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Purification and properties of mouse thymus thymidylate synthase. comparison of the enzyme from mammalian normal and tumour tissues
In/. J. Bioch~n~. Vol. IX. Nu. 4. ,,p. 361 36X. 1986 Printed in Grrar Britain.All riphci reserved
Copyright
0020-7 I I X’X6 53.00 + 0.00 c’ 1986 Pergamon Press L.rd
PURIFICATTON AND PROPERTIES OF MOUSE THYMUS THYMIDYLATE SYNTHASE. COMPARISON OF THE ENZYME FROM MAMMALIAN NORMAL AND TUMOUR TTSSUES WOJCIE(.H RODE, JOANNA CIESLA, ZBIGNIEW ZIELINSKI and BARBARA KFDZIERSKA Department
of Cellular
Biochemistry,
Nencki
Institute
of Experimental
Biology,
3 Pasteur St,
02-093 Warsaw, Poland (Rwriwd
24 Septenzher 1984)
Abstract-I. Mouse thymus thymidylate synthase has been purified to apparent electrophor~ti~ homogeneity and compared wtth the enzyme from mouse tumour LIZ10 and Ehrlich ascites carcinoma cells. 2. The enzyme is a dimer composed of 35.000mol. wt monomers. 3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. 4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34’C, reflecting a change of rate-limiting step in dTMP formation. 5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
INTRODUCTION
Thymus glands were isolated from 3-5 weeks old albino Swiss mice (Mus musculus), washed with ice-cold saline, frozen and stored at -30‘C. Calf thymus glands were frozen just after removal from the slaughtered animals and stored at -30°C. Mouse leukemia L1210 cells were maintained, harvested and stored as earlier described (Rode et al., 1984).
Thymidylate synthase (methylenetctrahydrofolate: deoxyuridine-S’-monophosphate ~-methyltransferase: EC 2.1.1.45) catalyzes methylation of dUMP to dTMP with concomitant conversion of the (-),L -N5x”)-methylenetetrahydroto the dihydro- derivative of pterolymonoor oligoglutamate (Blakley, 1969; Kisliuk et af., 1974; Dolnick and Cheng, 1978). Properties of this enzyme isolated from different mammalian normal and tumour tissues have been studied (Horinishi and Greenberg, 1970; Fridland et al., 1971; Langenbach et al.. 1972; Gupta and Meldrum, 1972; Dolnick and Cheng, 1977,1978; Lockshin et al., 1979; Rode et al., 1979, 1980; Slavik and Slavikova, 1980; Priest et cd., 1981; Jastreboff ef al., 1982, 1983; Bapat et al., 1983; Rode and JastrebofF, 1984; Lu et al., 1984) but comparison between thymidylate synthase isolated from normal and tumour tissues of the same specific origin is still lacking. Such a comparison should be of interest, since the enzyme is a target for cancer chemotherapy (Danenberg, 1977; Sdnti, 1980). We present here properties of mouse thymus thymidylate synthase purified and studied by the same methods as applied earlier to the enzyme from several mouse tumours (Rode et al., 1979; Jastreboff rt al., 1982, 1983; Rode and Jastreboff, 1984). Some properties of thymidylate synthase isolated from Ehrlich ascites carcinoma, L12lO cells or calf thymus and studied in parallel are also described.
The previously described procedure was employed (Jastreboff et al., 1982; Rode et al., 1984). Unless otherwise indicated, the reaction mixture, in a total volume of 40 ~1, contained: 2 nmol [5--‘H]dI-JMP (- 6 x IO’ cpm/~fmol), 25 nmol ( F ),L-tetrahydrofolate, 0.1 pmol formaldehyde. 4 pmol 2-mercaptoethanol, 2 pmol Tris-HCI buffer pH_7.5, 0.4 pmol ascorbate buffer nH 7.5.0.025% Triton X-100 and enzyme (co.5 pmol). It was incubated at 37°C not longer than 30 min. All samples were done at least in duplicate. The enzyme activity is expressed in units defined as the amount required to release I pg equivalent of tritium from [5-‘HIdUMP (equivalent to formation of I pmol of dTMP) per min under conditions of the assay.
Mouse thymus glands (from -200 mice) were thawed with 3 vol of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KC1 and 0.01 M 2-mercaptoethanol, and homogenized in a ground glass homogenizer. The resulting mixture was centrifuged at 20,OOOg for 30 min at 4°C and the supernatant, fraction further referred to as the crude extract, was saved. Ammonium sulfate fractionation of the crude extract and affinity chromatography on the N”‘formyl-5,8-dideazafolate-ethyl-Sepharose column (Rode ef nl., 1979) were done as previously described (Jastreboff er a/., 1982), except that Buffer B was substituted with Buffer A. Calf thymus glands were thawed with I vol of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KCI and 0.01 M Z-mer~apt~thanoi, and homogenized in a Waring blender-type homogenizer. The homogenate was centri-
MATERIALS AND METHODS ( &),L-5, 6, 7, 8Tetrahydrofolate was prepared according to Lorenson er al. (1967). and other reagents were as previously described (Jastreboff et al., 1982). 361
fuged at 20,OOOg for 30 min at 4 C, the supernatant treated at 4 C. with a tenth of its volume of 25% (w:v) aqueous streptomycin sulfate and centrifuged at 20,000 g for I5 min at 4 C. The resulting supernatant underwent ammonium sulfate fractionation and affinity chromatography on N”1-formyl-5.8-dideazafolate-ethyl-Sepharose as previously described (Rode PI cl/., 1980). Specific activity of the final preparation (electrophoretically heterogeneous) was 0.21 units:‘mg protein. Electrophoretically homogeneous preparations of the enzyme from Ehrlich ascites carcinoma and Ll210 cells were obtained as presented earlier (Rode e/ r/l., 1979; Jastreboff <‘I crl.. 1982). Protein contents of thymidylate synthase preparations was determined by the procedure of Sedmak and Grossberg (1977) with bovine serum albumin as a standard.
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to Webcr and Osborn (1969) was employed to test homogeneity of thymidylate synthase preparations and to determine molecular weight of the denatured enzyme. Identification of thymidylate synthase on gel was accomplished through testing its ability to form a ternary complex with 5-fluoro-dUMP and WV‘I”methylenetetrahydrofolate (Fig. 1) as elsewhere described (Rode c/ crl., 1979). Bovine serum albumin (mol. at = f&000), ovalbumin (mol. wt =45,000), pepsin (mol. wst = 34,700), trypsinogen (mol. wt = 24.000), P-lactoglobulin (subunit mol. wt = 18.4000) and lysozyme (mol. wt = 14.300). components of “Dalton Mark VI” (Sigma Chemical Co.. St Louis. MO). were used as molecular weight standards. Samples for electrophoresis were prepared as previously described (Rode er ol.. 1979).
Molecular weight of native thymidylate synthase was determined by gel filtration of the enzyme complex with 5-fluoro[‘H]dUMP and N’.‘“-methylenetetrahydrofolate on a Sephadex G-100 column as described earlier (Jastreboff e/ N/., 1982). Cytochrome c (mol. wt = 12,400). ribonuclease (mol. wt = 13.700), ovalbumin and bovine serum albumin were used as molecular weight standards.
Parameters of the Hill equation were determined using a pocket computer program described by Pinto and Oestreicher (1984). Values of the overall velocity instead of the initial velocity and those of the arithmetical mean substrate concentration over the course of the assay instead of the initial substrate concentration were used (Lee and Wilson. 197 I). Kinetic parameters of thymidylate synthase inhibition by 5-fluoro-dUMP were determined as a result of analysis of the loss in enzyme activity over a given time interval with preincubation of the enzyme with N’,‘“-methylenetetrahydrofolate, a low concentration of dUMP (to prevent thermal inactivation of the enzyme), and different concentrations of the inhibitor (Brouillette et al.. 1979).
Sratrsricul methods Linear plots were fitted using the linear gram of the “Sharp” EL-5100 calculator.
regression
pro-
Statistically evaluated results arc presented as means I SEM, followed by the number of experiments (.NI in parentheses. KESC!LTS
Results of the purification of mouse lhymus thymidylate synthase are presented in Table I. The enzyme could be purified by the same affinity chromatography method, applied earlier to mouse tumour (Ll210 and Ehrlich ascites carcinoma) thymidylate synthase (Rode e’t al.. 1979: Jastreboff ef trl., 1982, 1983), based on the dUMP-dependent binding of this enzyme with the immobilized analogue of NL.“‘-methylenetetrahydrofolate. ,I;‘“formyl-5,8_dideazafolatc. The final preparation appeared to be homogeneous under the conditions of sodium dodecyl sulfate polyacrylamide gel clectrophoresis (Fig. 1). Molecular weight of the denatured mouse thymu\ enzyme was found to be 35,000 (Fig. 2; results of two determinations were 34.400 and 35,200). Parallel experiments showed molecular weight of denatured calf thymus thymidylate synthase to be 33,800 (rcsuits of two determinations were 32,500 and 35.000: not shown). Molecular weight of native thymidylate synthasc was determined to be 59,900 + 1000 (N = 4) for the mouse thymus enzyme (Fig. 2) and 61,400 f 6800 (N = 3) for the calf thymus enzyme (not shown). Based on specific activity of the purified preparation of mouse thymus thymidylate synthase and its molecular weight. molecular activity has been calculated (molecular activity = specific activity expressed in units/mg protein x mol. wt/lOOO) as 8.0 min ‘_ Fat the reasons given earlier (Jastreboff ct d.. 1983) doubled value of the subunit molecular weight was used in calculation as molecular weight of the native enzyme. The dependence of the reaction catalyzed by mouse thymus thymidylate synthase on temperature. L’Ypressed as an Arrhenius plot, was linear (Fig. 3. the correlation coefficient was always r 2 0.99). The activation energy was 10.52 & 0.78 kcal,imol (N = 3) Similar linear Arrhenius plot characterized also the calf thymus enzyme (r 2 0.99; not shown). The actlvation energy values, determined in two experiment5 were 9.57 and 12.44 kcal/mol (mean EA = I I .Ol kcal mol). In contrast, the Arrhenius plot for the I.1210 enzyme was biphasic (Fig. 3) with a transitional temperature at 33.7 C (34.6 and 32.8 C in two csperiments). Values for the activation energy belou the transitional temperature, determined in two experiments, were 13. I I and 8.85 kcal/mol (mean E,%,= 10.98 kcal/mol). Above the transitional temperature they decreased to 5.34 and 4.58 kcalimol. respectively (mean E,, = 4.9 I kcal.‘mol)
15
IO
5
0
00
02
04
06
0.8
10
Mobility
Fig. 1. Sodium dadecyl sulfate polyacrylamide gei electrophoresis of mouse thymus thymidylate synrhase preparation after second affinity chromatography (see Table if. Before electrophoresis, samples were treated with S-~uoro-[3H]dUMP and ~s~io-methylenetetrahydrofolate and prepared as previously described (Rode YI al., 1979). Upper gel was sliced and assayed for radioactivity.
363
=
Thymidylate synthase
365
E, 10 12 kcal/ mol \
37.0:
350:\ 31 9**
.
300’ \ 27 2’
l
250..
I
3 315
320
7 c Cylochmme
0.
c
Cytochrome
-\
3.25
= 5 34kcal
I
I
3 30
3 35
/mol
oh.
5 .
P \
E,z
l1. 42 Oio
6
Rlbonuclwse
20
I
3700.*348v,,=13
c ldlmer
llkcol/mol
Ovalbum!” TSlnatlvel
--t ‘\
.
4 /
BSA
32 O’\
3
29.5’*\
BSA
D
4 10
270**\
Idlmerl
24 6’. 21 3 15
I 3 20
I
Molecular
weight
Fig. 2. Molecular weight determinations of denatured (upper panel) and native (lower panel) mouse thymus thymidylate synthase by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration, respectively. For details see “Materials and Methods”. BSA, bovine serum albumin.
Dependence of velocity of the reaction catalyzed by mouse thymus, Ll210 and Ehrlich ascites carcinoma thymidylate synthase preparations on dUMP concentration, analyzed in terms of the Hill equation, indicated cooperative binding of the substrate by two sites present on the enzyme molecule, reflected by the slope (n,,,) of the Hill plot (Fig. 4; Table 2). The same results, describing the reaction of mouse thymus enzyme. plotted as the LineweaverrBurk plots yielded the apparent K, = 4.2 k 0.8 PM (N = 3). SFluoro-dUMP inhibited mouse thymus thymidylate synthase in a time-dependent manner (Fig. 5), pointing to tight binding of the inhibitor to the enzyme. Dependences of inactivation of the enzyme on time were always biphasic, suggesting different interaction of the inhibitor with two binding sites on the enzyme molecule. The inhibition constants, calculated using rates of inactivation determined during either early (0.0-1.5 min) or late (4-10 min) preincubation period, were remarkably different and indicated higher affinity of the inhibitor towards the first (K, = 2.6 nM) than towards the second (K, = 61 nM) binding site (Fig. 5; duplicate experiment yielded K, and k, values within 5% of the presented ones). An experiment analogous to that presented on Fig. 5 showed similar pattern of Ehrlich ascites carcinoma thymidylate synthase inhibition by
3 25
l/T(K-‘1
105
lo4
I
I
I
330
335
x~O-~
Fig. 3. Dependences of the reaction catalyzed by mouse thymus (upper panel) and L1210 (lower panel) thymidylate synthase on temperature (Arrhenius plot). S-fluoro-dUMP with K, values of 5.5 and 71 nM and k, values of 0. I5 and 0.17 min’ for early and late incubation periods, respectively (not shown). MgCl, (0.1&60 mM) did not influence activity of mouse thymus thymidylate synthase (not shown). In the presence of 30 or 50 mM MgC12 ATP at millimolar concentrations inhibited the enzyme down to
o-
O-
3-
;:: 02
05
10 [dUMPI
Fig. 4. Dependence thymus thymidylate
50
100
(PM)
of the reaction catalyzed by mouse synthase on dUMP concentration (Hill plot).
Table
2. Dependence
synthase
Mouse Ll210 Ehrlich
on dUMP
the
rcac~~on
caAyted
(parameters
hy
thymidqkw
of ihc Hill
plot)
thymus cells ascites
carcinoma
about bition
of
concentration
ceils
50% of its original activity (Fig. 6). No inhicould be observed in the absence of MgCI,.
E 2
I
w
20 1
DISCUSSION
I
1
/
I
5
10
50
loo
[ATPI
Fig. 6. Efkct of ATP on mouse thymus thymidylate synthase activity in the presence of 30 mM (0) or SOmM (0) MgCi,.
Mouse thymus thymidylate synthase was found to be a dimer composed of 35,000 mol. wt subunits. Similar structure was found to be common for the enzyme from neonatal mouse liver (Priest et rr/.. 1981), Ehrlich ascites carcinoma parental and 5-~uoro-dUrd-resistant cells (~ngenb~~ch (‘I nl., 1972; Jastreboff ef al.. 1982, 1983) L1210 cells (Rode et al., 1979) as well as from several human and rat tumours (Lockshin rf al., 1979: Rode ct rtf.. 1980: Slavik and Slavikova, 1980). We confirmed here an existence of such a structure also in case of the enzyme from calf thymus, since conflicting reports concerning this material were published (Slavik and Molecular Slavikovi. 1980; Dwivedi et al.. 1983). activity (at 37%) of the enzyme from mouse thymus (8 min-‘) was distinctly lower than that of mouse tumour L1210 (29 min -I), Ehrlich ascites parental (31-33 min’) or 5-fluoro-dUrd-resistant (at least
91 min. ‘) cell thymidylate synthase (Fridiand et uf., 1971; Rode et al., 1979; Jastroboff et al.. 1982. 1983). Dcpendences of the reaction catalyzed by thyisolated from synthase preparations midylate different human (Dolnick and Cheng, 1977; Rode L’I ui., 1980) and mouse (Jastrebofl’ e? al., 1982, 1983) tumours on temperature, expressed as the Arrhenius plots, were biphasic with activation energies sharply decreasing at 34-35 C. A similar temperaturedependent change of activation energy, indicating a different reaction step to become rate-limiting, was exhibited by the reaction catalyzed by the L12lO enzyme (Fig. 3). On the other hand, activation energies of the reaction catalyzed by the enzyme from
60
20
,L-----0
2
4
Prelncubotlon
6
e
nme
(m In)
10
‘0
01 1 /IFdUMP
02 (PM-’
i
Fig. 5. Inhibition of mouse thymus thymidylate synthase by 5-Ruoro-dUMP. k/f’: Semi-log plot ot percentage of remaining enzyme activity vs time of preincubation at 37 C in the presence of 3.2iiM dUMP, I mM ( ~),L-~~5,‘*-methyI~netetrahydrofoIat~ and varying con~ntratio~s of inhibitor (4.1, 0; 8.11 *; 16.3, lJ; and 24.4nM, n ). After preincubation 25 PM [5-‘HIdUMP was added and tritium released during 4 min determined. Right: Double reciprocal plot of apparent rate of inactivation of enzyme vs concentration of inhibitor, reflecting the dependence (Brouillette CI al., 1979):
where k, is the inactivation rate constant, Closed and open symbols describe apparent inactivation rates during O-I.5 and 4-10 min preincubation, respectively. Inhibition parameters determined: K, = 2.6 nM. k2=0.24min-’ (0) and K,=61 nM, kz=0.44min’ (0).
Thymidylate
mouse or calf thymus were invariably constant throughout the temperature range studied (Fig. 3; Results; Horinishi and Greenberg, 1970). Thus, thymidylate synthase from normal tissue may be structurally different than that of tumour origin. The difference could be of physiological importance, since comparison of activation energies determined at physiological temperatures for the reaction catalyzed by thymidylate synthases from normal and tumour sources reveals higher values for the former than for the latter (Horinishi and Greenberg, 1970; Dolnick and Cheng, 1977; Rode ef al., 1980; Jastreboff et al., 1982, 1983; Results). Of particular interest is such a comparison of mouse thymidylate synthases from thymus. Ll210, Ehrlich ascites carcinoma parental and 5-fluoro-dUrd-resistant cells, where there is a correlation (correlation coefficient r = - 0.78) between molecular activity at 37°C (8, 29, 31 and 91 min ‘, respectively) and activation energy (10.5, 5.0. 2.6 and 1.4 kcal/mol, respectively) (Rode et al., 1979; Jastreboff et al., 1983; Results). This correlation suggests that thymidylate synthase in tumour cells may be modified in comparison with the enzyme from normal cells of the same specific origin. Such modifications seem to enable more efficient lowering of activation energy of the reaction and increase the enzyme molecular activity. Mouse thymidylate synthases from thymus and both tumour lines appear to bind dUMP cooperatively at two binding sites present on enzyme molecule, as indicated by slopes of the Hill plots between (1 < %pp < 2: Table 2). An interaction dUMP-binding sites may affect kinetics of thymidylate synthase inhibition by dUMP analogues. In keeping with this notion are results describing inhibition of the enzyme from mouse thymus and Ehrlich ascites carcinoma cells by 5-fluoro-dUMP, an analogue active in cancer chemotherapy (Danenberg, 1977). The biphasic dependence of the enzyme inactivation rate on time of the inhibitor action (Fig. 5) suggests cooperative binding. K, increase, dependent on time of the inhibitor binding, indicates negative cooperativity, with interaction factor (ratio of K, describing binding at the second to that describing binding at the first site) of 24 and I3 for mouse thymus and Ehrlich ascites carcinoma thymidylate synthases, respectively. Cooperative binding of two other dUMP analogues, 5-ethyl-dUMP and 5-propyl-dUMP, by L12lO and Ehrlich ascites carcinoma thymidylate synthases has been also described (Rode et al., 1984). Mouse thymus thymidylate synthase was inhibited in the presence of MgClz by ATP at millimolar concentrations (Fig. 6). Since neither MgClz nor ATP alone influenced activity of the enzyme, then MgATP-complex had to be either
the inhibitor. MgATP influence in various
as well as Mg’*
were
found
to
ways activity of thymidylate synthase preparations from different mouse tumours (Rode and Jastreboff, 1984). Various patterns of sensitivity towards Mg2+ and MgATP found for mouse thymidylate synthases from normal and different tumour tissues present one more evidence that they are different enzyme forms. However, explanation of a possible physiological meaning of the effect of MgATP on thymidylate synthase activity demands further studies.
367
synthase REFERENCES
Bapat A. R., Zarow C. and Danenberg P. V. (1983) Human leukemic cells resistant to 5-fluoro-2’-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides. J. biol. Chem. 258, 413&4136. Blakley R. L. (1969) The Biochemistry qf Folk Acid and Related Pteridines, pp. 23 l-266. Wiley, New York. Brouillette C. B., Chang C. T.-C. and Mertes M. P. (1979) 5’-phosphate: an 5-(a-Bromoacetyl)-2’-deoxyuridine affinity label for thymidylate synthetase. J. med. Chem. 22, 1541-1544. Danenberg P. V. (1977) Thymidylate synthetase-a target enzyme in cancer chemotherapy. Biochim. hiophys. Actu 413, 73-92. Dolnick, B. .I. and Cheng Y.-C. (1977) Human thymidylate synthetase derived from blast cells of patients with acute myelocytic leukemia. J. biol. Chem. 252, 7697-7703. Dolnick B. J. and Cheng Y.-C. (1978) Human thymidylate synthetase. II. Derivatives of pteroylmonoand polyglutamates as substrates and inhibitors. J. biol. Chem. 253, 3563-3567. Dwivedi C. M.. Kisliuk R. L. and Maley G. F. (1983) Structural studies of calf thymus thymidylate synthase. In Chemistry and Biology of Pteridines (Edited by Blair J. A.), pp. 639444. Waiter de Gruyter, Berlin. Fridland A.. Langenbach R. J. and Heidelberger C. (1971) Purification ofyhymidylate synthetase from Ehrlich as: cites carcinoma cells. J. biol. Chem. 246, 71 l&71 14. Gupta V. S. and Meldrum J. B. (1972) Purification and properties of thymidylate synthetase from pig thymus. Can. J. Biochem. 50, 352-362. Horinishi H. and Greenberg D. M. (1970) Purification and properties of thymidylate synthetase from calf thymus. Biochim. biophys. Acta 258, 741-752. Jastreboff M., Kedzierska B. and Rode W. (1982) Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2+ and MgATP2-. Biochem. Pharmacol. 31, 217-223. Jastreboff M. M., Kedzierska B. and Rode W. (1983) Altered thymidylate synthetase in 5-fluorodeoxyuridineresistant Ehrlich ascites carcinoma cells. Biochem. Pharmacol. 32, 2259-2267. Kisliuk R. L., Gaumont J. and Baugh C. M. (1974) Polyglutamyl derivatives of folate as substrates and inhibitors of thymidylate synthetase. J. biol. Chem. 249, 410&4103. Langenbach R. J., Danenberg P. V., Heidelberger C. (1972) Thymidylate synthetase: mechanism of inhibition by 5-fluoro-2’-deoxyuridylate. Biochem. Biophys. Res. Commun. 48, 1565-1571. Lee H.-J. and Wilson J. B. (1971) Enzymic parameters: measurement of V and K,,,. Biochim. biophys. Acta 242, 519-522. Lockshin A., Moran R. G. and Danenberg P. V. (1979) Thymidylate synthetase purified to homogeneity from human leukemic cells. Proc. natn. Acad. Sci. U.S.A. 76, 750-754. Lorenson M. Y., Maley G. F. and Maley F. (1967) The purification and properties of thymidylate synthetase from chick embryo extracts. J. biol. Chem. 242, 3332-3344. Lu Y.-Z., Aiello P. D. and Matthews R. G. (1984) Studies on the polyglutamate specificity of thymidylate synthetase from fetal pig liver. Biochemistry 23, 687M876. Pinto G. F. and Oestreicher E. G. (1984) Pocket computer program for fitting the Hill equation. Comput. Biol. Med. 14, 507-511. Priest D. G., Doig M. T. and Hynes J. B. (1981) Purification of mouse liver thymidylate synthetase by affinity chromatography using lo-methyl-5,8-dideazafolate as the affinant. Experientia 37, 119-120. Rode W., Dolnick B. J. and Bertino J. R. (1980) Isolation
368
W~JCIECH RODE er ul.
of a homogeneous preparation of human thymidyiate synthetase from HeLa cells. Biochem. Pharmacol. 29, 723-726. Rode W. and Jastreboff M. M. (1984) EtTects of Mg’+ and adenine nucleotides on thymidylate synthetase from different mouse tumors. Molec. Cell. Biochem. 60, 13 76. Rode W.. Kulikowski T.. Kedzierska B.. Jastreboff M. and Shugar D. (1984) Inhibition of mammalian tumour thymidylate synthetase by S-alkylated 2’-deoxyuridine 5’-phosphates. Biochem. Pharmacol. 33, 2699-2705. Rode W.. Scanlon K. J.. Hynes J. and Bertino J. R. (1979) Purification of mammalian tumor (Ll210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents. J. hiol. Chem. 254, I 1538 I 1543.
Santi D. V. (1980) Perspectives on the design and biochemical pharmacology of inhibitors of thymidylate synthetase. J. med. Chem. 23, 103-I I I. Sedmak J. J. and Grossberg S. E. (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Analyt. Biochem. 79, 544552. Slavik K. and Slavikova V. (1980) Purification of thymidylate synthetase from enzyme-poor sources by affinity chromatography. In Methods in En:_vmolog,v (Edited by McCormick D. B. and Wright L. D.), Vol. 66. Part E. pp. 709-723. Academic Press, New York. Weber K. and Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 440664412.
Copyright
0020-7 I I X’X6 53.00 + 0.00 c’ 1986 Pergamon Press L.rd
PURIFICATTON AND PROPERTIES OF MOUSE THYMUS THYMIDYLATE SYNTHASE. COMPARISON OF THE ENZYME FROM MAMMALIAN NORMAL AND TUMOUR TTSSUES WOJCIE(.H RODE, JOANNA CIESLA, ZBIGNIEW ZIELINSKI and BARBARA KFDZIERSKA Department
of Cellular
Biochemistry,
Nencki
Institute
of Experimental
Biology,
3 Pasteur St,
02-093 Warsaw, Poland (Rwriwd
24 Septenzher 1984)
Abstract-I. Mouse thymus thymidylate synthase has been purified to apparent electrophor~ti~ homogeneity and compared wtth the enzyme from mouse tumour LIZ10 and Ehrlich ascites carcinoma cells. 2. The enzyme is a dimer composed of 35.000mol. wt monomers. 3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. 4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34’C, reflecting a change of rate-limiting step in dTMP formation. 5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
INTRODUCTION
Thymus glands were isolated from 3-5 weeks old albino Swiss mice (Mus musculus), washed with ice-cold saline, frozen and stored at -30‘C. Calf thymus glands were frozen just after removal from the slaughtered animals and stored at -30°C. Mouse leukemia L1210 cells were maintained, harvested and stored as earlier described (Rode et al., 1984).
Thymidylate synthase (methylenetctrahydrofolate: deoxyuridine-S’-monophosphate ~-methyltransferase: EC 2.1.1.45) catalyzes methylation of dUMP to dTMP with concomitant conversion of the (-),L -N5x”)-methylenetetrahydroto the dihydro- derivative of pterolymonoor oligoglutamate (Blakley, 1969; Kisliuk et af., 1974; Dolnick and Cheng, 1978). Properties of this enzyme isolated from different mammalian normal and tumour tissues have been studied (Horinishi and Greenberg, 1970; Fridland et al., 1971; Langenbach et al.. 1972; Gupta and Meldrum, 1972; Dolnick and Cheng, 1977,1978; Lockshin et al., 1979; Rode et al., 1979, 1980; Slavik and Slavikova, 1980; Priest et cd., 1981; Jastreboff ef al., 1982, 1983; Bapat et al., 1983; Rode and JastrebofF, 1984; Lu et al., 1984) but comparison between thymidylate synthase isolated from normal and tumour tissues of the same specific origin is still lacking. Such a comparison should be of interest, since the enzyme is a target for cancer chemotherapy (Danenberg, 1977; Sdnti, 1980). We present here properties of mouse thymus thymidylate synthase purified and studied by the same methods as applied earlier to the enzyme from several mouse tumours (Rode et al., 1979; Jastreboff rt al., 1982, 1983; Rode and Jastreboff, 1984). Some properties of thymidylate synthase isolated from Ehrlich ascites carcinoma, L12lO cells or calf thymus and studied in parallel are also described.
The previously described procedure was employed (Jastreboff et al., 1982; Rode et al., 1984). Unless otherwise indicated, the reaction mixture, in a total volume of 40 ~1, contained: 2 nmol [5--‘H]dI-JMP (- 6 x IO’ cpm/~fmol), 25 nmol ( F ),L-tetrahydrofolate, 0.1 pmol formaldehyde. 4 pmol 2-mercaptoethanol, 2 pmol Tris-HCI buffer pH_7.5, 0.4 pmol ascorbate buffer nH 7.5.0.025% Triton X-100 and enzyme (co.5 pmol). It was incubated at 37°C not longer than 30 min. All samples were done at least in duplicate. The enzyme activity is expressed in units defined as the amount required to release I pg equivalent of tritium from [5-‘HIdUMP (equivalent to formation of I pmol of dTMP) per min under conditions of the assay.
Mouse thymus glands (from -200 mice) were thawed with 3 vol of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KC1 and 0.01 M 2-mercaptoethanol, and homogenized in a ground glass homogenizer. The resulting mixture was centrifuged at 20,OOOg for 30 min at 4°C and the supernatant, fraction further referred to as the crude extract, was saved. Ammonium sulfate fractionation of the crude extract and affinity chromatography on the N”‘formyl-5,8-dideazafolate-ethyl-Sepharose column (Rode ef nl., 1979) were done as previously described (Jastreboff er a/., 1982), except that Buffer B was substituted with Buffer A. Calf thymus glands were thawed with I vol of ice-cold 0.05 M phosphate buffer, pH 7.5, containing 0.1 M KCI and 0.01 M Z-mer~apt~thanoi, and homogenized in a Waring blender-type homogenizer. The homogenate was centri-
MATERIALS AND METHODS ( &),L-5, 6, 7, 8Tetrahydrofolate was prepared according to Lorenson er al. (1967). and other reagents were as previously described (Jastreboff et al., 1982). 361
fuged at 20,OOOg for 30 min at 4 C, the supernatant treated at 4 C. with a tenth of its volume of 25% (w:v) aqueous streptomycin sulfate and centrifuged at 20,000 g for I5 min at 4 C. The resulting supernatant underwent ammonium sulfate fractionation and affinity chromatography on N”1-formyl-5.8-dideazafolate-ethyl-Sepharose as previously described (Rode PI cl/., 1980). Specific activity of the final preparation (electrophoretically heterogeneous) was 0.21 units:‘mg protein. Electrophoretically homogeneous preparations of the enzyme from Ehrlich ascites carcinoma and Ll210 cells were obtained as presented earlier (Rode e/ r/l., 1979; Jastreboff <‘I crl.. 1982). Protein contents of thymidylate synthase preparations was determined by the procedure of Sedmak and Grossberg (1977) with bovine serum albumin as a standard.
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate according to Webcr and Osborn (1969) was employed to test homogeneity of thymidylate synthase preparations and to determine molecular weight of the denatured enzyme. Identification of thymidylate synthase on gel was accomplished through testing its ability to form a ternary complex with 5-fluoro-dUMP and WV‘I”methylenetetrahydrofolate (Fig. 1) as elsewhere described (Rode c/ crl., 1979). Bovine serum albumin (mol. at = f&000), ovalbumin (mol. wt =45,000), pepsin (mol. wst = 34,700), trypsinogen (mol. wt = 24.000), P-lactoglobulin (subunit mol. wt = 18.4000) and lysozyme (mol. wt = 14.300). components of “Dalton Mark VI” (Sigma Chemical Co.. St Louis. MO). were used as molecular weight standards. Samples for electrophoresis were prepared as previously described (Rode er ol.. 1979).
Molecular weight of native thymidylate synthase was determined by gel filtration of the enzyme complex with 5-fluoro[‘H]dUMP and N’.‘“-methylenetetrahydrofolate on a Sephadex G-100 column as described earlier (Jastreboff e/ N/., 1982). Cytochrome c (mol. wt = 12,400). ribonuclease (mol. wt = 13.700), ovalbumin and bovine serum albumin were used as molecular weight standards.
Parameters of the Hill equation were determined using a pocket computer program described by Pinto and Oestreicher (1984). Values of the overall velocity instead of the initial velocity and those of the arithmetical mean substrate concentration over the course of the assay instead of the initial substrate concentration were used (Lee and Wilson. 197 I). Kinetic parameters of thymidylate synthase inhibition by 5-fluoro-dUMP were determined as a result of analysis of the loss in enzyme activity over a given time interval with preincubation of the enzyme with N’,‘“-methylenetetrahydrofolate, a low concentration of dUMP (to prevent thermal inactivation of the enzyme), and different concentrations of the inhibitor (Brouillette et al.. 1979).
Sratrsricul methods Linear plots were fitted using the linear gram of the “Sharp” EL-5100 calculator.
regression
pro-
Statistically evaluated results arc presented as means I SEM, followed by the number of experiments (.NI in parentheses. KESC!LTS
Results of the purification of mouse lhymus thymidylate synthase are presented in Table I. The enzyme could be purified by the same affinity chromatography method, applied earlier to mouse tumour (Ll210 and Ehrlich ascites carcinoma) thymidylate synthase (Rode e’t al.. 1979: Jastreboff ef trl., 1982, 1983), based on the dUMP-dependent binding of this enzyme with the immobilized analogue of NL.“‘-methylenetetrahydrofolate. ,I;‘“formyl-5,8_dideazafolatc. The final preparation appeared to be homogeneous under the conditions of sodium dodecyl sulfate polyacrylamide gel clectrophoresis (Fig. 1). Molecular weight of the denatured mouse thymu\ enzyme was found to be 35,000 (Fig. 2; results of two determinations were 34.400 and 35,200). Parallel experiments showed molecular weight of denatured calf thymus thymidylate synthase to be 33,800 (rcsuits of two determinations were 32,500 and 35.000: not shown). Molecular weight of native thymidylate synthasc was determined to be 59,900 + 1000 (N = 4) for the mouse thymus enzyme (Fig. 2) and 61,400 f 6800 (N = 3) for the calf thymus enzyme (not shown). Based on specific activity of the purified preparation of mouse thymus thymidylate synthase and its molecular weight. molecular activity has been calculated (molecular activity = specific activity expressed in units/mg protein x mol. wt/lOOO) as 8.0 min ‘_ Fat the reasons given earlier (Jastreboff ct d.. 1983) doubled value of the subunit molecular weight was used in calculation as molecular weight of the native enzyme. The dependence of the reaction catalyzed by mouse thymus thymidylate synthase on temperature. L’Ypressed as an Arrhenius plot, was linear (Fig. 3. the correlation coefficient was always r 2 0.99). The activation energy was 10.52 & 0.78 kcal,imol (N = 3) Similar linear Arrhenius plot characterized also the calf thymus enzyme (r 2 0.99; not shown). The actlvation energy values, determined in two experiment5 were 9.57 and 12.44 kcal/mol (mean EA = I I .Ol kcal mol). In contrast, the Arrhenius plot for the I.1210 enzyme was biphasic (Fig. 3) with a transitional temperature at 33.7 C (34.6 and 32.8 C in two csperiments). Values for the activation energy belou the transitional temperature, determined in two experiments, were 13. I I and 8.85 kcal/mol (mean E,%,= 10.98 kcal/mol). Above the transitional temperature they decreased to 5.34 and 4.58 kcalimol. respectively (mean E,, = 4.9 I kcal.‘mol)
15
IO
5
0
00
02
04
06
0.8
10
Mobility
Fig. 1. Sodium dadecyl sulfate polyacrylamide gei electrophoresis of mouse thymus thymidylate synrhase preparation after second affinity chromatography (see Table if. Before electrophoresis, samples were treated with S-~uoro-[3H]dUMP and ~s~io-methylenetetrahydrofolate and prepared as previously described (Rode YI al., 1979). Upper gel was sliced and assayed for radioactivity.
363
=
Thymidylate synthase
365
E, 10 12 kcal/ mol \
37.0:
350:\ 31 9**
.
300’ \ 27 2’
l
250..
I
3 315
320
7 c Cylochmme
0.
c
Cytochrome
-\
3.25
= 5 34kcal
I
I
3 30
3 35
/mol
oh.
5 .
P \
E,z
l1. 42 Oio
6
Rlbonuclwse
20
I
3700.*348v,,=13
c ldlmer
llkcol/mol
Ovalbum!” TSlnatlvel
--t ‘\
.
4 /
BSA
32 O’\
3
29.5’*\
BSA
D
4 10
270**\
Idlmerl
24 6’. 21 3 15
I 3 20
I
Molecular
weight
Fig. 2. Molecular weight determinations of denatured (upper panel) and native (lower panel) mouse thymus thymidylate synthase by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration, respectively. For details see “Materials and Methods”. BSA, bovine serum albumin.
Dependence of velocity of the reaction catalyzed by mouse thymus, Ll210 and Ehrlich ascites carcinoma thymidylate synthase preparations on dUMP concentration, analyzed in terms of the Hill equation, indicated cooperative binding of the substrate by two sites present on the enzyme molecule, reflected by the slope (n,,,) of the Hill plot (Fig. 4; Table 2). The same results, describing the reaction of mouse thymus enzyme. plotted as the LineweaverrBurk plots yielded the apparent K, = 4.2 k 0.8 PM (N = 3). SFluoro-dUMP inhibited mouse thymus thymidylate synthase in a time-dependent manner (Fig. 5), pointing to tight binding of the inhibitor to the enzyme. Dependences of inactivation of the enzyme on time were always biphasic, suggesting different interaction of the inhibitor with two binding sites on the enzyme molecule. The inhibition constants, calculated using rates of inactivation determined during either early (0.0-1.5 min) or late (4-10 min) preincubation period, were remarkably different and indicated higher affinity of the inhibitor towards the first (K, = 2.6 nM) than towards the second (K, = 61 nM) binding site (Fig. 5; duplicate experiment yielded K, and k, values within 5% of the presented ones). An experiment analogous to that presented on Fig. 5 showed similar pattern of Ehrlich ascites carcinoma thymidylate synthase inhibition by
3 25
l/T(K-‘1
105
lo4
I
I
I
330
335
x~O-~
Fig. 3. Dependences of the reaction catalyzed by mouse thymus (upper panel) and L1210 (lower panel) thymidylate synthase on temperature (Arrhenius plot). S-fluoro-dUMP with K, values of 5.5 and 71 nM and k, values of 0. I5 and 0.17 min’ for early and late incubation periods, respectively (not shown). MgCl, (0.1&60 mM) did not influence activity of mouse thymus thymidylate synthase (not shown). In the presence of 30 or 50 mM MgC12 ATP at millimolar concentrations inhibited the enzyme down to
o-
O-
3-
;:: 02
05
10 [dUMPI
Fig. 4. Dependence thymus thymidylate
50
100
(PM)
of the reaction catalyzed by mouse synthase on dUMP concentration (Hill plot).
Table
2. Dependence
synthase
Mouse Ll210 Ehrlich
on dUMP
the
rcac~~on
caAyted
(parameters
hy
thymidqkw
of ihc Hill
plot)
thymus cells ascites
carcinoma
about bition
of
concentration
ceils
50% of its original activity (Fig. 6). No inhicould be observed in the absence of MgCI,.
E 2
I
w
20 1
DISCUSSION
I
1
/
I
5
10
50
loo
[ATPI
Fig. 6. Efkct of ATP on mouse thymus thymidylate synthase activity in the presence of 30 mM (0) or SOmM (0) MgCi,.
Mouse thymus thymidylate synthase was found to be a dimer composed of 35,000 mol. wt subunits. Similar structure was found to be common for the enzyme from neonatal mouse liver (Priest et rr/.. 1981), Ehrlich ascites carcinoma parental and 5-~uoro-dUrd-resistant cells (~ngenb~~ch (‘I nl., 1972; Jastreboff ef al.. 1982, 1983) L1210 cells (Rode et al., 1979) as well as from several human and rat tumours (Lockshin rf al., 1979: Rode ct rtf.. 1980: Slavik and Slavikova, 1980). We confirmed here an existence of such a structure also in case of the enzyme from calf thymus, since conflicting reports concerning this material were published (Slavik and Molecular Slavikovi. 1980; Dwivedi et al.. 1983). activity (at 37%) of the enzyme from mouse thymus (8 min-‘) was distinctly lower than that of mouse tumour L1210 (29 min -I), Ehrlich ascites parental (31-33 min’) or 5-fluoro-dUrd-resistant (at least
91 min. ‘) cell thymidylate synthase (Fridiand et uf., 1971; Rode et al., 1979; Jastroboff et al.. 1982. 1983). Dcpendences of the reaction catalyzed by thyisolated from synthase preparations midylate different human (Dolnick and Cheng, 1977; Rode L’I ui., 1980) and mouse (Jastrebofl’ e? al., 1982, 1983) tumours on temperature, expressed as the Arrhenius plots, were biphasic with activation energies sharply decreasing at 34-35 C. A similar temperaturedependent change of activation energy, indicating a different reaction step to become rate-limiting, was exhibited by the reaction catalyzed by the L12lO enzyme (Fig. 3). On the other hand, activation energies of the reaction catalyzed by the enzyme from
60
20
,L-----0
2
4
Prelncubotlon
6
e
nme
(m In)
10
‘0
01 1 /IFdUMP
02 (PM-’
i
Fig. 5. Inhibition of mouse thymus thymidylate synthase by 5-Ruoro-dUMP. k/f’: Semi-log plot ot percentage of remaining enzyme activity vs time of preincubation at 37 C in the presence of 3.2iiM dUMP, I mM ( ~),L-~~5,‘*-methyI~netetrahydrofoIat~ and varying con~ntratio~s of inhibitor (4.1, 0; 8.11 *; 16.3, lJ; and 24.4nM, n ). After preincubation 25 PM [5-‘HIdUMP was added and tritium released during 4 min determined. Right: Double reciprocal plot of apparent rate of inactivation of enzyme vs concentration of inhibitor, reflecting the dependence (Brouillette CI al., 1979):
where k, is the inactivation rate constant, Closed and open symbols describe apparent inactivation rates during O-I.5 and 4-10 min preincubation, respectively. Inhibition parameters determined: K, = 2.6 nM. k2=0.24min-’ (0) and K,=61 nM, kz=0.44min’ (0).
Thymidylate
mouse or calf thymus were invariably constant throughout the temperature range studied (Fig. 3; Results; Horinishi and Greenberg, 1970). Thus, thymidylate synthase from normal tissue may be structurally different than that of tumour origin. The difference could be of physiological importance, since comparison of activation energies determined at physiological temperatures for the reaction catalyzed by thymidylate synthases from normal and tumour sources reveals higher values for the former than for the latter (Horinishi and Greenberg, 1970; Dolnick and Cheng, 1977; Rode ef al., 1980; Jastreboff et al., 1982, 1983; Results). Of particular interest is such a comparison of mouse thymidylate synthases from thymus. Ll210, Ehrlich ascites carcinoma parental and 5-fluoro-dUrd-resistant cells, where there is a correlation (correlation coefficient r = - 0.78) between molecular activity at 37°C (8, 29, 31 and 91 min ‘, respectively) and activation energy (10.5, 5.0. 2.6 and 1.4 kcal/mol, respectively) (Rode et al., 1979; Jastreboff et al., 1983; Results). This correlation suggests that thymidylate synthase in tumour cells may be modified in comparison with the enzyme from normal cells of the same specific origin. Such modifications seem to enable more efficient lowering of activation energy of the reaction and increase the enzyme molecular activity. Mouse thymidylate synthases from thymus and both tumour lines appear to bind dUMP cooperatively at two binding sites present on enzyme molecule, as indicated by slopes of the Hill plots between (1 < %pp < 2: Table 2). An interaction dUMP-binding sites may affect kinetics of thymidylate synthase inhibition by dUMP analogues. In keeping with this notion are results describing inhibition of the enzyme from mouse thymus and Ehrlich ascites carcinoma cells by 5-fluoro-dUMP, an analogue active in cancer chemotherapy (Danenberg, 1977). The biphasic dependence of the enzyme inactivation rate on time of the inhibitor action (Fig. 5) suggests cooperative binding. K, increase, dependent on time of the inhibitor binding, indicates negative cooperativity, with interaction factor (ratio of K, describing binding at the second to that describing binding at the first site) of 24 and I3 for mouse thymus and Ehrlich ascites carcinoma thymidylate synthases, respectively. Cooperative binding of two other dUMP analogues, 5-ethyl-dUMP and 5-propyl-dUMP, by L12lO and Ehrlich ascites carcinoma thymidylate synthases has been also described (Rode et al., 1984). Mouse thymus thymidylate synthase was inhibited in the presence of MgClz by ATP at millimolar concentrations (Fig. 6). Since neither MgClz nor ATP alone influenced activity of the enzyme, then MgATP-complex had to be either
the inhibitor. MgATP influence in various
as well as Mg’*
were
found
to
ways activity of thymidylate synthase preparations from different mouse tumours (Rode and Jastreboff, 1984). Various patterns of sensitivity towards Mg2+ and MgATP found for mouse thymidylate synthases from normal and different tumour tissues present one more evidence that they are different enzyme forms. However, explanation of a possible physiological meaning of the effect of MgATP on thymidylate synthase activity demands further studies.
367
synthase REFERENCES
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368
W~JCIECH RODE er ul.
of a homogeneous preparation of human thymidyiate synthetase from HeLa cells. Biochem. Pharmacol. 29, 723-726. Rode W. and Jastreboff M. M. (1984) EtTects of Mg’+ and adenine nucleotides on thymidylate synthetase from different mouse tumors. Molec. Cell. Biochem. 60, 13 76. Rode W.. Kulikowski T.. Kedzierska B.. Jastreboff M. and Shugar D. (1984) Inhibition of mammalian tumour thymidylate synthetase by S-alkylated 2’-deoxyuridine 5’-phosphates. Biochem. Pharmacol. 33, 2699-2705. Rode W.. Scanlon K. J.. Hynes J. and Bertino J. R. (1979) Purification of mammalian tumor (Ll210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents. J. hiol. Chem. 254, I 1538 I 1543.
Santi D. V. (1980) Perspectives on the design and biochemical pharmacology of inhibitors of thymidylate synthetase. J. med. Chem. 23, 103-I I I. Sedmak J. J. and Grossberg S. E. (1977) A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Analyt. Biochem. 79, 544552. Slavik K. and Slavikova V. (1980) Purification of thymidylate synthetase from enzyme-poor sources by affinity chromatography. In Methods in En:_vmolog,v (Edited by McCormick D. B. and Wright L. D.), Vol. 66. Part E. pp. 709-723. Academic Press, New York. Weber K. and Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 440664412.