Abbreviations dNTP, deoxyribonucleoside triphosphate; dThd, thymidine; hTK1, human cytosolic thymidine kinase 1; NaP, sodium orthophosphate; NaPP, sodium dipolyphosphate; NaPPP, sodium t
Trang 1catalytic efficient form is induced by pyrophosphate,
in addition to tripolyphosphates, or high enzyme
concentration
Birgitte Munch-Petersen
Department of Science, Systems and Models, Roskilde University, Denmark
For decades, it has been the general belief that the
building blocks of DNA, the deoxyribonucleoside
triphosphates (dNTPs), play a central role in
maintai-ning correct DNA synthesis Recent investigations of
DNA synthetic processes in yeast and human cells
have indicated that initiation and progress of DNA
replication are closely associated with the cellular
dNTP concentration [1–3]
The level of the dNTPs is strictly controlled and
fluctuates during the cell cycle, in close correlation
with the rate of DNA synthesis, with low dNTP levels
in G1 cells increasing during S phase, generally with
dTTP being the most abundant and dGTP the least
[4–6] In quiescent cells, dNTP levels are several-fold
lower [7], and in non-proliferating human lymphocytes,
which are G0 cells, the dTTP pool is many times smaller than the other dNTP pools [4]
In most cells and organisms except for a few para-sites, the dNTPs are provided by two main routes, the
de novoand the salvage pathways The central enzyme
in the de novo route, ribonucleotide reductase, cata-lyzes reduction of ribonucleotides to the corresponding 2¢-deoxyribonucleotides, after which they are phos-phorylated to the triphosphate level by nucleoside diphosphate kinase The specificity of ribonucleotide reductase is controlled by the concentration of the end-products dATP, dTTP and dGTP, where dTTP is the key regulator switching the specificity from reduc-tion of pyrimidine ribonucleotides to reducreduc-tion of purine ribonucleotides [8] Therefore, the cellular dTTP
Keywords
ATP; gel filtration; kinetics; tetramerization;
thymidine kinase
Correspondence
B Munch-Petersen, Department of Science,
Systems and Models, Universitetsvej 1,
Building 18.1, Roskilde University, DK-4000
Roskilde, Denmark
Fax: +45 4674 3011
Tel: +45 4674 2419
E-mail: bmp@ruc.dk
Website: http://www.ruc.dk/nsm/
(Received 5 August 2008, revised 5
November 2008, accepted 17 November
2008)
doi:10.1111/j.1742-4658.2008.06804.x
Thymidine kinase (TK1) is a key enzyme in the salvage pathway of deoxy-ribonucleotide metabolism, catalyzing the first step in the synthesis of dTTP by transfer of a c-phosphate group from a nucleoside triphosphate
to the 5¢-hydroxyl group of thymidine, forming dTMP Human TK1 is cytosolic and its activity is absent in resting cells, appears in late G1, increases in S phase coinciding with the increase in DNA synthesis, and disappears during mitosis The fluctuation of TK1 through the cell cycle is important in providing a balanced supply of dTTP for DNA replication, and is partly due to regulation of TK1 expression at the transcriptional level However, TK1 is a regulatory enzyme that can interchange between its dimeric and tetrameric forms, which have low and high catalytic effi-ciencies, respectively, depending on pre-assay incubation with ATP Here, the part of ATP that is necessary for tetramerization and how the reaction velocity is influenced by the enzyme concentration are determined The results show that only two or three of the phosphate groups of ATP are necessary for tetramerization, and that kinetics and tetramerization are closely related Furthermore, the enzyme concentration was found to have
a pivotal effect on catalytic efficiency
Abbreviations
dNTP, deoxyribonucleoside triphosphate; dThd, thymidine; hTK1, human cytosolic thymidine kinase 1; NaP, sodium orthophosphate; NaPP, sodium dipolyphosphate; NaPPP, sodium tripolyphosphate; rhTK1, recombinant human TK1; TmTK, TK from Thermotoga maritima.
Trang 2level is critical for maintaining a proper balance
between the dNTPs In addition to the ribonucleotide
reductase-controlled pathway, the dTTP level is
con-trolled by thymidine kinases and TMP nucleotidases,
forming a substrate cycle [8,9]
A crucial step in dTTP synthesis is phosphorylation
of thymidine (dThd) to dTMP Two thymidine kinases
catalyze this step, the cytosolic TK1 and the
mitochon-drial TK2 (EC 2.7.1.21 for both TK1 and TK2),
encoded by two nuclear genes TK1 is cell-cycle-specific
and is not expressed in quiescent cells, in which only the
constitutively expressed TK2 is present The complex
transcriptional and translational regulation of TK1
ensures that the increase in TK1 activity coincides with
an increase in the DNA synthesis rate and dNTP pools
[10] TK1 is degraded to undetectable levels during
mitosis by means of the anaphase-promoting complex
APC⁄ C-Cdh1, which recognizes a KEN box in the
C-terminus [11] Human TK1 (hTK1) is a regulatory
enzyme that can occur in two forms, a dimer with low
activity and a tetramer with high activity The
conver-sion between the two forms is reversible and depends on
enzyme concentration and the presence of ATP [12]
When hTK1 purified from human lymphocytes was
incubated with ATP prior to assay, the kinetics is
hyper-bolic, with a Kmof approximately 0.5 lm and a Vmaxof
10 lmolÆmin)1Æmg)1 Without pre-assay incubation with
ATP, the Vmaxis the same but the kinetics is
non-hyper-bolic, with an apparent Kmof 15–17 lm and a Hill
coef-ficient less than one, indicating negative co-operativity
This behavior means that the catalytic efficiency
(kcat⁄ Km) is approximately 30-fold higher for hTK1 that
had been incubated with ATP This ‘ATP effect’ on the
kinetics apparently depends on the enzyme
concentra-tion in a linear manner, and no transiconcentra-tion to the
catalyti-cally highly active form was observed at concentrations
of hTK1 below 10 ngÆmL)1 (0.4 nm) [12] Therefore,
transition does not occur at the low assay concentration
of TK1 (< 3 ngÆmL)1) This also explains why both
enzyme forms showed linear progress curves for product
versus time
It is very likely that the ‘ATP effect’ is a fine tuning
of the hTK1 activity during the cell cycle When hTK1
is degraded in G2⁄ M phase, and given that ATP is
fairly constant during the cell cycle, the initial low
hTK1 concentration in the following G1 phase implies
predominance of the low-activity dimer form As the
hTK1 concentration increases during S phase, more
and more enzyme will be in the high-active tetramer
form Recently, phosphorylation of hTK1 at serine 13
has been proposed to be involved in this regulation,
preventing ATP-induced transformation to the
high-active tetramer [13]
The structure of human TK1 was solved in 2004 [14], and it is closely related to several bacterial TK1 structures but is fundamentally different from the structures of the non-TK1 like kinases deoxycytidine kinase [15], deoxyguanosine kinase and Drosoph-ila melanogaster multi-substrate kinase [16] This indi-cates a different evolutionary origin of the two classes of deoxyribonucleoside kinases However, the exact binding of ATP is not clear, as the enzyme is a tetramer with dTTP in the active site for all TK1 structures except the structure for TK1 from Thermo-toga maritima (TmTK) which has the inhibitor TP4A bound to the tetrameric enzyme [17] The structure
of hTK1 with TP4A has also been solved, but here
no electron density was seen with adenosine In TmTK, the adenosine moiety was bound at the a-helix dimer interface, and this form is more open than hTK1 Therefore, at present, it appears that the adenosine group is very loosely bound to hTK1
In the present work, the part of the phosphate donor that is necessary for the dimer–tetramer transi-tion of native hTK1 purified from human lymphocytes was identified Further, the effect of the concentration
of the recombinant enzyme on its oligomerization behaviour was investigated The results show that the dipolyphosphate group is sufficient for inducing transi-tion to the high-active tetramer, and that kinetics and oligomerization are closely related In addition, the results show a clear relationship between the enzyme concentration and the catalytically high-active tetra-meric form, and that the tetramer dissociates into dimers very slowly
Results and Discussion
Identification of the group inducing tetramerization of human TK1 Human TK1 has 234 amino acids and a subunit size
of 25.5 kDa [18] Several reports have shown by gel filtration that native as well as recombinant hTK1 elutes as a dimer in the absence of ATP (1–5 mm) and
as a tetramer in its presence [12,13,19,20] The recently solved structures of a number of TK1-like enzymes from human, bacteria and vaccinia virus all show tet-rameric forms [14,17,21–23] As the adenosine moiety does not show electron density in any of the human TK1 structures, it may be that the adenosine moiety is
of no significance for inducing the reversible dimer– tetramer transition Therefore, the present study aimed
to identify the part of the nucleotide molecule that triggers tetramerization Figure 1A–C shows the elution profiles of native TK1 from human
Trang 3lympho-cytes in the presence of the ribonucleoside
triphos-phates GTP, CTP and UTP For comparison, elution
profiles with and without ATP are shown in
Fig 1D,E With all NTPs, hTK1 elutes as tetramers
with apparent molecular masses of approximately 100–
120 kDa (see Table 1) Therefore, it can be concluded
that the nature of the base is insignificant for the
tetra-merization effect
The next goal was to determine the role played by
the sugar and phosphate groups As seen in Fig 1F,
ADP was able to induce the tetramer, whereas, in the
presence of AMP, the majority of the enzyme eluted as
a dimer with a size of approximately 53 kDa A minor shoulder is seen at approximately 115 kDa (Fig 1G) (Table 1) This suggested that the phosphate part of the nucleotide is more important for tetramerization than the sugar and base Indeed, as seen in Fig 1H, hTK1 elutes as a tetramer in the presence of sodium tripolyphosphate (NaPPP)
In all these elutions, 2 mm MgCl2 was present in the elution buffers To determine the effect of sodium dipolyphosphate (NaPP), the gel filtration has to be performed in absence of MgCl2, as the combination
of MgCl2 and Chaps causes a heavy precipitate
Fig 1 Effect of nucleotides and
polyphos-phates on oligomerization of native hTK1.
Approximately 10 ng native TK1 purified
from human lymphocytes in a total volume
of 200 lL was injected into a Superdex 200
column (10 · 300 mm) together with 0.1
mg Blue Dextran used as an internal
standard for determination of the void
volume, V 0 , in the individual experiments.
Prior to injection, hTK1 was diluted to 6
lgÆmL)1and incubated with 3 m M of the
indicated nucleotides or polyphosphates at
4 C for 2 h, and stored for at least 2 weeks
at )80 C Fractions (200 lL) were collected
into 100 lL column buffer containing 30%
glycerol and 2 m M ATP The fractions were
assayed for thymidine kinase activity under
standard assay conditions with 100 l M
dThd The molecular markers (vertical bars)
are (from left to right): b-amylase (200 kDa),
BSA (66 kDa), ovalbumin (45 kDa), carbonic
anhydrase (30 kDa) and cytochrome
c (12.4 kDa) Veis the elution volume The
standard variation for Ve⁄ V 0 of the marker
proteins was below 2% (CV) for more than
20 independent experiments.
Trang 4However, that the presence of MgCl2 is insignificant
can be seen in Fig 2A, where hTK1 elutes as a
tetra-mer whether NaPPP is present with or without
MgCl2 (Fig 1H) Figure 2B,C shows that TK1 also
elutes as a tetramer in the presence of NaPP,
whereas the elution profile with sodium
orthophos-phate (NaP) indicates a dimer of approximately
48.5 kDa with a shoulder at approximately 100 kDa
In all of these elutions, the same amount of hTK1
was applied (10 ng) and recovery of activities was
approximately 20–40% The lower activity seen in
the elution with AMP (Fig 1G) is due to the
inhibi-tory effect of AMP in the assay The average mass
of the eight tetrameric hTK1s was estimated as
103.7 ± 3.2 (SEM) kDa (Table 1)
Are the oligomerization pattern and kinetics
related?
The kinetics of hTK1 is complex and deviates from
hyperbolic kinetics, with apparent negative
co-oper-ativity and a K0.5(substrate concentration at
half-max-imal velocity) of approximately 15 lm [12] However,
when hTK1 was incubated with ATP prior to the
assay, it showed hyperbolic kinetics with a Km of
approximately 0.5 lm Both enzyme forms have the
same Vmax, meaning that the catalytic efficiency of
ATP-incubated hTK1 is approximately 30-fold higher
than that of non-incubated hTK1 The two TK1 forms
can therefore be referred to as the high- and
low-efficiency forms To explain the apparent negative
co-operativity, a model has been proposed whereby the
dimer has high Kmand the tetramer has low Km, and
the ratio between the two forms depends on the dThd
concentration [24] According to this model, the simul-taneous presence in the assay of the two forms will result in the apparent negative co-operative behavior
To further elucidate this, the relationship between the oligomerization status and the kinetic behaviour was investigated, i.e whether the tetrameric and dimeric forms in Figs 1 and 2 exhibited low or high catalytic efficiency Therefore, the various incubated hTK1 forms from Figs 1 and 2 were analyzed for their kinetic behavior with dThd, and the results are pre-sented in Figs 3 and 4 Only in cases where TK1 was incubated prior to the assay with the compounds pro-ducing the dimer, i.e AMP (Fig 3F) and NaP (Fig 4D), did the enzyme exhibit low catalytic effi-ciency like non-incubated TK1 (Fig 3D), i.e with apparent negative co-operativity as indicated by con-cave Hofstee plots of v versus v⁄ s (insets to the kinetic plots), Hill coefficients < 1 and high K0.5 values (Table 1) All of the tetrameric forms showed approxi-mately hyperbolic Michaelis–Menten kinetics, with low
Kmvalues between 0.51 and 0.95 lm [mean tetrameric
Km value is 0.73 lm ± 0.05 (SEM); Table 1] These results clearly show that the high-efficiency hyperbolic kinetics with low Kmis associated with the tetrameric form and that the low-efficiency negative
co-operativi-ty kinetics with high apparent Km is associated with the dimeric form of TK1
Phosphate donor specificity The results from Figs 1–4 showed that inorganic di- and tripolyphosphates were able to induce tetra-merization and hyperbolic kinetics with low Km values similar to the nucleoside di- and tri-phosphates, and
Table 1 Native molecular size and kinetic parameters.
Incubation conditions
Phosphate donor capacityb(%)
a Values are means ± SEM, with the number of independent experiments in parentheses b Phosphate donor capacity as a percentage of the activity with ATP measured under standard assay conditions with 2.5 m M of the respective donor replacing ATP. cMeasured with 2.5 m M MgCl2in the assay.
Trang 5therefore the potential capacity of these compounds
for phosphate transfer was compared to those of the
other nucleotides The results are presented in Table 1
and show that the inorganic polyphosphates are not
able to act as phosphate donor This also shows that
phosphate donor capacity and the tetramerization
effect are two independent events
Impact of enzyme concentration on the oligomerization of hTK1
The above-described experiments were all performed with the native enzyme purified from human lympho-cytes to a final concentration of approximately
5 lgÆmL)1 [25], and the concentration of the applied enzyme in the gel filtration experiments in Figs 1 and
2 was 50 ngÆmL)1 (10 ng applied) Using recombinant techniques, concentrations of pure hTK1 more than 1000–10 000-fold higher can be obtained, enabling considerably higher concentrations during gel filtra-tion This may explain the appearance of both dimer and tetramer peaks during gel filtration of non-incu-bated recombinant human TK1 (rhTK1), although the tetramer peak is the smallest [19,20] In these studies, TK1 was applied at a concentration of approximately 3 lgÆmL)1 Recently, it was reported that human TK1 elutes exclusively as a tetramer when applied at a concentration range of 0.4–
20 mgÆmL)1 [26] The authors suggest that the high-level expression of TK1 obtained in their work may influence the oligomerization pattern of the enzyme However, the more than 100-fold higher concentra-tion used in the experiments by Birringer et al [26] compared to those used by Berenstein et al [19] and Frederiksen et al [20] may also explain the different elution profiles
To further clarify this issue and the effect of enzyme concentration on the oligomerization status, the elution profile of rhTK1 was analyzed under the conditions and at the concentrations outlined in Fig 5 In Fig 5A, rhTK1 was applied at a concen-tration of 0.2 mgÆmL)1 As seen from the elution profile, rhTK1 elutes exclusively as a tetramer at this enzyme concentration, similar to the elution pattern reported by Birringer et al [26] This shows that, at high concentrations, TK1 is a tetramer independent
of the presence of ATP or phosphate groups In Fig 5B, rhTK1 was diluted to 6 lgÆmL)1 immediately before gel filtration Here, the enzyme eluted as both
a dimer and a tetramer, with approximately 40% of the enzyme activity in the tetrameric form In Fig 5C, the enzyme was treated as in previous stud-ies [19,20], i.e diluted to 6 lgÆmL)1, allowed to stand
at 4C for 2 h, and then stored at )80 C for at least 2 weeks before gel filtration This treatment did not affect the enzyme activity, as the same Vmax was obtained before and after the treatment As seen from Fig 5C, only a minor part of the enzyme is in the tetramer form This elution profile is very similar
to those previously reported by Berenstein et al and Frederiksen et al [19,20] In their gel-filtration
Fig 2 Effect of orthophosphate and di- and tri-polyphosphates on
oligomerization of native hTK1 hTK1 was diluted and incubated
with 3 m M of the indicated nucleotides or phosphate compound
without MgCl 2, injected onto the Superdex 200 column, eluted with
column buffer without MgCl2 containing 2 m M of the respective
nucleotide or phosphate compound, and assayed as described for
Fig 1.
Trang 6experiments on recombinant human TK1, Li et al.
[13] diluted and treated the enzyme as in Fig 5C and
also found a similar elution profile Together, these
observations show that rhTK1 behaves as a tetramer even in the absence of phosphate groups when applied at concentrations of 200 lgÆmL)1 or higher,
Fig 3 Effect of nucleotides on hTK1 dThd substrate kinetics Native human TK1 (hTK1) was incubated with 3 m M of the indicated nucleotide for 2 h at 4 C, and stored for at least 2 weeks at )80 C The initial velocity with the indicated dThd concentrations was determined as described in Experimental procedures Open symbols; incubation with nucleotide Closed symbols; incubation without nucleotide Inset, Hofstee plots of the data.
Fig.4 Effect of NaP, NaPP and NaPPP on hTK1 dThd substrate kinetics Native human TK1 (hTK1) was incubated pre-assay with
3 m M of the indicated compound with or without MgCl 2 , and the initial velocity with the indicated dThd concentrations was determined as described in Experimental procedures Inset, Hofstee plots of the data.
Trang 7and a substantial amount of enzyme is still observed
as a tetramer, even when diluted to 6 lgÆmL)1, when
gel filtration commences immediately after dilution
Further, the time- and storage-dependent differences
in behaviour after dilution to 6 lgÆmL)1 indicate that dissociation of the tetramer to the dimer is a slowly progressing process This is supported by the kinetic behaviour of the recombinant enzyme as shown in Fig 5D, where the enzyme was diluted from 0.5 mgÆmL)1 immediately before the kinase assay Under these conditions, the kinetic behaviour was essentially like that of the tetramer form, exhibiting hyperbolic kinetics with a Km of 0.7 lm This also indicates slow dissociation of the tetramer form, and may explain why linear progress curves are always obtained with all forms of the enzyme and under all incubation conditions When rhTLK1 is diluted from high storage concentrations to low assay concentra-tions of 2–3 ngÆmL)1, which is below the limit for the ATP tetramerization effect, the enzyme would be expected to dissociate to the dimer form with higher
Km during the assay, and this would result in non-linear progress curves However, slow dissociation from tetramer to dimer will result in linear progress curves, as consistently observed with this enzyme Such a slow dissociation may indicate that hTK1 is a hysteretic enzyme
The finding that the two linked phosphate groups
in pyrophosphate are sufficient for formation of the tetramer clearly shows that neither the base nor the sugar plays a role in the oligomerization process This appears to agree with the structural conditions for ATP binding to human TK1 In the first crystal structure of TK1-type enzymes of human and myco-plasmic origin [14], the feedback inhibitor dTTP was bound in the substrate pocket, similar to the binding
of dTTP to the D melanogaster multi-substrate deoxyribonucleoside kinase [16], despite the funda-mental differences between the two structures The three phosphate groups bind backwards, and the thy-mine group is buried in a cleft between the a⁄ b domain and the so-called lasso domain, a domain that is unique to TK1-type enzymes The same pattern is seen with other TK1 types of bacterial
Fig 5 Effect of concentration of recombinant human TK1 on oligo-merization and kinetics The column and dilution buffer used and the assay performed are described in Fig 1 (A) 40 lg was applied
at a concentration of 0.6 mgÆmL)1 (B, C) 1 lg was applied at a concentration of 6 lgÆmL)1 In (B), the enzyme was diluted immedi-ately before application, whereas in (C), the enzyme was diluted, incubated for 2 h at 4 C, and stored at )80 C for more than
2 weeks (D) dThd substrate kinetics with recombinant human TK1 (0.1 ng in 50 lL assay reaction volume) diluted from 0.6 mgÆmL)1
to 0.01 lgÆmL)1 immediately before assay Inset, Hofstee plot of the data.
Trang 8origin [21,22] In a recent study, the bi-substrate
inhi-bitor P1-(5¢-adenosyl)P4-[5¢-(2¢-deoxy-thymidyl)]
tetra-phosphate (AP4dT) was crystallized together with
hTK1 and TmTK [17,23] In both structures,
thy-mine and the three phosphates were bound in the
lasso motif, essentially as for dTTP in the previous
structures The authors conclude that the fourth
phosphate, which is analogous to the a-phosphate in
ATP, is observed in both structures, whereas electron
density is obtained only with the adenine group in
the TmTK structure [23] Moreover, with the
ana-logue bound, the TmTK structure appears more
open than the hTK1 structure This indicates that
the adenine group in the hTK1 structure makes only
a few, if any, contacts with the enzyme It may also
explain at least partly why the kinetic and oligomeric
effects can be exhibited by only two phosphate
groups, which probably are analogous to the a and
b phosphate groups in the nucleotide ADP On the
other hand, the large difference in phosphate donor
capacity, only 4% with ADP and no activity with
NaPPP and NaPP, indicates that the base part of
the phosphate donor must play an essential role in
the catalytic process
The physiological TK1 concentration is estimated
to increase from approximately 0.03–0.09 lgÆmL)1
(1.2–3.6 nm) in G0 and G1 cells to approximately
4–6 lgÆmL)1 (160–240 nm) in peak S-phase cells [12],
assuming equal distribution throughout the cytoplasm
This indicates that, in G1⁄ early S phase, TK1 will be
in the dimer form, irrespective of the cellular ATP
con-centration, due to the low enzyme concentration As
the TK1 concentration increases during S phase, more
and more of the enzyme will be in the tetramer form
as previously proposed [12] Further, as shown here,
high-efficiency kinetics with low Km values is
exclu-sively displayed by the tetramer forms, and
low-efficiency kinetics with high Km values is displayed by
the dimer forms These observations strengthen the
previous hypothesis that the dimer⁄ tetramer
inter-change of TK1 with low⁄ high catalytic efficiency is a
fine-tuning mechanism that may serve to provide a
bal-anced supply of dTTP throughout the cell cycle,
adjusted to the need for DNA synthesis [12,13,24] As
dTTP is a key regulator of ribonucleotide reductase,
higher dTTP concentrations will result in unbalanced
dNTP pools, which are known to be mutagenic [27–
29] In the light of these effects, the complex regulatory
and structural properties of hTK1 may be important
for maintaining a balanced supply of the DNA
precur-sor This underlines the importance of elucidating the
molecular and structural background of the enzymatic
and catalytic properties of human thymidine kinase
Experimental procedures
Superdex 12, Glutathione–Sepharose, pGEX-2T vector, thrombin, [methyl-3H]dThd (25 CiÆmmol)1) and the Esc-herichia coli strains XL Gold and BL21 were purchased from Amersham Biosciences (now part of GE Healthcare Bio-Sciences, Hillerod, Denmark) Strains XL Gold and BL21 were used to propagate and express, respectively, the recombinant thymidine kinase Chaps was purchased from Roche A/S (Copenhagen, Denmark) Triton X-100, dithiotreitol, non-radioactive nucleosides and molecular mass markers were purchased from Sigma-Aldrich (Copen-hagen, Denmark) Materials for cloning, PCR, DNA seq-uencing and assays were standard commercially available products
Enzyme preparation
Native human TK1 (hTK1) was purified from human lymphocytes as previously described [25] Briefly, superna-tant from streptomycin-precipitated crude cellular homoge-nate was precipitated with ammonium sulfate, desalted on Sephadex G-25, separated from other deoxynucleoside kinases by ion-exchange chromatography on a DEAE column, and further purified by affinity chromatography
on a 3¢-dTMP Sepharose column dThd from the affinity chromatography step was removed, and hTK1 was con-centrated on a carboxymethyl-Sepharose column as described previously [12]
Recombinant human TK1 (rhTK1) was expressed using the pGEX-2T-LyTK1val106 vector [19], the bacteria were harvested after induction with 0.1 mm isopropyl-1-thio-b-d-galactopyranoside for 6 h at 25C, rhTK1 was purified by glutathione–Sepharose chromatography, and the thrombin cleavage fractions were further purified
by carboxymethyl chromatography as previously des-cribed [19]
Pre-assay incubation and storage of enzymes
Both native and recombinant hTK1 were diluted to a con-centration of 6 lgÆmL)1in Superdex column buffer (50 mm imidazole⁄ HCl pH 7.5, 5 mm MgCl2, 0.1 m KCl, 2 mm Chaps and 5 mm dithiothreitol), incubated with or without
3 mm of the respective nucleotide or phosphate compound for 2 h at 4C, and stored for at least 2 weeks at )80 C before use for kinetic and molecular mass analyses The activity at saturating conditions was similar before and after dilution, incubation and storage
Native molecular size
The apparent molecular size was determined by gel filtra-tion on a Superdex 12 (10· 300 mm) column connected to
a Gradifrac automatic sampler (Amersham Biosciences) as
Trang 9described previously [19] The column was pre-equilibrated
in column buffer (50 mm imidazole⁄ HCl pH 7.5, 5 mm
MgCl2, 0.1 m KCl, 2 mm Chaps and 5 mm dithiothreitol)
containing two milimolar of the respective nucleotide or
phosphate compounds In each experiment, 0.2 mL enzyme
dilution containing 0.1 mg Blue Dextran 2000
(Sigma-Aldrich) was applied Blue dextran was used as an internal
standard for determination of the void volume V0 of the
column This value was used for calculation of Ve⁄ V0 The
column was standardized using the following marker
pro-teins: b-amylase, 200 kDa; BSA, 66 kDa; ovalbumin,
45 kDa; carbonic anhydrase, 30 kDa; cytochrome c,
12.4 kDa This approach ensures high reproducibility in
determination of the molecular mass, as the standard
varia-tion in Ve⁄ V0for the markers was less than 2% (coefficient
of variation) from 20 separate marker elution profiles
Fractions (200 lL) were collected into 100 lL column
buf-fer containing 30% glycerol and 2 mm ATP for
preserva-tion of enzyme activity The fracpreserva-tions were assayed for
thymidine kinase activity under standard assay conditions
with 100 lm dThd
Thymidine kinase assay
Thymidine kinase activity was assayed by measuring
ini-tial velocities using the DE-81 filter paper method as
described previously [12,19] Standard assay conditions
were 50 mm Tris⁄ HCl pH 7.5, 2.5 mm ATP, 2.5 mm
MgCl2, 10 mm dithiothreitol, 0.5 mm Chaps, 3 mgÆmL)1
BSA, 3 mm NaF and the indicated concentration of
[methyl-3H]dThd in a final volume of 50 lL The reaction
was started by adding approximately 0.1 ng enzyme
diluted from 6 lgÆmL)1 in ice-cold enzyme dilution buffer
(50 mm Tris⁄ HCl pH 7.5, 1 mm Chaps, 3 mgÆmL)1 BSA)
immediately before the start of the reaction During the
first 15 min of the reaction, four samples of 10 lL each,
taken at various time points 3, 6, 9 and 12 min after the
start of the reaction, were applied to the DE-81 filters
The filters were washed three times for 5 min each in
5 mm ammonium formate and once for 5 min in water,
and the nucleotides were eluted from the DE-81 filters
by shaking for 30 min in 0.2 m KCl⁄ 0.1 m HCl, after
which the radioactivity was determined by scintillation
counting
Analysis of kinetic data
Kinetic data were fitted by non-linear regression analysis to
the Michaelis–Menten equation v¼ Vmax ½S=ðKmþ ½SÞ
or the Hill equation v¼ Vmax ½Sn=ðK0:5 nþ ½SnÞ using
prism 5 from GraphPad Software Inc (La Jolla, CA,
USA; http://www.graphpad.com/), where Kmis the Michaelis
constant and K0.5 is the substrate concentration where
v= 0.5 Vmax When n = 1, there is no co-operativity, and
K0.5= Km
Acknowledgements
This work was supported by grants from the Danish Research council and the Novo Nordic Research Council The skilful technical assistance of Marianne Lauridsen is gratefully acknowledged
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