[go: up one dir, main page]

WO2012161688A1 - Essai de sulfotransférase - Google Patents

Essai de sulfotransférase Download PDF

Info

Publication number
WO2012161688A1
WO2012161688A1 PCT/US2011/037543 US2011037543W WO2012161688A1 WO 2012161688 A1 WO2012161688 A1 WO 2012161688A1 US 2011037543 W US2011037543 W US 2011037543W WO 2012161688 A1 WO2012161688 A1 WO 2012161688A1
Authority
WO
WIPO (PCT)
Prior art keywords
sulfotransferase
assay
phosphate
reaction
gpapp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/037543
Other languages
English (en)
Inventor
Zhenliang L. Wu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research and Diagnostics Systems Inc
Original Assignee
Research and Diagnostics Systems Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research and Diagnostics Systems Inc filed Critical Research and Diagnostics Systems Inc
Priority to PCT/US2011/037543 priority Critical patent/WO2012161688A1/fr
Publication of WO2012161688A1 publication Critical patent/WO2012161688A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase

Definitions

  • Sulfation is a common modification that affects the biological activity of a wide variety of substrates. Sulfation reactions are catalyzed by sulfotransferases. Sulfotransferases are a large group of enzymes that transfer a sulfate from a donor substrate to an acceptor. Many sulfotransferases exist in nature, but in humans, the sulfotransferases are of two types. Cystosolic sulfotransferases (SULTs) are mainly involved in modifying small molecules such as steroids, neurotransmitters, and xenobiotics, and in drug detoxification. Golgi- resident sulfotransferases are involved in modifying glycans and proteins on cell membranes and within the extracellular matrix. Sulfated glycans such as
  • glycosaminoglycans and numerous O- and N- glycans play roles in maintaining biochemical and biophysical properties.
  • Sulfated proteins such as leukocyte adhesion molecule PSGL-1, play roles in protein-protein and cellular interactions.
  • sulfotransferases may be ideal targets for drug intervention.
  • Assays for detecting sulfotransferase activity exist, but they have significant drawbacks.
  • Some sulfotransferase assays uses a radioisotope, 35 S. Such assays require separation steps such as HPLC, TLC, filter blinding, or electrophoresis to separate substrates from products. As such, in addition to the difficulties associated with the use of radioisotopes, the need for separation makes such assays very time consuming.
  • Other methods which can be used include mass spectrometry, fluorescent detection and colorimetric detection.
  • One fluorescent method uses 4-methylumbellifery sulfate as the ultimate sulfate donor and measures the fluorescence of 4-methylumbelliferone.
  • one colorimetric detection method uses p-nitrophenyl sulfate as the ultimate sulfate donor and measures the color intensity of the generated p-nitrophenol.
  • Embodiments of the invention include assays, methods and kits for detecting and quantifying sulfotransferase activity.
  • Embodiments of the invention use a phosphatase, Golgi-resident PAP-specific 3 '-phosphatase (gPAPP), which selectively removes a phosphate from the 3 '-phosphoadenosine-5' -phosphate (PAP) produced by the sulfotransferase reaction.
  • PAP 3 '-phosphoadenosine-5' -phosphate
  • the free phosphate released by gPAPP can then be detected and quantified, with the amount of free phosphate correlating to the activity of the
  • an assay for detecting activity of a sulfotransferase of interest which can be referred to as a test sulfotransferase, includes gPAPP and a free phosphate detector.
  • the free phosphate detector may be a colorimetric assay.
  • the free phosphate detector includes a first reagent including ammonium molybdate and the second reagent including malachite green oxalate.
  • the assay may further include a control sulfotransferase such as SULT1C4 or CHST3.
  • the assay may also include a free phosphate standard.
  • the assay may also include 3 '-phosphoadenosine-5 '-phosphosulfate (PAPS) and/or PAP.
  • the assay includes a buffer, which may include magnesium.
  • an assay for detecting activity of a test sulfotransferase includes gPAPP, a 5' nucleotidase, and a free phosphate detector.
  • the assay may further include PAPS and/or PAP.
  • the assay also includes a control
  • Embodiments of the invention also include methods for detecting and/or quantifying sulfotransferase activity.
  • the method includes combining a sulfotransferase, gPAPP, a substrate of the sulfotransferase, and PAPS under conditions to produce PAP as a first reaction, and measuring free phosphate.
  • the method may further include calculating sulfotransferase activity using the measured amount of free phosphate.
  • measuring the free phosphate include measuring optical density.
  • the method further includes conducting a second reaction, including combining the substrate of the sulfotransferase and PAPS with gPAPP in the absence of the sulfotransferase and measuring free phosphate, wherein the second reaction provides a background control for the first reaction (which included the sulfotransferase).
  • the method can include reducing the measured phosphate of the first reaction by the measured phosphate of the second reaction to calculate the amount of phosphate resulting from the sulfotransferase reaction.
  • measuring free phosphate includes applying a colorimetric free phosphate detection assay to the first reaction.
  • measuring free phosphate includes adding a first reagent including ammonium molybdate to the reaction, and then adding a second reagent including malachite green oxalate to the reaction.
  • a method of detecting sulfotransferase activity includes conducting a first reaction comprising combining a sulfotransferase, gPAPP, a 5 '-nucleotidase, a substrate of the sulfotransferase, and PAPS under conditions to produce PAP and measuring free phosphate.
  • Figure 1 is a representative sulfotransferase reaction
  • Figure 2 is a sulfotransferase reaction including release of free phosphate according to embodiments of the invention
  • Figure 3 is another sulfotransferase reaction including release of free phosphate according to embodiments of the invention.
  • Figure 4 is a graph of gPAPP activity versus MgCl 2 concentration
  • Figure 5 is a graph of the ratio of free phosphate released by gPAPP and/or CD73 (Pi) to the free phosphate released by TNAP (PITNAP) from 3'-AMP, 5'-AMP and PAP;
  • Figure 6 is a graph of phosphate released from PAPS and PAP versus gPAPP
  • Figure 7 is a graph of phosphate released from PAP by gPAPP in the presence or absence of PAPS versus PAP concentration
  • Figure 8 is a graph of the rate of phosphate release versus SULTl Al concentration under various conditions
  • Figure 9 is a graph of the activity of SULTl Al versus concentration of pNP
  • Figure 1 OA is a graph of activity of CHST3 versus concentration of CS
  • Figure 10B is a graph of activity of CHST3 versus concentration of PAPS.
  • Figure 11 is a graph of CHST3 activity versus CHST3 concentration.
  • Embodiments of the invention provide methods and assays for quickly and easily detecting and quantifying the activity of sulfotransferase enzymes. Embodiments of the invention are useful for high throughput testing of drugs to evaluate their effect upon sulfotransferases. Embodiments of the invention utilize the nearly universal use of PAPS as the sulfate donor in sulfotransferase reactions, with ultimate release and detection of the phosphate in the PAPS using a colorimetric assay.
  • a generic sulfation reaction is shown in Figure 1.
  • the sustrate is shown as R.
  • the sulfated product is shown as S-R.
  • the sulfate donor is PAPS, and this sulfate donor is shown.
  • the PAPS looses its sulfate and becomes PAP.
  • Each sulfation reaction therefore results in the production of 1 molecule of PAP.
  • Embodiments of the invention rely upon this one to one nature of the relationship between PAP production and sulfation, and the nearly universal nature of PAPS as the sulfate donor, to provide a method to detect and quantify sulfation as further described below. It can also be seen that the removal of the sulfate from PAPS results in the exposure of the 3' phosphate of PAP, which makes the 3' phosphate available for release by a gPAPP.
  • Figure 2 shows how embodiments of the invention can be used to assay sulfotransferase activity by utilizing the production of PAP from PAPS.
  • the sulfotransferase reaction proceeds as shown in Figure I, with the production of PAP in direct correlation to sulfotransferase activity.
  • the PAP then reacts with a PAP specific phosphatase, gPAPP, to release the 3' phosphate from the PAP and generate 1 molecule of 5-adenosine monophosphate (5' -AMP) and one molecule of free phosphate.
  • the quantity of the free phosphate produced can then be detected by one of various known free phosphate detectors, and this amount directly correlates to the activity of the sulfotransferase.
  • the free phosphate detector is a colorimetric assay, which is particularly useful for high throughput testing.
  • Embodiments of the invention may be used with any sulfotransferase which uses PAPS as the sulfate donor.
  • PAPS as the sulfate donor.
  • Examples of specific sulfotransferases which can be used in embodiments of the invention include carbohydrate sulfotransferases (such as CHSTl, CHST2, CHST3, CHST4, CHST5, CHST6, CHST7, CHST8, CHST9, CHST10, CHSTl 1, CHST12, CHS 13, CHS 14 and CHS 15), galactose-3-o-sulftotransferases (such as GAL3ST1, GAL3ST2, GAL3ST3, and GAL3ST4), heparin sulfate 2-O-sulfotransferases (such as HS2ST1), heparin sulfate 3-O-sulfotransferases (such as HS3ST1, HS3ST2, HS3ST3A1, HSEST3A2, HS3ST3B1, HS3ST3B2, HS3ST4, HS3ST5, and HS3ST6), heparin sulfate 6- O-sulf
  • Embodiments of the invention can also be used with microbial sulfotransferases, such as Nod factor H, a sulfotransferase of Rhizobium melioti involved in establishing nitrogen-fixing symbiosis between rhizobia and leguminous plants, and StaL, a glycopeptide antibiotic sulfotransferase from Nod factor H, a sulfotransferase of Rhizobium melioti involved in establishing nitrogen-fixing symbiosis between rhizobia and leguminous plants, and StaL, a glycopeptide antibiotic sulfotransferase from
  • the substrate used in embodiments of the invention is any substrate which is acted upon by the sulfotransferase used in the assay.
  • the substrate may be a protein, carbohydrate, lipid or steroid, for example.
  • Other examples of substrates include peptides,
  • oligosaccharides drugs and xenobiotics.
  • sulfation by cytosolic sulfation by cytosolic
  • sulfotransferases can be one step in the metabolism of certain drugs or xenobiotics.
  • a drug or xenobiotic may be used as a substrate and the assay may be used to analyze the rate of sulfation of the drug or xenobiotic.
  • a drug or xenobiotic may be used as a substrate and an additional agent may be added to the reaction, such as a potential promoter or inhibitor of the sulfotransferase, to determine the effect of the additional agent upon the metabolism of the drug or xenobiotic by the sulfotransferase.
  • Embodiments of the invention take advantage of the specificity of gPAPP as a coupling phosphatase.
  • PAP and PAPS include phosphate moieties that could be removed by phosphatases.
  • gPAPP is specifically active on PAP and not on PAPS. As such, all phosphate released by gPAPP is from PAP and not from PAPS. Because PAP is produced by the sulfotransferase reaction, the production of PAP directly correlates to the activity of the sulfotransferase. The specificity of gPAPP therefore allows phosphate to be released from PAP only, and this phosphate can then be measured, such that the measured amount of phosphate directly correlates to the activity of the sulfotransferase.
  • the phosphatase gP APP may be isolated from naturally occurring sources or may be produced recombinantly.
  • the phosphatase gPAPP useful in embodiments of the invention also include any homologue proteins from different organisms and any mutational variations described herein.
  • On method of obtaining gPAPP which may be used in embodiments of the invention is by using recombinant mouse gPAPP from E51 to K356 (Gene ID: 242291) which can be expressed in CHO cells as anN-terminal His-tagged recombinant protein and purified using nickel affinity resin and Superdex-200 from GE Healthcare (Pittsburgh, PA). Further description of how to make gPAPP which can be used in embodiments of the invention can be found in Frederick, J.
  • PAPS One method of obtaining PAPS which can be used in embodiments of the invention it by using recombinant S. cerevisiae ATP sulfurylase and P.chrysogenum APS kinase, then purigying the PAPS using a DEAE Sepharose fast-flow column from GE Healthcare, for example. Further description of how to produce PAPS which may be used in
  • PAP such as including PAP
  • PAP which can be used in embodiments of the invention is commercially available from Sigma Aldrich (St Louis, MO).
  • the free phosphate may be readily detected and/or measured by any means.
  • Several methods are known for measuring free phosphate, any of which may be used.
  • the free phosphate may be detected and/or measured using a colorimetric assay. Examples of colorimetric assays for measurement of free phosphate which may be used in embodiments of the invention include the Malachite Green
  • the free phosphate may be detected and/or measured by
  • free phosphate may be selectively detected by a fluorescent sensor as described in U.S. Pat. No. 7,521,250, the disclosure of which is hereby incorporated by reference.
  • free phosphate may be detected using a recombinant E. coli phosphate-binding protein labeled with the fluorophore MDCC known as Phosphate Sensor and available from Invitrogen (Carlsbad, CA).
  • the Malachite Green Phosphate Detection Kit is one method that may be used to detect free phosphate and is based on the malachite green-molybdate binding reaction, and the kit itself, or the components or variations thereof, may be used in embodiments of the invention.
  • the Malachite Green assay includes a first reagent, Malachite Green Reagent A, which includes ammonium molybdate and sulfuric acid, and a second reagent,
  • Malachite Green Reagent B which includes malachite green oxalate and polyvinyl alcohol.
  • the Malachite Green assay further includes a phosphate standard, KH 2 P0 4 .
  • the phosphate standard may be used to create a standard curve of absorbance at 620 nm for interpretation of sample assay results.
  • the use of the assay includes incubating a sample with Malachite Green Reagent A for 10 minutes at room temperature, then adding
  • the Malachite Green Phosphate Detection kit itself, or components or variations thereof, may therefore be used to detect levels of free phosphate released from PAP, according to embodiments of the invention.
  • PAPS, substrate, sulfotransferase, assay buffer, and gPAPP are combined to produce a sample for testing.
  • the sample may further include an additional component or test agent, such as a potential sulfotransferase inhibitor or promoter.
  • the resulting sample may be combined with Malachite Green Reagent A to stop the reaction, then incubated 10 minutes, and then combined with Malachite Green Reagent B and incubated an additional 20 minutes as described above.
  • the absorbance may then be read at 620 nm using a spectrometer, and the reading may be correlated to a phosphate standard curve and/or a control (including all reaction components except the
  • sulfotransferase enzyme to determine the amount of free phosphate released by gPAPP. This amount may be compared to the initial quantity of PAPS and/or sulfotransferase present in the sample to determine the activity of the sulfotransferase. When a test agent is used, this amount may be compared to the amount of phosphate produced in a reaction including all of the same components except the test agent, to determine the effect of the test agent upon the sulfotransferase activity.
  • the PAP produced by the sulfatase reaction includes two phosphate molecules, which are known as the 3' and 5' phosphates.
  • the phosphatase gPAPP is not only specific for PAP (as compared to PAPS) but also is specific for the 3' phosphate of PAP, such that gPAPP only releases the 3' phosphate from PAP while the 5' phosphate is unaffected.
  • the 5' phosphate remains bound to the PAP and is not detected by the free phosphate detector.
  • the 5' phosphate can also be removed from PAP through the use of an additional specific phosphatase (a 5 '-nucleotidase).
  • reaction An example of a reaction in which two phosphates are released is shown in Figure 3.
  • the first portion of this reaction is the same as the reaction shown in Figure 2, with PAPS converted to PAP during the sulfotransferase reaction, and gP APP releasing one phosphate (the 3' phosphate) from each PAP produced. Removal of the 3' phosphate from PAP produces 5 '-AMP.
  • the reaction also includes a 5 '-nucleotidase which removes the remaining phosphate from 5 'AMP to produce an additional molecule of phosphate and an adenosine for each sulfotransferase reaction.
  • 5 '-nucleotidases examples include
  • 5 '-nucleotidase IB (NT5C1B), cytosolic 5 '-nucleotidase II (NT5C2), cytosolic 5'- nucleotidase III (NT5C3), NT5C3L, NT5DC4.
  • the 5'-nucleotideases may be isolated from naturally occurring sources or may be produced recombinantly and many are commercially available.
  • the 5 '-nucleotidases useful in embodiments of the invention also include any homologue proteins from different organisms and any mutational variations of any of the phosphatases described herein.
  • PAP has been reported to be a potent inhibitor of sulfotransferases and as such has the potential to inhibit the same reaction which is being assayed and in which it is produced. This aspect of sulfotransferase reactions makes development of an assay more complicated. However, because embodiments of the invention promptly degrade PAP to 5 '-AMP using gPAPP, this potential inhibition does not occur. As such, in addition to providing a way to detect and quantify sulfotransferase reactions, embodiments of the invention avoid the inhibition of sulfotransferase reactions which can be caused by the production of PAP.
  • Embodiments of the invention include assays, kits and methods for detecting and measuring sulfotransferase activity.
  • the assay includes one or more of the following components: PAPS; gPAPP; a buffer; and a control sulfotransferase.
  • the assay may optionally include free phosphate detection reagents, such as a first reagent comprising molybdate and a second reagent comprising malachite green.
  • the assay may also include PAP.
  • the kit may include gPAPP and a free phosphate detection assay.
  • the kit may include PAPS and gPAPP.
  • the kit may include gPAPP, a free phosphate detection assay, and PAPS.
  • the kit may further include an assay buffer, PAP and/or a phosphate standard.
  • the phosphate detection assay may be a Malachite Green detection assay.
  • the PAPS and/or PAP may be supplied in the assay buffer.
  • the gPAPP may also be supplied in the assay buffer.
  • the sulfotransferase assay kit includes an assay buffer, gPAPP, PAPS, Malachite Green Reagent A, Malachite Green Reagent B, and a phosphate standard, such as K3 ⁇ 4P0 4 .
  • the kit may further include a sulfotransferase to be used as a positive control for the various components of the kit.
  • kits further includes a 5' -nucleotidase.
  • a sulfotransferase may be provided as a control in the kit, or a control sulfotransferase may be supplied by the user of the kit.
  • the control sulfotransferase serves to assure proper functioning of the assay.
  • the assay may be performed using the control sulfotransferase and the results may be compared to known expected results for the sulfotransferase. If the results are within the expected range, the assay can be considered to be functioning properly. In this way, when the assay is performed using a sulfotransferase of interest, the results may be considered reliable.
  • a control sulfotransferase provided in a kit is preferably stable over time and has a known activity, and the data regarding the control sulfotransferase activity and expected results may be provided with the kit.
  • Examples of sulfotransferases which may be provided in kits to serve as controls include any stable sulfotransferase having a high specific activity and an available substrate, such as
  • the use of the kit may include first creating a free phosphate standard curve.
  • the phosphate standard curve may be created using serial dilution of a phosphate standard in the assay buffer, and followed by measuring the absorbance using a phosphate detection reagents. For example, each dilution may be combined with
  • a phosphate standard curve may be produced.
  • the phosphate standard curve may be made using serial dilutions, such as 2-fold serial dilutions, of a phosphate solution such as the phosphate standard, in the assay buffer.
  • serial dilutions may be as in Table 1, below.
  • the serial phosphate dilutions may be added to a clear 96-well plate and may be performed in triplicate.
  • the Malachite Green Reagent A is first added to each well, followed by the Malachite Green Reagent B. After 20 minutes, the optical density (OD) is read at 620 nm for each well using a microplate reader or spectrophotometer. The average OD for each dilution may be obtained.
  • the phosphate input may be plotted against the OD, or the average of the OD for each dilution, to create a standard curve, such as by using linear regression or a computer generated four parameter logistic (4-PL) curve fit.
  • a similar curve may be obtained using other free phosphate detection methods or other phosphate sources.
  • the slope of the linear regression line may be used as the conversion factor, i.e. the amount of phosphate corresponds to an absorbance unit. This conversion factor may then be used to calculate the amount of free phosphate from the measured absorbance for each reaction.
  • a single buffer is used which is the assay buffer.
  • the assay buffer should allow the sulfotransferase, and preferably also gPAPP and the 5 '-nucleotidase, if used, to function normally.
  • the assay buffer may contain about 10 mM MgCl 2 and may have a pH of about 7.0 to about 8.0.
  • the assay buffer may comprise 25 mM Tris and 10 mM MgCl 2 at pH 7.5.
  • two or more buffers may be used.
  • the first buffer may be an assay buffer and the second buffer may be a phosphatase buffer.
  • the phosphatase may be Mg dependent, and therefore the phosphatase buffer should include Mg + , while the assay buffer might not have this component.
  • the buffer and other reagents have divalent cations such as calcium, magnesium and manganese.
  • gPAPP (and the 5 '-nucleotidase, if used) may be active in the assay buffer used with or provided with the assay. In such embodiments, gPAPP may be combined with the PAPS, the substrate, and the
  • gPAPP is not active in the assay buffer used with or provided with the assay.
  • PAPS, the substrate, and the sulfotransferase may first be combined in a first buffer which is the assay buffer and allowed to react.
  • a second buffer which is the gPAPP buffer may then be added after the completion of the first reaction.
  • the gPAPP may be added with the gPAPP buffer or may be added after the addition of a sufficient amount of the gPAPP buffer.
  • the gPAPP buffer may be stronger than the sulfotransferase assay buffer, such that the conditions provided by the gPAPP buffer will overwhelm those provided by the assay buffer, with the resulting mixture being more similar to the gP APP buffer and therefore being favorable for gPAPP activity.
  • the assays described herein do not involve radioisotope usage and do not require chemical separation, like previous methods. Furthermore, the assays described herein can be performed using multi-well high throughput techniques.
  • Embodiments of the invention further include methods of detecting and/or quantifying the activity of a sulfotransferase.
  • the method includes performing a first reaction including combining a sulfotransferase, which may be considered a test sulfotransferase, with a substrate of the sulfotransferase and PAPS in a reaction buffer.
  • the method further includes combining the reactants with gPAPP.
  • the gPAPP may be combined simultaneously or approximately simultaneously with the other reactant.
  • the sulfotransferase, substrate and PAPS are combined first and allowed to react, and then the gPAPP is added to the reaction after the reaction has progressed for a certain amount of time, such as about 20 minutes.
  • the method includes performing a first reaction including combining a sulfotransferase which is a test sulfotransferase with a substrate of the sulfotransferases and PAPS in a reaction buffer.
  • the method further includes combining the reactants with gPAPP and a 5 '-nucleotidase.
  • the gPAPP and the 5 '-nucleotidase may be combined simultaneously or approximately simultaneously with the other reactants.
  • the gPAPP and the 5 'nucleotidase are combined with the other reactants after the sulfotransferase reaction has progressed for a certain amount of time, such as about 20 minutes.
  • the gPAPP is added to the reaction first either simultaneously or after allowing the sulfotransferase reaction to progress and then the 5 '-nucleotidase is added after allowing time for the gPAPP to react.
  • the next step in any of the embodiment is measuring the level of free phosphate.
  • the reaction is tested using a free phosphate detector such as the Malachite Green assay as described above.
  • malachite reagent A is added first, followed by the addition of malachite reagent B.
  • measuring the level of free phosphate may include measuring OD and comparing the measured OD to a free phosphate standard curve or applying a conversion factor which may be determined using a free phosphate standard curve to the measured OD, to convert the measured OD to a free phosphate level.
  • the method may further include preparing the free phosphate standard curve.
  • the method may further include performing a second reaction which is a control reaction.
  • performing the control reaction includes combining each of the reactants as in the first reaction (the test reaction) in the same manner and under the same conditions as the test reaction but in the absence of the sulfotransferase.
  • performing the second reaction may include combining the substrate of the sulfotransferase with PAPS, and either concurrently or subsequently adding gPAPP, depending upon how the test reaction was performed. If used in the test reaction, the 5 '-nucleotidase would also be combined with the reactants, in the same manner as in the test reaction.
  • the method further includes measuring the level of free phosphate in the second reaction, and this step would be performed in the same manner as in the test reaction. Finally, the method may include subtracting the measured free phosphate of the second reaction from the measured free phosphate of the first reaction.
  • the results can be adjusted to compensate for any free phosphate, PAP and 5'- AMP that may be present in the reagents and are not caused by the sulfotransferase reaction.
  • some amount of PAPS may spontaneously degrade to PAP even in the absence of a sulfotransferase, making the PAP available to the gPAPP and resulting in some amount of free phosphate.
  • By adjusting (reducing) the level of the free phosphate measured in the sulfotransferase test reaction by the amount of free phosphate present in the control a more accurate measurement of sulfotransferase activity can be obtained which does not depend upon the stability of PAPS.
  • the assay is used to evaluate the effect of a test agent on the sulfotransferase.
  • the sulfotransferase, substrate, PAPS and test agent are combined in a test reaction, and gPAPP may be added simultaneously or subsequently.
  • a 5 'nucleotide may also be added to the reaction.
  • the phosphate level is measured as in the other methods.
  • the phosphate level (or the activity of the sulfotransferase which may be determined using the phosphate level) is compared to the phosphate level (or sulfotransferase activity) for a separate reaction (a control reaction) including the same reactants but without the test agent.
  • the control reaction may either be performed or its values (phosphate level and/or sulfotransferase activity) may already be known and used for comparison to the test reaction.
  • the gPAPP used was recombinant mouse gPAPP expressed in CHO cells as an N-terminal His-tagged recombinant protein containing amino acids from E51 to K356, created by R & D Systems.
  • the sequence was based on accession number NP 808398 from the National Center for Biotechnology Information.
  • the chondroitin sulfate (CS) and ?-nitrophenol (pNP) were obtained from Sigma Aldrich.
  • the PAPS was prepared by R & D Systems using recombinant S. cerevisiae ATP sulurylase and P.
  • chrysogenum APS kinase was also prepared by R & D Systems.
  • the activity of gPAPP in the presence of Mg 2+ was tested by combining 0.025 ⁇ g gPAPP, 25 nmol PAP and increasing amounts of Mg 2+ in 50 ⁇ , of assay buffer of 25 mM Tris at H 7.5.
  • the results are shown in Figure 4.
  • gPAPP was found to be active in the presence of Mg 2+ , and a maximum activity was achieved when the Mg 2+ concentration was greater than 10 mM.
  • the magnesium dependence of gPAPP is consistent with its localization in the Golgi apparatus and the presence of magnesium transporters in the Golgi apparatus.
  • the activity of gPAPP was evaluated at various pH levels.
  • gPAPP The activity of gPAPP in the presence of 10 mM Mg 2+ was tested by combining 0.025 ⁇ g gPAPP, 25 nmol PAP in 50 ⁇ , of buffers at different pH levels. It was found that gPAPP was active at a pH from 4.0 to 9.0, with optimal activity around pH 7.5.
  • gPAPP tissue-non-specific alkaline phosphatase
  • gPAPP can be used as a coupling phophatase in sulfotransferase assays to release the 3 '-phosphate from PAP, and that CD73 can be used as a secondary coupling phosphatase to further release the 5 '-phosphate from 5 '-AMP produced in a gPAPP- coupled sulfotransferase reaction.
  • the coupling capacity of gPAPP was determined.
  • the coupling capacity of a coupling enzyme is the amount of product that can be completely converted into signal by one ⁇ g of the enzyme under specific conditions.
  • the coupling capacity is the amount of PAP (product) that can be converted by gP APP into signal
  • the coupling capacity of gPAPP was determined by combining 1 ⁇ g gPAPP with increasing amounts of PAP, in the presence or absence of 0.5 mM PAPS in 50 assay buffer containing 10 mM Mg 2+ at pH 7.5 for either 20 or 40 minutes. The released phosphate was then plotted versus PAP input as shown in Figure 7. It was found that 1 ⁇ g PAPP was able to complete the hydrolysis of 8 nmol and 15 nniol of PAP in 20 and 40 minutes, respectively. The coupling capacity for gPAPP was therefore 8 nmol in a 20 minute reaction and 15 nmol in a 40 minute reaction.
  • ?-nitrophonol pNP
  • pNP ?-nitrophonol
  • SULT1 Al is known to have a high affinity for pNP, with a half maximal velocity (Km) reported below 1 ⁇ . Measuring the Km of SULT1 Al for pNP therefore requires a highly sensitive test. Because of this, CD73 was used in the reaction in addition to gPAPP, to increase the assay sensitivity.
  • Carbohydrate sulfotransferase 3 is a sulfotransferase which is known to catalyze the sulfation of N-acetylgalactosamine at the 6-0 position.
  • CHST3 Carbohydrate sulfotransferase 3
  • 1 g CHST3 was combined with 1 ⁇ g gPAPP and increasing concentrations of CS in 50 ⁇ assay buffer containing 0.8 mM PAPS, 10 mM MgCl 2 at pH 7.5.
  • the optimal concentration for CS was visually determined to be > 5 mg/ml and the optimal concentration for PAPS was visually determined to be about 0.5 mM using Figs. 10A and 10B by finding the substrate concentration corresponding to the highest point in the curves. An enzyme dose curve was then created for CHST3 at the optimal
  • Embodiments of the invention includes various aspects, some of which are described in the numbered paragraphs below.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP and a free phosphate detector.
  • the free phosphate detector may be a colorimetric assay, such as a free phosphate detector including a first reagent and a second reagent, wherein the first reagent comprises ammonium molybdate and the second reagent comprises malachite green oxalate.
  • the assay may further include a source of free phosphate for use as a phosphate standard and/or PAPS and/or PAP.
  • the assay may further include a buffer, which may include magnesium.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP and a control sulfotransferase.
  • the control sulfotransferase may be SULT1C4 or CHST3, for example.
  • the assay may further include a source of free phosphate for use as a phosphate standard and/or PAPS and/or PAP.
  • the assay may further include a buffer, which may include magnesium.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP; a control sulfotransferase; and a free phosphate detector.
  • the free phosphate detector may be a colorimetric assay, such as a free phosphate detector including a first reagent and a second reagent, wherein the first reagent comprises ammonium molybdate and the second reagent comprises malachite green oxalate.
  • the control sulfotransferase may be
  • the assay may further include a source of free phosphate for use as a phosphate standard and/or PAPS and/or PAP.
  • the assay may further include a buffer, which may include magnesium.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP; a 5' nucleotidase; and a free phosphate detector.
  • the assay may further include PAPS and/or PAP.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP; a 5' nucleotidase; and a control sulfotransferase.
  • the assay may further include PAPS and/or PAP.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP; a 5' nucleotidase; a control sulfotransferase, and a free phosphate detector.
  • the assay may further include PAPS and/or PAP.
  • An assay for detecting activity of a test sulfotransferase comprising: gPAPP, a control sulfotransferase, a free phosphate detector, PAPS and a buffer.
  • the assay may further include PAP and/or a 5' nucleotidase.
  • a method of detecting sulfotransferase activity comprising conducting a first reaction comprising: combining a sulfotransferase, gPAPP, a substrate of the
  • the first reaction can further include a buffer, which may include magnesium.
  • Measuring free phosphate includes applying a colorimetric assay to the first reaction and measuring optical density.
  • a method of detecting sulfotransferase activity comprising conducting a first reaction comprising: combining a sulfotransferase, gPAPP, a substrate of the
  • the first reaction can further include a buffer, which may include magnesium.
  • Measuring free phosphate includes applying a colorimetric assay to the first reaction and measuring optical density.
  • a method of detecting sulfotransferase activity comprising conducting a first reaction and a second reaction.
  • the first reaction comprises combining a sulfotransferase, gPAPP, a substrate of the sulfotransferase, and PAPS under conditions to produce PAP; and measuring free phosphate.
  • Measuring free phosphate includes applying a colorimetric assay to the first reaction and measuring optical density.
  • the first reaction can further include a buffer, which may include magnesium.
  • the second reaction comprises combining the substrate of the sulfotransferase and the PAPS with the phosphatase in the absence of the sulfotransferase and measuring free phosphate, such as under the same conditions as the first reaction (except for the absence of sulfotransferase), wherein the second reaction provides a background control for the first reaction.
  • the second reaction can include the same buffer as the first reaction.
  • the method can further include reducing the measured phosphate of the first reaction by the measured phosphate of the second reaction to calculate the amount of phosphate correlated to the sulfotransferase reaction.
  • a method of detecting sulfotransferase activity comprising conducting a first reaction comprising: combining a sulfotransferase, gPAPP, 5 '-nucleotidase, a substrate of the sulfotransferase, and PAPS under conditions to produce PAP; and measuring free phosphate.
  • Measuring free phosphate includes applying a colorimetric assay to the first reaction and measuring optical density.
  • the reaction can further include a buffer, which may include magnesium.
  • Measuring free phosphate includes applying a colorimetric assay to the first reaction and measuring optical density.
  • the method can further include performing the same reaction in the absence of the agent, and comparing the results of the two reactions to determine the effect of the agent upon sulfotransferase activity.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne des essais et des procédés de détection et/ou de quantification d'un essai de sulfotransférase comprenant la libération d'un phosphate libre à partir de PAP produit par la réaction de la sulfotransférase et la détection du phosphate libre. L'essai peut inclure gPAPP, un détecteur de phosphate libre et une sulfotransférase de contrôle facultative. Le procédé inclut la combinaison d'un essai de sulfotransférase avec le substrat de la sulfotransférase, PAPS et gPAPP et la détection du phosphate libre. Le taux de phosphate libre est directement corrélé à l'activité de la sulfotransférase. L'essai et les procédés peuvent être utilisés pour cribler des agents quant à leur effet sur l'activité de la sulfotransférase.
PCT/US2011/037543 2011-05-23 2011-05-23 Essai de sulfotransférase Ceased WO2012161688A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2011/037543 WO2012161688A1 (fr) 2011-05-23 2011-05-23 Essai de sulfotransférase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2011/037543 WO2012161688A1 (fr) 2011-05-23 2011-05-23 Essai de sulfotransférase

Publications (1)

Publication Number Publication Date
WO2012161688A1 true WO2012161688A1 (fr) 2012-11-29

Family

ID=44626835

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/037543 Ceased WO2012161688A1 (fr) 2011-05-23 2011-05-23 Essai de sulfotransférase

Country Status (1)

Country Link
WO (1) WO2012161688A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030109501A1 (en) * 2001-10-05 2003-06-12 Guchen Yang High throughput assay for N-sulfotransferase activity of glucosaminyl N-deacetylase/N-sulfotransferases
US20030211562A1 (en) * 2002-05-13 2003-11-13 Carolyn Bertozzi Method for identifying modulators of sulfotransferase activity
WO2006017342A2 (fr) * 2004-07-13 2006-02-16 Y's Therapeutics, Inc. Procédés de mesure de l’activité enzymatique associée à la sulfatation
EP1923402A1 (fr) * 2005-06-28 2008-05-21 Seikagaku Corporation Procédé de détermination de l'activité enzymatique
US20080233592A1 (en) * 2003-01-30 2008-09-25 Robert Lowery Assay Method for Group Transfer Reactions
US7521250B2 (en) 2002-02-22 2009-04-21 Japan Science And Technology Agency Fluorescent sensor for phosphate ion and phosphorylated peptide

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030109501A1 (en) * 2001-10-05 2003-06-12 Guchen Yang High throughput assay for N-sulfotransferase activity of glucosaminyl N-deacetylase/N-sulfotransferases
US7521250B2 (en) 2002-02-22 2009-04-21 Japan Science And Technology Agency Fluorescent sensor for phosphate ion and phosphorylated peptide
US20030211562A1 (en) * 2002-05-13 2003-11-13 Carolyn Bertozzi Method for identifying modulators of sulfotransferase activity
US20080233592A1 (en) * 2003-01-30 2008-09-25 Robert Lowery Assay Method for Group Transfer Reactions
WO2006017342A2 (fr) * 2004-07-13 2006-02-16 Y's Therapeutics, Inc. Procédés de mesure de l’activité enzymatique associée à la sulfatation
EP1923402A1 (fr) * 2005-06-28 2008-05-21 Seikagaku Corporation Procédé de détermination de l'activité enzymatique

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Toxicology", part Unit 4.5 1 January 2001, JOHN WILEY & SONS, INC., Hoboken, NJ, USA, ISBN: 978-0-47-114085-6, article JONATHAN J. SHENG ET AL: "Measurement of Aryl and Alcohol Sulfotransferase Activity", XP055016337 *
BRADFORD, M. M.: "A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle ofprotein-dye binding", ANAL BIOCHEM., vol. 72, 1976, pages 248 - 254
CHEN W-T ET AL: "Fluorometric assay for alcohol sulfotransferase", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS INC, NEW YORK, vol. 339, no. 1, 1 April 2005 (2005-04-01), pages 54 - 60, XP004781299, ISSN: 0003-2697, DOI: 10.1016/J.AB.2004.12.016 *
FREDERICK J. P. ET AL.: "A role for a lithium-inhibited Gulgi nucleotidase in skeletal development and sulfation", PROC NATL ACAD SCI U SA., vol. 105, 2008, pages 11605 - 11612
WU ZHENGLIANG L ET AL: "A versatile polyacrylamide gel electrophoresis based sulfotransferase assay", BMC BIOTECHNOLOGY, BIOMED CENTRAL LTD. LONDON, GB, vol. 10, no. 1, 10 February 2010 (2010-02-10), pages 11, XP021067394, ISSN: 1472-6750 *
WU, Z. L. ET AL.: "A versatile polyacrylamide gel electrophoresis based sulfotransferase assay", BMC BIOTECHNOLOGY, vol. 10, pages 11, XP021067394

Similar Documents

Publication Publication Date Title
CN101900735B (zh) 用于测定高半胱氨酸的方法
JPH0642839B2 (ja) 体液中のカリウムイオンを測定する方法及びそのための組成物
EP2235201A1 (fr) Procédés et compositions pour le dosage de l'homocystéine
Pazmiño et al. Thiol S‐methylation in uremia: Erythrocyte enzyme activities and plasma inhibitors
US9096888B2 (en) Sulfotransferase assay
US20140273036A1 (en) Luminescent detection of inorganic phosphate and coupled reactions
EP2771480B1 (fr) Procédés de détection d'adénosine monophosphate dans des échantillons biologiques
Prather et al. Golgi-resident PAP-specific 3′-phosphatase-coupled sulfotransferase assays
CN111057746B (zh) 一种肌酸激酶同工酶测定试剂盒
Smith et al. An Adenosine Triphosphate-dependent Deoxyribonuclease from Hemophilus influenzae Rd: II. ADENOSINE TRIPHOSPHATASE PROPERTIES
WO2012161688A1 (fr) Essai de sulfotransférase
CN102575281B (zh) 脱氢酶或对应的底物的测定方法及测定用试剂盒
Ogawa et al. A new enzymatic method for the measurement of creatinine involving a novel ATP-dependent enzyme, N-methylhydantoin amidohydrolase
JP6522466B2 (ja) オートタキシンの活性測定方法
JP6487711B2 (ja) プリンヌクレオシドホスホリラーゼを用いた、オルトリン酸、アルカリホスファターゼ、及びピロリン酸等の新規な測定方法、並びに組成物
CN111139284B (zh) 一种高准确度的5’-核苷酸酶测定试剂盒
CN113957120B (zh) 一种胆碱酯酶测定试剂盒
CN110684822A (zh) 一种基于丙酮酸激酶检测样品中微生物的方法及试剂盒
US20250236866A1 (en) Euglobulin-based method for determining the biological activity of defibrotide
Bovet et al. Determination of active enzyme concentration using activity-based probes and direct mass spectrometric readout
Sillero et al. Polyphosphates strongly inhibit the tRNA dependent synthesis of poly (A) catalyzed by poly (A) polymerase from Saccharomyces cerevisiae
JPH04252200A (ja) Ｎａｄｈキナーゼを用いる高感度定量法
EP1874953B1 (fr) Procedes de determination de l'activite d'une phosphoryltransferase au moyen d'adenosine 5'-triphosphate (atp)-gamma-s
JP2003274996A (ja) 夾雑物質による影響回避方法
AU662516B2 (en) Determination of ions in fluids

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11724343

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11724343

Country of ref document: EP

Kind code of ref document: A1