhibited ATP-dependent phosphorylation of thymidine, this does not appear to be due to a unique mitochondrial TK, since it is not demonstrably sensitive to dTTP, but is sensitive to NaF. Thus, the combined actions of ATPase and NPT (both are inhibited by NaF (7, 15)) are probably responsible for the ATP-dependent phosphorylation by the crude mitochondrial fraction. The electrophoresis study indicated, however, that a relatively small TK peak migrated at the same rate as that of the postmitochondrial supernatant, suggesting cytoplasmic contamination of the mitochondrial preparation. In Acetabularia, the TK-like activity which was found recently (19) is mainly bound to the particulate fraction (but outside mitochondria or chloroplasts), and the Triton X-100 does not solubilize the TKlike activity. This is probably not the case with the TK activity of Tetrahymena, since it appears to migrate freely in the 5% gel. These observations, of course do not exclude the presence of a unique TK in mitochondria of Tetrahymena. The characteristics of NPT in Tetrahymenu agree well with the other reports of this enzyme (3-7, 9, 10). It is partially inhibited by NaF and ATP, but dTTP and Mg”+ have little effect. Although the specific activity of NPT of the mt-lysate was 1.6 to 1.8 times
greater
than
that
of cyto-
lysate, it is not known whether they represent different enzymes.
IN Tetrahymena
69
pyriformis ACKNOWLEDGMENTS
This work was supported by grants from the National Research Council of Canada to S.Y. and to
P.G.v.
REFERENCES
2. 3. 4. 5. 6.
KIT, S. (1970) in Metabolic Pathways (Greenberg, D. M., ed.), pp. 69-275, Academic Press, New York. ANDEWSON, E. (1973) in The Enzymes (Bayer, P. D., ed.), pp. 49-96, Academic Press, New York. BHAWERMAN, G., AND CHARGAFF, E. (1954) Biochim. Biophys. Acta 15, 549-559. BHUNNGRABER, E. G., AND CHAHGAFF, E. J. (1967) J Biol. Chem. 242, 4834-4840. D&NC, Q., AND IVES, 0. H. (1977) Biochim. Biophys. Acta 277, 235-244. KIT, S., LEKJNG, W., THKULA, D., AND D~JBBS, D. R. (1975) Arch. Biochem. Biophys. 169, 66-76.
7. BOI,S, N. C., CORFF, S. C., AND YUYAMA, S. (1977) J. Cell Physiol. 90, 271-280. 8. SHOIJP, G. D., PRESCOTT, D. M., ANI) WYKES, J. R. (1966) J. Cell Biol. 31,295-300. A. M. (1977) 9. Bor.s, N. C., AND ZIMMEIZMAN, Comp. Biochem. Physiol. 57B, 31-35. 10. AHIMA, T., MASAKA, M., SHIOSAKA, T., OKIIIIA, H., AND FUJII, S. (1971) Biochim. Biophys. Acta
246, 184-193. N. F., ANI) 11. NEFF, R. J., RAY, S. A., BENTON, WII.ROHN, M. (1964) in Methods in Cell Physi-
ology (Prescott, D. M., ed.), Vol. I, pp. 55-83. Academic Press, New York. 12. IVES,
D. H., DUIIHAM,
J. P., ANII
TIJCKF,H,
J. S.
(1969) Anal. Biochem. 28, 192-205. 13. KIT, S., LEIJNG, W., AND TIIK~JI,A, D. (1973) Arch. Biochem. Biophys. 158, 503-513. 14. LOWRY, 0. H., ROSERHOUCH, N. J., FAHK, A. L., ANI) RANIIAM., R. J. (1951) J. Biol. Chem. 193, 265-275. 15. AFUMA, T., SIUOSAKA, T., OK~DA, H., AND FUJII, S. (1972) Biochim. Biophys. Acta 277, 15-24. 16 KIT, S., LEIJN~:, W., TRKIJI.A, D., AND DUBRS, R. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,703-715. 17 RANDEHATH, K., ANII RANDF,WATH, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 13, pp. 323-347, Academic Press, New York/London. 151 YoI?N(:, P., CORFF, S., ANII YIJYAMA, S. Cytobios, -1’ in press. 19. BANNWAI~TH. H., IKEHAHA, N., AND SCF~WEIGEH, H. G. (1977) Proc. Roy. Sot. London, Ser. B 198, 155-176.
Identification SHIJHEI *Department
of True Thymidine YIJYAMA,*
SONDRA
of Zoology, University of Toronto, Biology, Queen’s Uniuemty, Received
Kinase
October
in Tetrahymena
CORFF,*
AND
PAUL
Pyriformis G. YOTJNGT
Toronto, Ontario, Canada ML5 IAI, and tZ)epartrnent Kingston, Ontorio, Canada K71 3N6 31, 1977; revised
January
ST
of
2, 1978
Thymidine kinase is present in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis. Previous workers have been unable to find a specific thymidine kinase activity in this organism. The cytoplasm of Tetrahymena contained a thymidine phosphorylating activity which was ATP dependent, was stimulated by Mg’+, and was inhibited by dTTP. This activity was also partly inhibited by dCTP. Althbugh the mitochondrial fraction also by Mg”+ and not signifiexhibited ATP-dependent phosphorylation, it is not stimulated cantly inhibited by dTTP. Nucleoside phosphotraniferase activity is detectable both in cytoplasmic and mitochondrial fractions, although it is not clear whether they represent separate enzymes. Nucleoside phosphotransferase activity is inhibited both by NaF and by ATP. Thymidine kinase and nucleoside phosphot.ransferase activit.ies were separated hy establishing the presence of both enzymes in this polyacrylamide gel electrophoresis, organism. Both crude mitochondrial lysate and postmitochondrial supernatant samples exhibited similar gel electrophoretic patterns for thymidine kinase and nucleoside phosphotransferase activities. The former, however, exhibited a relatively small peak of thymidine kinase migrating at the same rate as that of the postmitochondrial supernatant. A separate peak of thymidine kinase was not found in the mitochondria of Tetrahymena.
Thymidine is phosphorylated to 2’-deoxythymidine 5’-monophosphate (dTMP) by either thymidine kinase (TK,’ EC 2.7.1.75) or nucleoside phosphotransferase (NPT, EC 2.7.1.77). Thymidine kinase utilizes nucleoside triphosphates (preferentially ATP) as phosphate donors, is very specific with regard to the substrate phosphorylated (thymidine or deoxyuridine only), requires Mg2+, and is feedback inhibited by dTTP (1, 2). Nucleoside phosphotransferase, on the other hand, utilizes a wide range of nucleoside mono- and diphosphates as phosphate donors and phosphorylates a wide range of nucleosides, is not dependent upon Mg”+, and is generally inhibited by NaF but not by dTTP (3-7). The mechanism of thymidine phosphorylation in Tetrahymena pyriformis has been controversial. An early report indi’ Abbreviations u.sed: TK, thymidine nucleoside phosphotransferase; DEAE, ethyl; ME, mercaptoethanob mt-lysate, lysate; cyto-lysate, cytoplasmic lysate; ethylimino.
kinase; NPT, diethylaminomitochondrial PEI, phospho64
0003.9861/78/1881-0064$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights ot reproduction m any form reserved.
cated that the thymidine kinase of this organism was unusual in that it was not feedback inhibited by dTTP (.%).The above conclusion was later disputed by other workers (9, 10). The ATP-dependent phosphorylation of thymidine by Tetrahymena extract was much less than the AMP-dependent phosphorylation of thymidine and the thymidine kinase-like activity could not be separated (by gel filtration and DEAESephadex chromatography) from ATPase and nucleoside phosphotransferase activities (10). Also, the activities of ATPand AMP-dependent phosphorylation increased at the same rate in synchronous cultures (9). It was therefore claimed that thymidine kinase was absent in Tetrahymena and that thymidine phosphorylation in the ATP-dependent reaction was catalyzed by the combined actions of ATPase and nucleoside phosphotransferase (9, 10). In this paper, we present evidence that demonstrates the presence of a true thymidine kinase (EC 2.7.1.75) in the cytoplasm (outside mitochondria) of Tetrahymena pyriformis ST.
THYMIDINE MATERIALS
AND
KINASE
METHODS
Organism and culture method. Tetrahymena pyriformis ST was grown in Neffs medium (11) to 1 to 3 X 10” cells/ml with vigorous shaking. In this medium, cells at this range of concentration are in early to midlogarithmic growth phase. Thymidine kinase and nucleoside phosphotransferase assays. The TK and NPT assays followed the method of Ives et al. (12) and have been reported previously (7). The complete TK assay contained, in a volume of 200 ~1, final concentrations as follows: 0.01 M ATP, 0.005 M MgCl,, 0.01 M mercaptoethanol (ME), 0.005 M NaF, 0.05 M Tris-HCl, pH 8.0, and 0.02 mM [methyl-,‘H]thymidine (0.25 &i/nmol, AmershamSearle). The complete NPT assay mixture was identical to that for TK except that 0.01 M AMP was substituted for ATI’, and NaF was omitted. The amount of cell extract was adjusted such that all of the data in this paper lie well within the linear range of the reaction. Data are expressed as nanomoles of dTMP formed per milligram of protein per minute. Preparation of cell extract. A crude cell lysate was prepared b,v washing cells once with distilled water and then lysing cells with 1% Triton X-100 in 0.01 M Tris. pH 8.0. A postmitochondrial supernatant and crude mitochondria were prepared as follows. Cells were homogenized in 0.35 M sucrose, 0.005 M MgCL, 0.01 M Tris, pH 7.0 using a hand emulsifier (Fisher). Whole cells, nuclei, and debris were removed by centrifugation at 600~ for 3 min. A postmitochondrial supernatant was then prepared by centrifugation at 77oOg for 10 min. Triton X-100 was added to 1%. The crude mitochondrial lysate was prepared by washing the above pellet once with the sucrose-Tris-MgL’ buffer using the same centrifugation procedure. The pellet was then resuspended in the sucrose-Tris-Mg” buffer and Triton X-100 was added to 1%. The mitochondrial suspension cleared in about 2 min, indicating complete lysis. All the above operations were performed at 0 to 4°C.J Polyacrylamide gel electrophoresrs. Polyacrylamide gel electrophoresis for TK using postmitochondrial supernatant or crude mitochondrial lysate was carried out following the method of Kit et al. (13). Following electrophoresis, the gels were sliced into 2mm sections and each section was incubated with 200 ~1 of the appropriate assay mixture. Incubation was for 2 h at 28°C with shaking. Other assays. Protein was determined by the method of Lowry et al. (14) using bovine serum albumin as standard. RESULTS
The experiments in this study were designed to identify thymidine kinase and to distinguish it from nucleoside phosphon transterase.
IN Tetrahymena
65
pyriformis
dTTP Sensitivity To examine whether or not a true TK is present in Tetrahymena, the sensitivity of the thymidine phosphorylating reaction to dTTP was examined. A crude mitochondrial lysate (mt-lysate) and a postmitochondrial supernatant (cyto-lysate) were prepared from logarithmically growing Tetrahymena cells and assayed for thymidine phosphorylating activity at different concentrations of dTTP under three different sets of conditions, i.e., ATP plus NaF, AMP alone, and AMP plus NaF. NaF is known to inhibit ATPase (15) as well as NPT. The results are shown in Figs. 1A and 1B. A
0
1
0 dTTP
c
“‘a....
._c 0 ‘0 ii
a..
2 mM
bL...~.
. ... . ..
...*. -....o. ~E” , o r ..% /-.-.. -L
/ T 0-
-......,,.
I 1 dTTP
I
.‘. . ... . ..
‘-....O ~
I
1 2
mM
. . . . 0 . . . . . . . .a . . . . . . . . . . . . . . . . . . . . . . . . . .
1
OO dCTP
2
mM
FIG. 1. Sensitivity of thymidine phosphorylating reaction to dTTP and dCTP. (A) dTTP sensitivity of ATP-dependent reaction (in presence of 5 mM NaF). V-V, cyto-lysate; U-U, mt-lysate. (B) dTTP sensitivity of AMP-dependent reaction in the absence or presence of NaF. Q-V, cyto-lysate (no NaF); Cl- --Cl, mt-lysate (no NaF); M, cyto-lysate (plus NaF); 0- - -0, mt-lysate (plus NaF). (C) dCTP sensitivity of ATP-dependent reaction. V-V, cytolysate; Cl- - 4, mt-Iysate. nm-nanomoles.
66
YUYAMA,
CORFF,
When ATP (plus NaF) was the phosphate donor, thymidine phosphorylation by the cyto-lysate was strongly inhibited by dTTP, whereas the reaction by the mt-lysate was not inhibited (Fig. 1A). In the absence of dTTP, the specific activity of the cyto-lysate was lo- to 12-fold greater than the mt-lysate (Fig. 1A). When AMP (without NaF) was the phosphate donor, phosphorylation by the cytolysate and by the mt-lysate were both slightly inhibited by dTTP (Fig. 1B) and the phosphorylation by the mt-lysate was slightly more sensitive to dTTP than that of the cyto-lysate (Fig. 1B). However, neither the degree nor the pattern of inhibition by dTTP was similar to that observed when using ATP as the phosphate donor. In the absence of dTTP, the specific activity of the cyto-lysate was about 60% of that of mt-lysate. When AMP (plus NaF) was the phosphate donor, the effect of dTTP on phosphorylation was similar to that observed when using AMP alone (no NaF) (Fig. 1B). Again, the phosphorylation reaction by the mt-lysate was slightly more sensitive to dTTP than that by the cytolysate (Fig. 1B). Since some TKs are also sensitive to dCTP (16), the effects of dCTP were examined using ATP (plus NaF) as the phosphate donor. With the cyto-lysate, 1 mM dCTP inhibited the phosphorylation by 30% and 2 mM dCTP, by 50%. Phosphorylation by the mt-lysate showed no dCTP sensitivity. The above data suggest that the cytoplasm of Tetrahymena contains typical TK and NPT. They also suggest that ATP-dependent phosphorylation of thymidine by mt-lysate is primarily not due to typical TK. NaF Sensitivity In the second series of experiments, the effects of NaF on the thymidine phosphorylating reaction were examined. The ATPdependent phosphorylation by the cyto-lysate showed little sensitivity to NaF (Fig. 2A), whereas it was markedly inhibited in the mt-lysate (at 5 mM, about 80% inhibition; at 10 mM, about 80%; Fig. 2A). NaF inhibited the AMP-dependent phosphoryl-
AND
YOUNG
lOI--
5p--.-.....y ......p......__. p._.._..._.__. 9 P, 2.5
‘j ii m
E \ D E
5.0
25
10
No F, mM
20 ....“*,....h ‘*., *.
t
1
I
I
I
2.5
5.0
7.5
10
NoF,
mM
FIG. 2. Sensitivity of thymidine phosphorylating reaction to NaF. (A) NaF sensitivity of ATP-dependent reaction. V-V, cyto-lysate; U - Xl, mt-lysate. (B) NaF sensitivity of AMP-dependent reaction. V- - -V, cyto-lysate; Cl- - -El, mt-lysate.
ation by either fraction (at 5 mM, 30-35% inhibition; at 10 mM, 55-60%; Fig. 2B). As mentioned earlier (in the dTTP sensitivity experiment), the specific activity of ATP-dependent phosphorylation by the cyto-lysate was greater than that by the mt-lysate, whereas the reverse was true for the specific activity of AMP-dependent phosphorylation. This observation was confirmed in the NaF sensitivity study. ibIg”+ Dependence The above observations showed that the characteristics of phosphorylation of thymidine by the cyto-lysate were those of either TK or NPT, depending upon the phosphate donor used, ATP or AMP, and that mt-lysate did not contain typical TK. In the third series of experiments, the Mg2+ dependence of the reaction was tested using the crude cell lysate. As seen in Fig. 3, Mg2+
THYMIDINE
3ot
IN Tetrahymena
rl
I
1
addition of AMP (Fig. 4B). This increased amount of phosphorylation was, at least in part, sensitive to dTTP. Although present data suggest a possible inhibitory effect of AMP on TK activity, it is not conclusive since there is no agent which inhibits NPT activity completely. Separation of TK and NPT by Polyacrylamide Gel Electrophoresis
; 5p-----T OO
67
pyriformis
0 _,,_.... ‘.‘.’ ..0,,,,,..,_....
,...,__... ... .; 25 ___,._..... .‘.,_......... 0 \ .cu ‘d ; 20 2 \
KINASE
/ 2.5 MgCI,,
I 5.0
I 7.5
1 10
mM
FIG. 3. Effects of Mg’+ on thymidine phosphorylating reaction using crude cell lysate. V-V, ATPdependent phosphorylation; Cl- - -Cl, AMP-dependent phosphorylation; O--O, ATP plus AMP-dependent phosphorylation.
concentrations of 2.5 to 5 mM stimulated the phosphorylation reaction 4- to 6-fold when ATP was the phosphate donor. When Mg”+ was absent and EDTA (10 mM) was added, there was a further 44% decrease in the phosphorylation of thymidine. No marked stimulation of phosphorylation by Mg2+ was observed when AMP (no NaF) was the phosphate donor (Fig. 3). Similarly, when both AMP and ATP were present as phosphate donors, Mg”+ did not stimulate the phosphorylation markedly (Fig. 3). Moreover, the presence of ATP greatly lowered the level of phosphorylation observed with AMP alone. ATP appeared, therefore, to inhibit the AMP-dependent phosphorylation. In order to verify this assumption, the effect of ATP on the NPT activity was examined in the presence of 1 mM dTTP, which completely inhibits the TK activity. As seen in Fig. 4A, phosphorylation of thymidine by AMP is strongly inhibited by ATP. In the reciprocal experiment, the effect of AMP on the ATP-dependent phosphorylation was tested. The amount of thymidine phosphorylation was increased by the
The results of our initial experiments provided evidence for the presence of TK in the cytoplasm of Tetrahymena. When the cyto-lysate was fractionated on 5% polyacrylamide gels, the TK was separated from the NPT (Fig. 5A). TK ran as a sharp peak and the activity was completely inhibited by 1 mM dTTP (Fig. 5A). NPT appeared to have two components, a sharp peak and a relatively broad peak (Fig. 5A). It is not clear whether the broad peak was an aggre-
ATP
0
(mM)
2
I AMP
3
4
(“‘M)
FIG. 4. Effects of ATP and AMP on the thymidine phosphorylating reaction using cell lysate. (A) Effects of ATP on AMP-dependent phosphorylation. Each reaction mixture contained 1 mM dTTP to inhibit TK activity. (B) Effects of AMP on ATP-dependent phosphorylation. X, the levels of phosphorylation in the presence of 1 mM dTTP.
68
YUYAMA,
CORFF,
AND
YOUNG
as the cytoplasmic enzyme and the broad peak migrated slightly slower than the corresponding peak. The product of the phosphorylation reaction was characterized to ensure that it was 5’-dTMP. Aliquots of reaction mixtures from the peak of TK activity in Fig. 5A and from the sharp peak of NPT in Fig. 5A were chromatographed against standards; PEI cellulose plates (BDH) were developed in one dimension using 0.25 M LiCl (17). In both cases, only thymidine (the substrate) and 5’-dTMP (the product) were radioactively labeled (Fig. 6). 20
IO
30
40
DISCUSSION
SI ice
Number
FIG. 5. Separation of TK and NPT activities of Tetratrymena extract by polyacrylamide gel electrophoresis. (A) TK and NPT activities of cyto-lysate. -, ATP-dependent phosphorylation; - - -, ATP-dependent phosphorylation in the presence of 1 mM dTTP; --, AMP-dependent phosphorylation. (B) TK and NPT activities of mt-lysate. F, ATP-dependent phosphorylation; - - -, ATP-dependent phosphorylation in the presence of 1 mM dTTP; --, AMP-dependent phosphorylation. Note the difference in the scales of TK and NPT. BPB = bromophenol blue marker.
gated form or a second enzyme. The high NPT activity seen on top of the gel (Fig. 5A) suggests the possibility of aggregation. The important point, however, is that TK and NPT are clearly separable on the gel, demonstrating the presence of both enzymes in the cyto-lysate of Tetrahymena pyriformis. When the mt-lysate was fractionated, the patterns of enzyme activities were quite similar to those of the cyto-lysate, although the NPT activity was much higher than that of TK, as expected (Fig. 5B). TK ran as a relatively small and sharp peak which migrated at the same rate as the cytoplasmic TK and was completely inhibited by dTTP (Fig. 5B). NPT had two components; the sharp peak migrated at the same rate
In this study, we have shown that thymidine kinase is present in Tetrahymena pyriformis ST. This is contrary to the previous reports that proposed the absence of TK in Tetrahymena (9, 10). Our conclusion is based on the fact that TK (thymidine phosphorylating enzyme which is ATP dependent and dTTP sensitive) is separated from NPT (AMP dependent) using electrophoresis. Since the ATP-dependent phosphorylation of thymidine, which is stimulated by Mg2+ and is inhibited by dTTP, is clearly demonstrable both with crude cell lysate and postmitochondrial supernatant of Tetrahymena, we are at a loss to explain earlier results (8, 9, 10). TK in Tetrahy-
‘:
‘.., r
dTMP
4r
w
z
3-
0 ; 0 ”
z-
l-
0
5
10
15
20
Fractions
FIG. 6. Identification of products of thymidine phosphorylating reaction using Tetrahymena extract. PEI plates were sliced transversely into 19 strips. The “H radioactivity of each strip was determined. p, ATP-dependent phosphorylation; - - -, AMP-dependent phosphorylation. Arrows with dTMP and dT indicate the center of the migrated position of the marker. 0 = origin.
THYMIDINE
KINASE
menu is a peak enzyme whose activity is highest during the S phase of the cell cycle (18), while NPT activity increases continuously during the cell cycle (9, 18). This observation also supports our conclusion that TK is present in Tetrahymena. The TK of Tetrahymena is similar to that found in the cytosol of other organisms (1, 16), except that it exhibits some degree of dCTP sensitivity which has previously only been reported for some mitochondrial TKs (16). However, TK of Tetrahymena is clearly not mitochondrial, because most of the TK activity is demonstrable in the postmitochondrial supernatant. Although the crude mitochondrial fraction exhibited ATP-dependent phosphorylation of thymidine, this does not appear to be due to a unique mitochondrial TK, since it is not demonstrably sensitive to dTTP, but is sensitive to NaF. Thus, the combined actions of ATPase and NPT (both are inhibited by NaF (7, 15)) are probably responsible for the ATP-dependent phosphorylation by the crude mitochondrial fraction. The electrophoresis study indicated, however, that a relatively small TK peak migrated at the same rate as that of the postmitochondrial supernatant, suggesting cytoplasmic contamination of the mitochondrial preparation. In Acetabularia, the TK-like activity which was found recently (19) is mainly bound to the particulate fraction (but outside mitochondria or chloroplasts), and the Triton X-100 does not solubilize the TKlike activity. This is probably not the case with the TK activity of Tetrahymena, since it appears to migrate freely in the 5% gel. These observations, of course do not exclude the presence of a unique TK in mitochondria of Tetrahymena. The characteristics of NPT in Tetrahymenu agree well with the other reports of this enzyme (3-7, 9, 10). It is partially inhibited by NaF and ATP, but dTTP and Mg”+ have little effect. Although the specific activity of NPT of the mt-lysate was 1.6 to 1.8 times
greater
than
that
of cyto-
lysate, it is not known whether they represent different enzymes.
IN Tetrahymena
69
pyriformis ACKNOWLEDGMENTS
This work was supported by grants from the National Research Council of Canada to S.Y. and to
P.G.v.
REFERENCES
2. 3. 4. 5. 6.
KIT, S. (1970) in Metabolic Pathways (Greenberg, D. M., ed.), pp. 69-275, Academic Press, New York. ANDEWSON, E. (1973) in The Enzymes (Bayer, P. D., ed.), pp. 49-96, Academic Press, New York. BHAWERMAN, G., AND CHARGAFF, E. (1954) Biochim. Biophys. Acta 15, 549-559. BHUNNGRABER, E. G., AND CHAHGAFF, E. J. (1967) J Biol. Chem. 242, 4834-4840. D&NC, Q., AND IVES, 0. H. (1977) Biochim. Biophys. Acta 277, 235-244. KIT, S., LEKJNG, W., THKULA, D., AND D~JBBS, D. R. (1975) Arch. Biochem. Biophys. 169, 66-76.
7. BOI,S, N. C., CORFF, S. C., AND YUYAMA, S. (1977) J. Cell Physiol. 90, 271-280. 8. SHOIJP, G. D., PRESCOTT, D. M., ANI) WYKES, J. R. (1966) J. Cell Biol. 31,295-300. A. M. (1977) 9. Bor.s, N. C., AND ZIMMEIZMAN, Comp. Biochem. Physiol. 57B, 31-35. 10. AHIMA, T., MASAKA, M., SHIOSAKA, T., OKIIIIA, H., AND FUJII, S. (1971) Biochim. Biophys. Acta
246, 184-193. N. F., ANI) 11. NEFF, R. J., RAY, S. A., BENTON, WII.ROHN, M. (1964) in Methods in Cell Physi-
ology (Prescott, D. M., ed.), Vol. I, pp. 55-83. Academic Press, New York. 12. IVES,
D. H., DUIIHAM,
J. P., ANII
TIJCKF,H,
J. S.
(1969) Anal. Biochem. 28, 192-205. 13. KIT, S., LEIJNG, W., AND TIIK~JI,A, D. (1973) Arch. Biochem. Biophys. 158, 503-513. 14. LOWRY, 0. H., ROSERHOUCH, N. J., FAHK, A. L., ANI) RANIIAM., R. J. (1951) J. Biol. Chem. 193, 265-275. 15. AFUMA, T., SIUOSAKA, T., OK~DA, H., AND FUJII, S. (1972) Biochim. Biophys. Acta 277, 15-24. 16 KIT, S., LEIJN~:, W., TRKIJI.A, D., AND DUBRS, R. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,703-715. 17 RANDEHATH, K., ANII RANDF,WATH, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 13, pp. 323-347, Academic Press, New York/London. 151 YoI?N(:, P., CORFF, S., ANII YIJYAMA, S. Cytobios, -1’ in press. 19. BANNWAI~TH. H., IKEHAHA, N., AND SCF~WEIGEH, H. G. (1977) Proc. Roy. Sot. London, Ser. B 198, 155-176.
