WO2004104192A1 - Procede pour isoler et purifier une ump kinase de staphylococcus - Google Patents

Procede pour isoler et purifier une ump kinase de staphylococcus Download PDF

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WO2004104192A1
WO2004104192A1 PCT/GB2004/002158 GB2004002158W WO2004104192A1 WO 2004104192 A1 WO2004104192 A1 WO 2004104192A1 GB 2004002158 W GB2004002158 W GB 2004002158W WO 2004104192 A1 WO2004104192 A1 WO 2004104192A1
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kinase
ump
enzyme
assay
ump kinase
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PCT/GB2004/002158
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English (en)
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Peter Clifford Doig
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Astrazeneca Ab
Astrazeneca Uk Limited
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Priority to EP04733841A priority Critical patent/EP1629094A1/fr
Priority to JP2006530519A priority patent/JP2007500512A/ja
Publication of WO2004104192A1 publication Critical patent/WO2004104192A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04014—UMP/CMP kinase (2.7.4.14), i.e. uridine monophosphate kinase

Definitions

  • the present invention relates to nucleoside monophosphate kinases and in particular to their purification, stabilisation and use in drug screening assays to identify kinase inhibitors.
  • Nucleoside monophosphate kinases are required for the biosynthesis of nucleic acids, of various key nucleotidyl intermediates and in energy metabolism.
  • One member of this kinase family uridine 5 '-monophosphate (UMP) kinase (PyrH) catalyzes the transfer of the ⁇ - phosphate of adenosine 5'-triphosphate (ATP) to UMP to produce the adensosine 5' ⁇ diphosphate (ADP) and uridine 5 '-diphosphate (UDP) and this function is essential for cellular survival.
  • UMP uridine 5 '-monophosphate
  • ATP adenosine 5'-triphosphate
  • ADP adensosine 5' ⁇ diphosphate
  • UDP uridine 5 '-diphosphate
  • a unique aspect in bacteria is that they possess separate UMP and cytidine 5'-monphos ⁇ hate (CMP) kinases whereas mammals and many higher eukaryotes possess a single enzyme that carries out both functions. These enzymes share little sequence homology and PyrH enzymes from bacteria possess unique structural and enzymological characteristics. The enzyme is ubiquitous, being found in all major pathogenic bacteria. The essential nature of this enzyme and its wide distribution make it an ideal target for intervention with an antibacterial agent.
  • CMP cytidine 5'-monphos ⁇ hate
  • WO-99/55729, WO-2001/12678 and corresponding US patent no. 6403337 disclose pyrH gene sequences from Staphylococcus aureus (S. aureus). We have cloned and purified S. aureus UMP kinase (PyrH) and the amino acid sequence is set out in Seq ID No. 2 hereinafter. When compared with the published amino acid sequence of this gene, it lacks Methionine as the first amino acid. Whilst we don't wish to be bound by theoretical considerations, this is not believed to have any material effect on the properties of the enzyme, for example when used in drug screening assays. Whilst purifying S.
  • aureus UMP kinase we found that it was necessary to add nucleosides or nucleotides to the purification procedure to provide a soluble, stable or active enzyme. Specifically we found that the addition of uridine 5'-triphosphate (UTP) results in stable protein that is soluble at high concentrations. We also found that guanosine 5'- triphosphate (GTP) and other nucleotides such as ATP are important to stabilize and/or activate the enzyme as well as acting as allosteric-like regulators of activity.
  • UTP uridine 5'-triphosphate
  • GTP guanosine 5'- triphosphate
  • ATP guanosine 5'- triphosphate
  • US patent application 2002/0119506 discloses the cloning and isolation of UMP kinase from three bacillus microorganisms, namely B. subtilis (Gram negative), M. tuberculosis (acid-fast) and H. influenzae (Gram negative). It reports GTP activation and UTP inactivation of the B. subt ⁇ lis enzyme. The degree of GTP activation for all three species is variable, a factor of 2 for M. tuberculosis, 3 for H. influenzae and between 10 and 20 for B. subtilis. However such effects are believed to be species specific ie. there is no anticipation that other enzymes will show significant activation. In particular there is no indication as to the properties of Gram positive staphylococcus UMP kinases.
  • Staphylococcus UMP kinase characterised in that during the process of isolation or purification the enzyme is stabilized by addition of a stabilisation species such that it can be stored for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity when compared with its initial stabilised activity.
  • Stabilization is conveniently effected by protecting the protein from degradation, modification or aggregation.
  • the enzyme is conveniently stabilized by use of a stabilisation species such as UTP or GTP or a functional analogue thereof.
  • concentration of stabilisation species such as UTP is conveniently at least 100 micromolar and the kinase is present at a concentration of up to 200% equivalent concentration.
  • UTP Ultrathyroxine triphosphate
  • “functional analogue” of UTP or GTP we mean a species that recognises and binds to the same binding site. Such species may include, by way of non-limiting example, nucleotide analogues such as 2'-Fluoro-uridine 5'-triphosphate, Guanosine 5'[gamma-thio]-triphosphate, and 273 ' -O-(N-Methyl-anthraniloyl)-guanosine-5 ' -triphosphate.
  • nucleotide analogues such as 2'-Fluoro-uridine 5'-triphosphate, Guanosine 5'[gamma-thio]-triphosphate, and 273 ' -O-(N-Methyl-anthraniloyl)-guanosine-5 ' -triphosphate.
  • the enzyme is any convenient Staphylococcus UMP kinase, such as any one of S. aerogenes, S. auricularis, S. arlettae, S. capitis, S. capitis subsp. ureolyticus, S. caprae, S. carnosus subsp. carnosus, S. chromogenes, S. cohnii subsp. cohnii, S. cohnii subsp. urealyticum, S. condimenti, S. delphini, S. epidermidis, S. equorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis subsp.
  • Staphylococcus UMP kinase such as any one of S. aerogenes, S. auricularis, S. arlettae, S. capitis, S. capitis subsp. ureolyticus, S
  • UMP kinase enzyme is provided in a form such that its specific activity over time is not unduly compromised and remains suitable for use in for example drug screening assays. Specifically, it can be stored for at least one week at between -20 and 4 degrees centigrade without more than 50% loss of specific enzyme activity when compared with its initial stabilised activity.
  • the stabilized enzyme is conveniently provided as storage solution made up of one or more of: a buffer, a nucleotide, glycerol, and a reducing agent.
  • the solution may contains a convenient buffer, preferably HEPES, PIPES or Tris-HCl, and a neutral or alkaline pH, preferably pH 8.0 through 8.5.
  • the solution contains a nucleotide, preferably UTP at a concentration of 0.5 to 2 millimolar, corresponding to a enzyme to nucleotide ratio of at least 50%.
  • the solution also contains glycerol (10% to 50% vol/vol), sodium chloride or similar salt at a concentration of 100 to 250 millimolar and a reducing agent such as dithiothreitol at a concentration between 100 micromolar to 5 millimolar.
  • the activation species is conveniently GTP or ATP or is a functional analogue or either species.
  • the nucleoside triphosphate(s) is conveniently present at a concentration of 100-1000 micromolar and the kinase is present at no more than 50% equivalent concentration.
  • the enzyme is any convenient Staphylococcus UMP kinase, such as any one of S. aerogenes, S. auricularis, S. arlettae, S. capitis, S. capitis subsp. ureolyticus, S. caprae, S. carnosus subsp. carnosus, S. chromogenes, S. cohnii subsp. cohnii, S. cohnii subsp. urealyticum, S. condimenti, S. delphini, S. epidermidis, S. equorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S. hominis subsp.
  • Staphylococcus UMP kinase such as any one of S. aerogenes, S. auricularis, S. arlettae, S. capitis, S. capitis subsp. ureolyticus, S
  • the activated enzyme is conveniently provided as a working solution.
  • the working solution may contain one or more of: a buffer, salts, nucleotides, and surfactants.
  • the working solution may contain any convenient buffer MES, PIPES, MOPS, HEPES, Tricine, Tris-HCl or Glycine and preferably in HEPES or PIPES buffer, at any convenient pH (pH 6
  • the solution may contain one or more salts such as, but not limited to NaCl, NH 4 CI, KC1, and/or MgCl 2 .
  • the working solution may also contain a surfactant, preferably a non-ionic surfactant, such as Brij 35, Triton X-100 or Tween 20 at a concentration near or below the critical micellar concentration.
  • a nucleotide preferably GTP and/or ATP, are added conveniently present at a concentration of
  • the activated UMP kinase is conveniently prepared from the stabilized UMP kinase of this invention, for example by displacement of the stabilisation species.
  • the enzyme is diluted into a working solution containing 5 ⁇ M to 5 mM GTP or 5 ⁇ M to 5 mM ATP or any combination of these nucleotides.
  • This may provide for example 4000-fold to 4,000,000-fold molar excess over PyrH enzyme.
  • Use of at least an 80,000-fold excess of GTP is preferred.
  • Stabilization at -80 to -30 degrees centigrade may be achieved by modifying the storage solution components.
  • the assay conveniently comprises a pyruvate kinase/lactate dehydrogenase
  • PK/LDH pyruvate kinase
  • ADP and UDP the products of the UMP kinase reaction
  • UDP the products of the UMP kinase reaction
  • phosphoenolpyruvate to ATP, UTP and pyruvate.
  • lactate dehydrogenase converts pyruvate to lactate and NADH to NAD + .
  • the decrease in NADH concentration as it is converted to NAD + may be monitored
  • the assay is conducted at any convenient pH (pH 6 through pH 9), and preferably conducted at a pH between 7 and 8.
  • the assay is conducted in any convenient buffer such as MES, PIPES, MOPS,
  • HEPES Tricine, Tris-HCl or Glycine and preferably conducted in HEPES or PIPES buffer.
  • a surfactant (Brij-35, Triton X-100 or Tween 20) is added to prevent protein from sticking to the reaction vessel or pipette tips. Brij-35 at a concentration of 0.01% v/v is the preferred surfactant.
  • the assay is conducted at any convenient temperature (15-30 degrees centigrade) and preferably conducted at a temperature between 20 and 30 degrees centigrade, especially room temperature (18 to 25 degrees Centigrade such as about 22 degrees centigrade).
  • the assay is conducted at any convenient UMP kinase concentration, such as between 0.1 and 300 nM, for example about 1.3 nM
  • the UMP kinase used in the assay is conveniently stabilized by GTP at a concentration between 50 uM - 10 mM GTP, preferably about 5 mM GTP.
  • Figure 1 shows the reaction pathway catalysed by UMP kinase
  • Figure 2 shows that GTP is required for full enzyme activity.
  • the graph shows the effect of pre-incubation of enzyme with varying concentration of GTP.
  • Figure 3 shows buffer and pH profiles.
  • the graph shows a comparison of buffers at various pH.
  • Figure 4 shows the temperature dependence of enzyme activity between 15 and 30 degrees centigrade.
  • Figure 5 shows the effects of salts on enzyme activity. Salts used were Na + , NH and SO 2" .
  • Figure 6 shows the optimisation of MgCl 2 concentration.
  • Figure 7 shows the effect of DMSO on enzyme activity-
  • Figure 8 shows the activity of various concentrations of enzyme.
  • Figure 9 shows the optimisation of coupler enzyme concentration in the UMP kinase assay.
  • FIG 10 shows enzyme stability over time with added GTP and Brij-35
  • Figure 11 shows enzyme stability in assay buffer.
  • Figure 12a shows the effect of varying concentrations of the UMP kinase inhibitor EDTA.
  • Figure 12b shows the effect of varying concentrations of the UMP kinase inhibitor AMP-para nitro phenol.
  • Figure 13a shows a time course for the UMP kinase assay in 384 well plates. NADH fluorescence was measured using an excitation/emission wavelength pair of 340/465nm.
  • Figure 13b shows a time course for the UMP kinase assay in 384 well plates. Absorbance was measured at 340nm
  • S. aureus pyrH was cloned from strain 601055. Primers INF 5174 (5') and INF 5 5183 (3') were used to obtain the S. aureus pyrH PCR product from the a cell lysate of S. aureus 601055.
  • the PCR product was purified using the Qiagen purification kit and ligated into the TOPO Invitrogen cloning vector. The ligated product was transformed into TOPO cells according to the manufacturers recommended method. Transformants were selected and the plasmid recovered using the Qiagen miniprep kit, according to the recommended method. Presence of the correct size DNA insert was verified by digestion with BamHl and Ndel 5 restriction enzymes. Clones with the correct size insert, containing S. aureus PyrH, were selected and DNA prepared using the Qiagen Maxiprep kit. Presence of the insert was rechecked by BamHl/Ndel digestion. The insert was sequenced to assure its identity.
  • the S. aureus PyrH insert was cloned into E. coli pT73.3 and transformed into BL21 (DE3). These cells were grown in LB media at 30°C, containing tetracycline (10 0 ⁇ g/ml) and expression induced by addition of ITPG to a final concentration of 1 mM. Induced cells expressed a protein with an apparent molecular mass of 26,000 as determined by SDS- polyacrylamide gel electrophoresis, consistent with the size expected for the polypeptide. Cell paste was collected by centrifugation and stored at -20°C until use.
  • the frozen cell paste was suspended in 50 ml of Lysis Buffer [50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP, 1 mM PMSF, 1 Protease inhibitor cocktail tablet (Roche Molecular Biochemical)]. Cells were disrupted by passing them twice through a French press operated at 18,000 psi, and the crude extract was centrifuged at 20,000 rpm 0 (45Ti rotor, Beckman) for 30 min at 4°C.
  • the supernatant was loaded at a flow rate of 1.5 rnl/min onto a 20 ml Q-Sepharose HP (HR16/10) column (Pharmacia) pre-equalibrated with Buffer A (50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP). The column was then washed with Buffer A, and the protein was eluted by a linear gradient from 0 to 1 M NaCl in Buffer A. Fractions containing PyrH were pooled, and solid (NH 4 ) 2 SO 4 (0.4 g/ml) was added to precipitate all the proteins and mixed on ice for 1 hour.
  • Buffer A 50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP.
  • Buffer A 50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT
  • the sample was centrifuged at 11,000 rpm for 30 min at 4°C (JA12 rotor, Beckman), the pellet was then dissolved in 5 ml of Buffer A.
  • the 5 ml sample was applied at a flow rate of 0.5 ml/min to a 5 320 ml Sephacryl S-300 (HR 26/60) (Pharmacia) pre-equalibrated with Buffer B (50 mM Tris-HCl, pH 8.5, 2 mM EDTA, 2 mM DTT, 2 mM UTP, 150 mM NaCl).
  • the fractions containing PyrH were pooled and dialyzed against 1 L Storage Buffer (50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA, 1 mM UTP, 150 mM NaCl, 2 mM DTT, 20% Glycerol.).
  • the protein was stored at -80°C.
  • UMP kinase catalyzes the transfer of the gamma phosphate of ATP to UMP, resulting in the formation of two products, ADP and UDP.
  • This activity can be monitored using a pyruvate kinase/lactate dehydrogenase (PK/LDH) coupling reaction.
  • PK/LDH pyruvate kinase/lactate dehydrogenase
  • Pyruvate kinase converts the products of the UMP kinase reaction, ADP and UDP, along with phosphoenolpyruvate, to ATP, UTP and pyruvate.
  • the second coupling enzyme lactate 0 dehydrogenase converts pyruvate to lactate and NADH to NAD + .
  • the decrease in NADH concentration as it is converted to NAD + can be monitored spectrophotometrically by either absorbance or fluorescence. It must be noted that the consumption of one mole of UMP results in the production of two moles of NAD + .
  • a 1.67X assay buffer stock solution was prepared such that 5 the reaction would contain final concentrations of 50 mM HEPES pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 6.6 units/mL PK/LDH, 0.4 mM ATP, 0.2 mM NADH, 1.29 ⁇ M S. aureus UMP kinase, 0.5 mM GTP and 0.001% Brij-35.
  • a 10X enzyme working stock solution containing 12.9 nM solution of S.
  • aureus UMP kinase in 50 mM HEPES pH 8.5, 100 M KCl, 5 mM GTP, 0.01% Brij-35 was prepared and incubated for 15 0 minutes at room temperature.
  • Assay plates were prepared by dispensing an appropriate volume of the 1.67X assay buffer stock to reaction vessels, adding compound DMSO stock solutions and incubating for at least 15 minutes at room temperature to allow equilibration of reagents before initiating reaction with a 3.33X UMP stock solution. For example, for a reaction volume of 100 ⁇ L, 2 ⁇ L of 50X compound in DMSO was added to 60 ⁇ L 1.67X buffer/enzyme mixture and mixed either by rotary shaking or pipetting.
  • reaction was initiated with the addition of 40 ⁇ L 0.67 mM UMP (final concentration was 0.2 mM).
  • Controls used were 2% DMSO (no inhibition) and 10 mM EDTA (100% inhibition).
  • Assay mixtures were mixed by rotary shaking for -20 seconds following addition of UMP. Blank reads were performed and reaction progress was monitored by following the decrease in NADH absorbance or fluorescence over time by either endpoint or continuous read methods. To facilitate testing large numbers of compounds, in some instances, reactions were quenched after 30 minutes with 10 mM EDTA and the final spectrophotomeric measurements made up to several hours later.
  • the assay was optimized to yield maximum specific activity within the constraints of desirable kinetic parameters. Assay parameters addressed included enzyme handling, substrate concentrations, salt requirements and effects, pH and buffer selection, temperature, stability of the reaction over time, linearity of the reaction over time, response of the assay to DMSO, coupler enzyme concentration and activity, and robustness of the assay.
  • S. aureus UMP kinase was found to have a pH optimum between 7.0 and 8.0.
  • the pH selected for the assay was 7.5 as changes in pH of less then 0.5 should not have a significant effect on the assay rate. Buffer effects were small but significant, with Tris and MOPS resulting in the lowest activities and HEPES and PIPES the highest enzyme activities.
  • the specific activity of the S. aureus UMP kinase was monitored over the temperature range 15-30°C. A generally linear increase in activity was observed with increase in temperature, with an overall increase in activity of 40% at 30°C.
  • NADH fluorescence (excitation emission wavelengths 340nm/465nm) was observed to be linear up to -200 ⁇ M. An increased linear range of up to -500 ⁇ M could be obtained using the Tecan Ultra in absorbance mode at 340 nm. An assay concentration of 200 ⁇ M NADH was chosen to ensure linearity of the monitored change in signal in the assay. A concentration of the coupling enzymes PK/LDH that would ensure their excess was chosen. The assay was observed to be under saturating conditions of PK/LDH at concentrations above 1.3 units/mL in a reaction mixture containing 150 ⁇ M UMP, 400 ⁇ M ATP and 0.000036 mg/mL PyrH.
  • the stock could be stored on ice for at least 7 hours without significant loss of activity. Stability and solubility of the enzyme was enhanced in the presence of NTPs (ATP, GTP, or UTP). GTP was found to be required for full enzyme activity. Stability at room temperature of S. aureus UMP kinase was investigated under conditions of varying concentrations of GTP and Brij-35 in the working solution containing a concentration of enzyme that was 10X that of the assay concentration. It was found that 5 mM GTP and a concentration of Brij-35 between 0.01 and 0.02% (wt/vol) resulted in maximum stability and highest activity of the enzyme.
  • NTPs ATP, GTP, or UTP
  • the working solution conditions chosen were 50 mM HEPES pH 8.5, 100 mM KCl, 5 mM GTP and 0.01% Brij-35. Stability of S. aureus UMP kinase in the assay buffer was also investigated. The higher pH for the working stock solution further increased the stability of the enzyme. Working stock solution enzyme was added to the assay buffer and incubated at room temperature for up to 6 hours before initiating the reactions with UMP. It was found that the enzyme retained >90% activity after incubation in assay buffer for 6 h. References
  • Serina L., et al., Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochemistry, 1995. 34(15): p. 5066-74.

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Abstract

Cette invention se rapporte à une UMP kinase de Staphylococcus isolée ou purifiée, qui se caractérise en ce que, pendant le processus d'isolation ou de purification, l'enzyme est stabilisée par addition d'une espèce de stabilisation permettant de conserver l'enzyme pendant au moins une semaine à une température comprise entre -20 et 4 °C sans perte supérieure à 50 % de l'activité enzymatique spécifique, par rapport à son activité stabilisée initiale.
PCT/GB2004/002158 2003-05-22 2004-05-19 Procede pour isoler et purifier une ump kinase de staphylococcus WO2004104192A1 (fr)

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EP04733841A EP1629094A1 (fr) 2003-05-22 2004-05-19 Procede pour isoler et purifier une ump kinase de staphylococcus
JP2006530519A JP2007500512A (ja) 2003-05-22 2004-05-19 スタフィロコッカスｕｍｐキナーゼの単離または精製の方法

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GB0311789A GB0311789D0 (en) 2003-05-22 2003-05-22 Method
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055729A1 (fr) * 1998-04-27 1999-11-04 Smithkline Beecham Corporation POLYPEPTIDES ET POLYNUCLEOTIDES pyrH DE STAPHYLOCOCCUS AUREUS
US6403337B1 (en) * 1996-01-05 2002-06-11 Human Genome Sciences, Inc. Staphylococcus aureus genes and polypeptides
US20020119506A1 (en) * 2000-12-18 2002-08-29 Institut Pasteur Genes encoding UMP kinase, methods for purifying UMP kinase and methods of characterizing UMP kinase
WO2003029484A2 (fr) * 2001-09-28 2003-04-10 Pharmacia & Upjohn Company Procedes et materiaux antimicrobiens
WO2003035858A2 (fr) * 2001-10-25 2003-05-01 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides purifies impliques dans le traitement des acides nucleiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6403337B1 (en) * 1996-01-05 2002-06-11 Human Genome Sciences, Inc. Staphylococcus aureus genes and polypeptides
WO1999055729A1 (fr) * 1998-04-27 1999-11-04 Smithkline Beecham Corporation POLYPEPTIDES ET POLYNUCLEOTIDES pyrH DE STAPHYLOCOCCUS AUREUS
US20020119506A1 (en) * 2000-12-18 2002-08-29 Institut Pasteur Genes encoding UMP kinase, methods for purifying UMP kinase and methods of characterizing UMP kinase
WO2003029484A2 (fr) * 2001-09-28 2003-04-10 Pharmacia & Upjohn Company Procedes et materiaux antimicrobiens
WO2003035858A2 (fr) * 2001-10-25 2003-05-01 Affinium Pharmaceuticals, Inc. Nouveaux polypeptides purifies impliques dans le traitement des acides nucleiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAGYI CRISTINA ET AL: "UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 270, no. 15, August 2003 (2003-08-01), pages 3196 - 3204, XP002299396, ISSN: 0014-2956 *
SERINA L ET AL: "ESCHERICHIA COLI UMP-KINASE, A MEMBER OF THE ASPARTOKINASE FAMILY, IS A HEXAMER REGULATED BY GUANINE NUCLEOTIDES AND UTP", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 34, no. 15, 1995, pages 5066 - 5074, XP002921636, ISSN: 0006-2960 *

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