Báo cáo khoa học: The role of Ureaplasma nucleoside monophosphate kinases in the synthesis of nucleoside triphosphates potx

To answer this question, nucleoside mono-phosphate kinases NMPKs from Ureaplasma were studied regarding their role in the synthesis of NTPs⁄ dNTPs.. The catalytic rates of dNTPs and dNDP

kinases in the synthesis of nucleoside triphosphates

Liya Wang

Department of Molecular Biosciences, Swedish University of Agricultural Sciences, The Biomedical Centre, Uppsala, Sweden

Mycoplasmas (Mollicutes) are wall-less bacteria and

phylogenetically belong to the gram-positive bacteria

Mollicutes are pathogens affecting humans, animals

and plants [1] Ureaplasma is a human pathogen

col-onizing the urogenital tract and is the most common

cause of nonchlamydial nongonococcal urethritis It

has also been implicated in infertility, spontaneous

abortion, stillbirth, and premature and perinatal

mor-bidity and mortality [2–4]

Mollicutes, in general, have low G + C content of

their genomes, and lack those genes necessary for

the synthesis of precursors for DNA, RNA and

pro-teins For example, no de novo purine and pyrimi-dine biosynthesis pathway exists [1] Of all the Mollicutes genomes sequenced to date, there is no annotated ndk gene, coding for nucleoside diphos-phate kinase, indicating that no homologue of any known ndk gene or catalytic domain is present in Mollicutes [5,6]

Nucleoside diphosphate kinase (NDK) catalyzes the final step in ribonucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) biosynthesis NDK is involved in multiple cellular processes including the control of cell growth and signalling by

Keywords

Mollicutes; nucleotide biosynthesis;

nucleoside diphosphate kinase; nucleoside

monophosphate kinase; Ureaplasma

Correspondence

L Wang, Department of Molecular

Biosciences, Section of Veterinary Medical

Biochemistry, Swedish University of

Agricultural Sciences, The Biomedical

Centre, PO Box 575, SE-751 23 Uppsala,

Sweden

Fax: +46 18550762

Tel: +46 184714119

E-mail: liya.wang@mbv.slu.se

(Received 11 January 2007, revised 5

February 2007, accepted 14 February 2007)

doi:10.1111/j.1742-4658.2007.05742.x

Mollicutes are wall-less bacteria and cause various diseases in humans, ani-mals and plants They have the smallest genomes with low G + C content and lack many genes of DNA, RNA and protein precursor biosynthesis Nucleoside diphosphate kinase (NDK), a house-keeping enzyme that plays

a critical role in the synthesis of nucleic acids precursors, i.e NTPs and dNTPs, is absent in all the Mollicutes genomes sequenced to date There-fore, it would be of interest to know how Mollicutes synthesize dNTPs⁄ NTPs without NDK To answer this question, nucleoside mono-phosphate kinases (NMPKs) from Ureaplasma were studied regarding their role in the synthesis of NTPs⁄ dNTPs In this work, Ureaplasma adenylate kinase, cytidylate kinase, uridylate kinase and thymidylate kinase were cloned and expressed in Escherichia coli The recombinant enzymes were purified and characterized These NMPKs are base specific, as indicated by their names, and capable of converting (d)NMPs directly to (d)NTPs The catalytic rates of (d)NTPs and (d)NDP synthesis by these NMPKs were determined using tritium-labelled (d)NMPs, and the rates for (d)NDP syn-thesis, in general, were much higher (up to 100-fold) than that of (d)NTP Equilibrium studies with adenylate kinase suggested that the rates of NTPs⁄ dNTPs synthesis by NMPKs in vivo are probably regulated by the levels of (d)NMPs These results strongly indicate that NMPKs could sub-stitute the NDK function in vivo

Abbreviations

AdK, adenylate kinase; CMPK, cytidylate kinase; GMPK, guanylate kinase; NDK, nucleoside diphosphate kinase; NMPK, nucleoside

monophosphate kinase; TMPK, thymidylate kinase; UMPK, uridylate kinase.

providing either all the NTPs or a specific NTP such

as GTP, CTP or UTP [7–9] NDK is a highly

con-served enzyme and is widely expressed In humans,

eight ndk genes have been reported and the

corres-ponding enzymes have different tissue distribution

and subcellular localization NDKs are also involved

in cell growth, differentiation and tumour metastasis,

etc [8,9] Null mutations in the ndk gene of

Droso-phila caused abnormalities in the development of the

larvae, leading to tissue necrosis and death at the

pre-pupal stage [10] In prokaryotes such as Escherichia

coli, deletion of the ndk gene led to a mutator

pheno-type due to abnormal dCTP and dGTP pools [11]

However, the growth rate was not affected, suggesting

that other enzymes may be able to substitute the

NDK activity [12,13]

So far, no NDK activity was detected in total cell

ex-tracts or the chromatographic fraction of Mycoplasma

pneumoniae in an attempt to isolate the NDK

enzyme [14] Thus, the question is how do Mollicutes

synthesize their dNTPs and NTPs without the NDK

enzyme?

Glycolytic enzymes such as pyruvate kinase and

phosphoglycerate kinase have been suggested to

replace the NDK activity in Mollicutes [14], and, in

that study, purine nucleoside diphosphates, e.g ADP

and GDP, were converted to triphosphates with

relat-ively good efficiency; however, pyrimidine nucleoside

diphosphates had very low relative activity Therefore,

there must be other alternative pathways that may

contribute to the synthesis of NTPs⁄ dNTPs, especially

with regard to pyrimidine nucleotides

Nucleoside monophosphate kinases (NMPK)

cata-lyze the reversible phosphorylation of a nucleoside

monophosphate (NMP) using a nucleoside

triphos-phate as phostriphos-phate donor, i.e N1MP + N2TP ‹fi

N1DP + N2DP Adenylate kinase (AdK) has been

suggested play a role in the synthesis of dNTPs and

NTPs using the reverse reaction and it was proposed

that AdK was the alternative enzyme in providing

nucleoside triphosphates in NDK-deficient E coli

[12,13,15]

All Mollicutes species sequenced to date possess five

nucleoside monophosphate kinases, which have been

assigned as AdK, thymidylate kinase (TMPK),

cyti-dylate kinase (CMPK), uricyti-dylate kinase (UMPK), and

guanylate kinase (GMPK) In this work, four of the

Ureaplasma parvum nucleoside monophosphate

kinases, i.e adenylate kinase, thymidylate kinase,

cyti-dylate kinase, and uricyti-dylate kinase, were cloned and

expressed in E coli The recombinant enzymes were

affinity purified and their role in dNTP and NTP

syn-thesis was investigated

Results

Ureaplasma nucleoside monophosphate kinases: cloning, expression and purification

Five NMPKs have been annotated in the genome

U parvum, i.e AdK (adk, UU251), TMPK (tmk, UU020), CMPK (cmk, UU342), UMPK (pyrH, UU513) and GMPK (gmk, UU213), based on the sequence homology with NMPKs from other organ-isms (GenBank accession number AF222894) There is

no experimental data regarding the functions of these genes reported [5]

Open reading frames coding for AdK (UU251), CMPK (UU342), UMPK (UU513), and TMPK (UU020) were PCR amplified using the U parvum genomic DNA as template and cloned into the pET-14b vector using the Nde I and BamH I restriction sites A 6-His tag and a thrombin cleavage site were introduced to the N-terminus of the recombinant pro-teins Tryptophans, coded by UGA codons in UMPK and AMPK, were mutated to UGG using the site-directed mutagenesis method in order to express these proteins in E coli Recombinant AdK, CMPK, UMPK and TMPK were expressed in E coli and puri-fied by metal affinity chromatography SDS⁄ PAGE analysis showed that the dominant band corresponded

to the subunit molecular mass of each enzyme (Fig 1) The purified enzymes were used directly in the assays without the removal of the His tag

Substrate specificity of Ureaplasma nucleoside monophosphate kinases

The substrate specificities of AdK, CMPK, UMPK and TMPK were explored using a phosphoryl transfer

Fig 1 SDS ⁄ PAGE analysis of purified recombinant Ureaplasma nucleoside monophosphate kinases Lanes 1–4, TMPK, UMPK, AdK and CMPK MW, molecular mass markers.

assay with 32P-labelled ATP as phosphate donor and

natural (deoxy)ribonucleoside monophosphates as

acceptors AdK used AMP and dAMP as the preferred

substrates (Fig 2); both CMP and dCMP were

effi-ciently phosphorylated by CMPK (Fig 2) UMPK

phosphorylated UMP and dUMP (Fig 2) while

TMPK phosphorylated dTMP and dUMP (Fig 2)

Products from the CMPK (with dCMP and CMP as

substrates) and the TMPK (with TMP as substrate)

reactions were re-analysed by TLC using a higher salt

concentration to separate the other nucleotides from

ATP As shown in Fig 3, substantial amounts of

dCTP, CTP and TTP were formed in these reactions

Nucleoside di- and triphosphate synthesis

catalyzed by AdK

When tritium-labelled AMP was used as substrate in

AdK catalyzed reactions, the reaction products, as

ana-lysed by the TLC, were found to be [3H]ADP and

[3H]ATP [3H]AMP was rapidly converted to [3H]ADP,

which reached a maximum after 3 min and then

declined, while the formation of [3H]ATP was increased

linearly with time within the first 15 min After 30 min,

the reaction reached equilibrium (Fig 4A) Further

experiments were designed to study the extent of

[3H]ATP and [3H]ADP synthesis using different

phos-phate donors The [3H]ATP synthesis rates were slightly

influenced by the phosphate donors used,

approxi-mately two-fold; the highest rate was found with ATP

as donor and the lowest with GTP as donor (Table 1)

In order to measure the rate of [3H]ADP synthesis, the reaction conditions was optimized, in terms of AdK concentration, so that a first order reaction was observed (Fig 4B) As shown in Table 1, the rates of [3H]ADP synthesis were approximately 40-fold higher than that of [3H]ATP As with [3H]ATP synthesis, phosphate donors did not have major impact on the

Fig 2 Substrate specificities of Ureaplasma

NMPKs The reactions were performed as

described in Experimental procedures and

the products were separated by TLC and

visualized by autoradiography The

concen-trations of nucleoside monophosphates and

[c-32P]ATP were as follows: 0.1 m M 1.

CMP; 2 dCMP; 3 AMP; 4 dAMP; 5 GMP;

6 dGMP; 7 UMP; 8 dUMP; 9 dTMP The

reaction products were marked as close to

the product spot as possible.

Fig 3 Direct formation of nucleoside triphosphates in NMPK cata-lyzed reactions Reaction mixtures 1 and 2 by CMPK and 9 by TMPK, described in Fig 2, were reanalysed by TLC (developed using 0.32 M NaH 2 PO 4 ) and the radiolabelled nucleoside triphos-phate products were separated from ATP.

rates of [3H]ADP synthesis, although the highest

[3H]ADP synthesis rate was obtained when ATP was

the phosphate donor (Table 1)

Reaction equilibrium for AdK

In the reactions described above, the concentration of phosphate donors was 1 mm, which was 10-fold higher than that of [3H]AMP; however, at equilibrium [3H]ATP was the dominant product Therefore, a ser-ies of reactions with varied ATP concentrations were carried out to study the equilibrium of labelled adenine nucleotides As shown in Table 2, at equilibrium the concentration of [3H]ATP was dependent on the con-centration of ATP used in the reaction; a higher ATP concentration yielded a higher concentration of [3H]ATP The equilibrium constants for labelled aden-ine nucleotides were lowest with highest ATP concen-tration tested (Table 2)

The equilibrium constants of labelled adenine nucleo-tides with different phosphate donors at 1 mm concen-tration was two-fold lower with ATP as compared with CTP, UTP or GTP as phosphate donor (Table 3)

0

20

40

60

80

100

Min

ATP

ADP

AMP

A

0

20

40

60

80

100

Min

ATP

AMP ADP

B

Fig 4 The synthesis of [3H]ADP and [3H]ATP from [3H]AMP

cata-lyzed by AdK (A) The conditions for linear rate of [ 3 H]ATP synthesis

were 0.2 l M AdK, 100 l M [ 3 H]AMP and 1 m M ATP ⁄ MgCl 2 in the

reaction buffer, as described in experimental procedures (B) The

conditions for linear rate [ 3 H]ADP synthesis were 1.0 n M AdK,

100 l M [ 3 H]AMP and 1 m M ATP ⁄ MgCl 2 in the reaction buffer, as

described in Experimental procedures.

Table 1 Rate of nucleoside di- and triphosphate synthesis by

Urea-plasma NMPKs (s)1) The concentrations of [3H]AMP, [3H]dCMP or

[ 3 H]UMP were 100 l M , the concentrations of phosphate donors

were 1 m M and the concentration of MgCl 2 was 10 m M The values

were the means of two to four measurements with < 10%

varia-tion.

[3H]ADP [3H]ATP [3H]dCDP [3H]dCTP [3H]UDP [3H]UTP

Table 2 The effect of ATP concentration on the equilibrium con-centration of labelled adenine nucleotides The initial concon-centration

of [ 3 H]AMP was 100 l M and the concentration of MgCl2 was

10 m M The values are the means of 2–4 measurements with

< 10% variations Equilibrium constants K eq of labelled adenine nucleotides were calculated according to the following equations: [ 3 H]ATP + [ 3 H]AMP fi 2 [ 3 H]ADP; Keq¼ ([ 3 H]ADP) 2 ⁄ ([ 3 H]ATP)([ 3 H] AMP) The theoretical K eq for AdK is close to unity, as determined

by the equation However, K eq values presented here refer to only the labelled adenine nucleotide at equilibrium but not the reaction equilibrium constants.

ATP (m M )

[ 3 H]ATP (l M )

[ 3 H]ADP (l M )

[ 3 H]AMP

Table 3 Equilibrium concentrations of labelled adenine nucleotides

in AdK-catalyzed reactions Equilibrium constants Keq of labelled adenine nucleotides were calculated as described for Table 2 The initial concentrations of phosphate donors were 1 m M and the initial concentration of phosphate acceptor [ 3 H]AMP was 100 l M and the concentration of MgCl 2 was 10 m M The values were the means of 2–4 measurements with < 10% variations.

[3H]ATP [3H]ADP [3H]AMP K eq

Nucleoside di- and triphosphate synthesis

catalyzed by CMPK

CMPK phosphorylated CMP and dCMP directly to

their triphosphate forms in assays with radiolabelled

ATP as phosphate donor (Fig 3) The rate of

[3H]dCDP and [3H]dCTP synthesis from [3H]dCMP

was further studied using ATP, UTP, GTP or CTP as

phosphate donors The rate for [3H]dCDP synthesis, in

general, is high, being 100-fold higher than that of

[3H]dCTP, except when CTP is used as a phosphate

donor (Table 1) ATP, UTP and GTP were good

phos-phate donors for both [3H]dCDP and [3H]dCTP

syn-thesis, while CTP was a poor phosphate donor, i.e

20-fold lower for [3H]dCDP synthesis and two-fold

lower for [3H]dCTP synthesis (Table 1)

Nucleoside triphosphate synthesis catalyzed

by UMPK

Using [3H]UMP as substrate, the rates of [3H]UDP

and [3H]UTP synthesis were determined Similar to

AdK and CMPK, the rate of [3H]UDP synthesis was

much higher than that of [3H]UTP (Table 1) ATP was

the most efficient phosphate donor for [3H]UDP

syn-thesis, while UTP, GTP and CTP were poor phosphate

donors There was detectable formation of [3H]UTP in

the reaction with GTP and CTP as phosphate donor,

but the rates of [3H]UTP synthesis were very low

(Table 1)

Mechanism of phosphoryl transfer

Using [c-32P]ATP as phosphate donor, the mechanism

of phosphoryl transfer during CTP synthesis by CMPK

was studied Initially, using either dCDP or CDP as the

substrate and [c-32P]ATP as the phosphate donor, no

radiolabelled dCTP formation was detected in the

reac-tion with dCDP as the substrate, but it was observed

when using CDP as the substrate CTP was also

formed in the control reaction using dCMP as

sub-strate However, as commercial CDP contains 4%

CMP, and the level of radiolabelled product (CTP) was

similar for both substrates (data not shown) and

cor-responded to the CMP content in the CDP solution

(4%), this accounted for the CTP product observed

When the reaction was repeated with purer CDP and

run at shorter time intervals (90 s), no CTP was formed

in the CDP reaction, while CTP was formed in the

con-trol reactions with CMP as substrate Thus, the CTP or

dCTP synthesis carried out by CMPK was not a direct

phosphorylation of CDP or dCDP by ATP, but rather

CTP or dCTP was formed in the reverse reaction in

two steps, i.e (d)CMP + ATP*fi (d)CDP* + ADP; and (d)CDP* + (d)CDP fi (d)CTP* + (d)CMP

Discussion

The aim of the present study was to define the role

of Ureaplasma NMPKs in the synthesis of nucleo-side triphosphates Four Ureaplasma NMPKs were cloned, expressed and the recombinant enzymes were affinity purified These NMPKs were shown to have narrow substrate specificity regarding the phosphate acceptors, i.e they are base specific and each enzyme has its own substrates sets (using the same nucleo-tides as their names indicated), with little overlapping activity AdK, CMPK and UMPK phosphorylated both ribos- respective deoxyribos- forms of nucleo-tides efficiently TMPK, however, was specific for dTMP and only dUMP had some activity At the phosphate donor site, however, the specificities were broader, e.g all natural nucleoside triphosphates were accepted as phosphate donors but with clear-cut pref-erences, especially in case of UMPK The less strin-gent requirement for phosphate donors may be an advantage, since the enzymes can use any phosphate donors available

It is known that reactions catalyzed by NMPKs are reversible, which means that these enzymes can also synthesize nucleoside triphosphates via the reverse reaction Using the tritium-labelled nucleoside mono-phosphates as substrates, the rates of (d)NDP and (d)NTP synthesis by these enzymes were investigated The results clearly showed that Ureaplasma NMPKs are able to synthesize NTP⁄ dNTPs via the reverse reaction, but not sequential phosphorylation of NMP

by NTP as demonstrated here This is also in agree-ment with a recent study using AdK, where it was clearly shown that the NDK-like activity of this enzyme is the result of the reverse reaction [11] The capacity of Ureaplasma NMPKs to synthesize nucleo-side triphosphates in general is lower than that of diphosphates, which may implicate that the conversion

of nucleoside diphosphates to triphosphates is the rate-limiting step

Equilibrium studies with AdK showed that the enzyme favours the reverse reaction, i.e the highest level of [3H]ATP formed from [3H]AMP was achieved with the highest ATP concentration used in the assay ATP was a better phosphate donor to bring about [3H]ATP formation from [3H]AMP as compared with other nucleoside triphosphates At physiologically rele-vant ATP concentrations (2–4 mm), the level of labelled [3H]AMP at equilibrium was the lowest among all labelled adenine nucleotides, suggesting that the

supply of nucleoside monophosphates may regulate the

synthesis of nucleoside di- and triphosphates

In Mollicutes, other enzymes exist that are capable

of synthesizing nucleoside triphosphates, e.g pyvuvate

kinase, phosphoglycerate kinase, etc [14] Together

with NMPKs, they can provide cells with precursors

for both DNA and RNA synthesis The relatively low

rates of (d)NTP synthesis may result in limited

(d)NTPs supply

In the literature there are only few earlier studies

regarding the level of ribonucleotides and

deoxyribo-nucleotides in Mycoplasmas [16–18] In Mycoplasma

mycoides subsp mycoides the levels of dNTPs were

100-fold lower as compared with NTPs [16–18]

Interestingly, the sum of ATP and UTP was two-fold

higher than that of CTP and GTP, and the ratio of

dATP and dTTP to dCTP and dGTP was also 2 : 1

[16,17] This fact is in accordance with the genome

composition of Mollicutes, which has a high A + T

content at > 70% Studies carried out in our

labora-tory showed that the dTTP and dCTP levels were

below the detection limit when Ureaplasma was

cul-tured in the presence of tritium-labelled deoxycytidine

or thymidine, even though > 20% of the

radioacti-vity was recovered in the DNA fraction, indicating a

very low dNTP pool in Ureaplasma [19] Similarly,

M mycoides subsp mycoides incubated with

radiola-belled thymidine monophosphate resulted also in a

high level of incorporation into DNA, but the level of

labelled dTTP was very low [18]

In organisms where NDK is present, the

rate-limit-ing step in the synthesis of dNTPs⁄ NTPs is not the

conversion of (d)NDPs to (d)NTPs, as the catalytic

efficiency of NDK is high [7–9] The regulatory

mech-anism in dNTPs production relies on allosteric

enzymes, e.g ribonucleotide reductase, which to a

large extent regulates dNDP production and thereby

dNTP pools [20,21] Disruption of the ndk gene in

E coli, Saccharomyces cerevisiae, and Saccharomyces

pombedid not affect growth or morphology [11,22,23],

suggesting that NDK is not essential Although

Molli-cutes lack the ndk gene, this work provided evidence

that NMPKs, probably together other enzymes such as

pyvuvate kinase [14], can replace NDK in providing

the cells with NTPs and dNTPs A recent study

sug-gests that cells have a mechanism for arresting DNA

synthesis when the dNTP pool size is limiting [24] The

doubling times for Mollicutes are usually much longer

when compared with other bacteria such as E coli,

and the limitation of dNTP levels for DNA synthesis

could be the reason

Mollicutes lack the de novo synthesis of purine and

pyrimidine bases and have to rely on salvage

path-ways for the biosynthesis of nucleotides required for cellular processes The work presented here clearly showed that NMPKs are base specific and highly efficient in the synthesis of NTPs and dNTPs NMPKs are essential enzymes for the survival of the organism, as demonstrated recently in Mycoplasma genitalium using transposon mutagenesis technique [25] Therefore, inhibition of these enzymes, especially TMPK or UMPK, will most probably impair the synthesis of both DNA and RNA precursors, which eventually leads to cell death Thus, these enzymes, especially TMPK and UMPK, are potential targets for future design of antibiotics against pathogenic Mycoplasmas

Experimental procedures

Materials Radioactive substances [c-32P]ATP (3000 CiÆmmol)1) were purchased from PerkinElmer LAS Inc (Boston, MA, USA) [2-3H]AMP (adenosine 5¢-monophosphate, 24.0 CiÆmmol)1) was from Amersham Biosciences (Uppsala, Sweden) [5-3H]dCMP (2¢-deoxycytidine 5¢-monophosphate, 21.9 CiÆmmol)1) and [5,6-3H]UMP (uridine 5¢-monophosphate,

32 CiÆmmol)1) were obtained from Moravek Biochemical, Inc (Brea, CA, USA) Non-radioactive nucleotides were from Sigma-Aldrich Sweden AB (Stockholm, Sweden)

Cloning, expression and purification of Ureaplasma nucleoside monophosphate kinases Primers used in PCR amplification of Ureaplasma nucleo-side monophosphate kinases were designed according to the DNA sequence of the respective enzyme in the database (GenBank accession number of AE002122) and restriction sites (Nde I or BamH I) were introduced to the 5¢-sequence

of the primers to facilitate subsequent cloning PCR reac-tions were carried out by a standard procedure using the

U parvum genomic DNA (ATCC # 700970D) as template The amplified PCR fragments were digested with Nde I and BamHI, purified on 1% agarose gel and cloned into the pET-14b vector (Novagen, Madison, WI, USA) that had been linearized with the same restriction enzymes The recombinant plasmids carrying the Ureaplasma nucleoside monophosphate kinase genes were sequence verified using the Bigdye terminator kit and ABI Prisma 310 genetic Ana-lyzer (Applied Biosystems, Foster City, CA, USA) In order

to express the recombinant protein in E coli UGA codons, coding for Trp in AdK and UMPK, were mutated to UGG using site-directed mutagenesis as described previously [26] and sequence verified Finally the plasmids carrying AdK, CMPK, UMPK and TMPK were transformed into the

E coliBL21 (DE3) bacteria for expression

For production of recombinant protein, E coli BL21

(DE3) harbouring the AdK, CMPK, UMPK or TMPK

plasmids was cultured in LB media with 50 lgÆmL)1

carbe-nicilin at 37C until the optical density at 600 nm reached

0.6 The cultures were then changed to the induction

temperature as indicated below and 0.1 mm isopropyl

b-d-1-thiogalactopyranoside was added and the cultures

were further incubated for 4 h The temperature for

induc-tion was 30C for AdK and CMPK; 37 C for UMPK;

28C for TMPK Bacteria were harvested by centrifugation

and the pellets were resuspended in buffer containing 50 mm

Tris⁄ HCl, pH 7.5, 0.2 m NaCl, 0.2 mm

phenylmethylsulfo-nyl fluoride and total proteins were extracted by sonication

at 18 W for a total of 3 min, with a pulse every 5 s The

lysates were centrifuged at 30 000 g at 4C for 30 min

(XL-70 ultracentrifuge, Beckman Coulter, rotor type

RT50Ti) and the supernatant was loaded onto a metal

affin-ity column (TALON resin, BD Biosciences Clontech, Palo

Alto, CA, USA), which was equilibrated with 50 mm

Tris⁄ HCl, pH 7.5, 0.2 m NaCl and 5 mm imidazole The

column was subsequently washed with the same buffer

con-taining 30 mm imidazole and the recombinant proteins were

eluted with 300 mm imidazole and 50 mm Tris⁄ HCl,

pH 7.5 Purified protein was analysed by SDS⁄ PAGE and

protein concentrations were determined by Bio-Rad protein

assay with BSA as standard Glycerol and dithiothreitol

were added to the purified enzymes to 10% and 2 mm,

respectively, and the enzymes were stored in aliquots at

)70 C

Enzyme assays

Phosphoryl transfer assays were performed essentially as

described previously [26] Briefly, each reaction was

per-formed in a total volume of 20 lL containing 50 mm

Tris⁄ HCl, pH 7.5, 0.5 mg.mL)1 BSA, 5 mm dithiothreitol,

2 mm MgCl2, 15 mm NaF, 0.1 mm nucleoside

monophos-phate, 0.1 mm [g-32P]ATP and 100 ng purified enzyme at

37C for 20 min and was stopped by heating at 70 C for

2 min After brief centrifugation (13 000 g, Biofuge 13,

Her-aeus Instruments, rotor type HFA 17.1, max 14 926 g),

1 lL of the supernatant was spotted onto a TLC plate

(PEI-cellulose; MERCK, VWR International AB, Stockholm,

Sweden) and dried Authentic markers were also applied

onto the same TLC plates The TLC plates were then

devel-oped in 0.2 m NaH2PO4for reactions with AdK and CMPK

and 0.1 m NaH2PO4 for UMPK and TMPK To separate

other nucleoside triphosphates from [g-32P]ATP, 0.32 m

NaH2PO4was used The reaction products were visualized

by phosphoimagine analysis (Fuji ImageGause V3.1, Fuji

Photo Film Co., Ltd., Tilburg, the Netherlands) Authentic

markers were visualized under UV light

NMPK assay with [3H]-labelled substrates were carried

out on the reaction mixture containing 50 mm Tris⁄ HCl,

pH 7.5, 0.5 mgÆmL)1 BSA, 5 mm dithiothreitol, 5 mm

MgCl2, 15 mm NaF, 0.1 mm3H-labelled substrate in a total volume of 20 lL The reaction was initiated by the addition the enzyme (100 ngÆreaction)1 for the determination of NTP synthesis rates and 0.5–1 ngÆreaction)1 for the deter-mination of NDP synthesis rates) and incubated at 37C

At each time point, 1 lL aliquot was withdrawn and spot-ted directly onto a TLC plate (PEI-cellulose) and dried The TLC plate was then developed in 0.2 m NaH2PO4for assays with AdK and CMPK and 0.1 m NaH2PO4 for assays with UMPK Non-radioactive markers were spotted onto the TCL plate and identified under UV light The reaction products were cut out and eluted with 0.5 mL of 0.1 m HCl and 0.2 m KCl, and then 2.5 mL of scintillation fluid was added and the radioactivity counted

The Rf values for ATP, ADP, AMP, dCTP, dCDP and dCMP in 0.2 NaH2PO4 were 0.01, 0.28, 0.61, 0.03, 0.39 and 0.67, respectively The Rfvalues for UMP, UDP, UTP, dTMP, dTDP and dTTP in 0.1 m NaH2PO4 were 0.37, 0.21, 0.05, 0.36, 0.22, and 0.05, respectively

Acknowledgements

This work was supported by grants from the Swedish research council for Environment, Agricultural Scien-ces, and Spatial Planning (FORMAS) and the Swedish Research Council (VR)

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