hymecromone and 6-8-difluoro-4-methylumbelliferyl-phosphate

Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

Topics: Fluoresceins; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Nitrophenols; Organophosphorus Compounds; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases

Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

Topics: Amino Acid Sequence; Binding Sites; Biocatalysis; Carrier Proteins; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors; Humans; Hymecromone; Kinetics; Marine Toxins; Metals; Models, Molecular; Mutation; Oxazoles; Phosphoprotein Phosphatases; Protein Domains; Protein Structure, Secondary; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Sulfates; Transcription Factors

Hydrogen sulfide (H2S) is known to induce persulfidation of protein thiols. However, the process of H2S-induced persulfidation is not fully understood as it requires an additional oxidant. There are several mechanistic possibilities and it is of interest to determine which pathway is kinetically most relevant. Here, we detail in vitro assays for the real-time monitoring of thiol redox states in two model proteins with oxidizable cysteines, PTEN, and roGFP2. These allow kinetic measurements of the response of defined protein thiols (or disulfides) to sulfide and sulfane sulfur species. The combination of these assays with cold cyanolysis reveals the role of intermediary sulfane sulfur species in H2S-induced protein thiol oxidation.

Topics: Cysteine; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Hydrogen Sulfide; Hymecromone; Kinetics; Oxidation-Reduction; PTEN Phosphohydrolase; Recombinant Proteins; Signal Transduction; Sulfides; Thiocyanates

Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control.. We conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production.. The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel targets to block egg formation in mosquitoes.

Topics: Aedes; Amino Acid Sequence; Animals; Fat Body; Female; Gene Expression Regulation; Genome, Insect; Hymecromone; Models, Molecular; Molecular Sequence Data; Ovary; Oviposition; Protein Structure, Tertiary; Protein Tyrosine Phosphatase, Non-Receptor Type 1; RNA, Small Interfering; Sequence Alignment; Sequence Homology, Amino Acid; Vanadates; Vitellogenins

Altered function of the protein tyrosine phosphatase (PTP) Lyp (PTPN22) has been implicated in the pathogenesis of a number of human diseases, and so accurate assessment of its functional activity is needed to further our understanding of its biology. We have developed an in vitro method to measure the specific catalytic activity of the Lyp phosphatase. Lyp is captured from cell lysates using an anti-Lyp monoclonal antibody coated 96-well plate, and activity measured by dephosphorylation of a fluorescent substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). The amount of protein is measured using an anti-Lyp HRP conjugate, with reference to a standard curve generated with purified Lyp. These two measurements are then used to calculate the specific phosphatase activity. We used this assay to show that the specific activity of the Lyp phosphatase is decreased by H(2)O(2) in Jurkat T cells and primary CD4+ T cells. We also modified this assay to measure the specific activity of CD45, the other main PTP regulating T cell receptor (TCR) signalling, in order to compare the relative susceptibility of CD45 and Lyp to oxidation by H(2)O(2). By measuring specific activity in Jurkat T cells and primary CD4+ T cells, we demonstrated that CD45 is more susceptible to oxidation by H(2)O(2) when compared to Lyp. Reduced function of CD45 and Lyp has been associated with human immune mediated inflammatory diseases, and a differential susceptibility to oxidation could be an important regulatory mechanism associated with both physiological and pathological changes in signalling through the TCR.

Topics: CD4-Positive T-Lymphocytes; Fluorometry; Humans; Hymecromone; Jurkat Cells; Leukocyte Common Antigens; Protein Tyrosine Phosphatase, Non-Receptor Type 22; Substrate Specificity

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with (19)Flourine magnetic resonance spectroscopic imaging ((19)FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using (19)Fluorine magnetic resonance spectroscopy ((19)FMRS) and (19)FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. (19)FMRS and (19)FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. (19)FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized (19)FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of (19)FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, (19)FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.

Topics: Alkaline Phosphatase; Animals; Bone and Bones; Cattle; Cell Line; Extracellular Space; Hydrolysis; Hymecromone; Magnetic Resonance Spectroscopy; Mice; Molecular Imaging; Rats

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.

Topics: Alkaline Phosphatase; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Gels; Hymecromone; Male; Nuclear Proteins; Phosphoprotein Phosphatases; Protein Phosphatase 2C; Rats; Rats, Wistar; Recombinant Proteins

Protein serine/threonine phosphatase (PP2A) is a major cellular enzyme implicated in the control of numerous signaling processes. The accurate measurement of PP2A activity in crude cell lysates, immune complexes, and purified preparations provides insight into the function and regulation of this essential enzyme, which, in turn, can lead to a better understanding of the signaling pathways that it modulates. The method presented here utilizes 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) and a FLEXstation for the continuous measure of PP2A activity associated with many different protein preparations. This automated fluorescence-based assay offers several distinct advantages over colorimetric and radioactive assays of phosphatase activity including (1) decreased substrate preparation time, (2) real-time kinetic data, (3) high sensitivity, and (4) the capability to analyze a wide variety of phosphatases.

Topics: Fluorescence; Hymecromone; Molecular Biology; Phosphoric Monoester Hydrolases; Reproducibility of Results

The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor constants for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates, para-nitrophenylphosphate and phosphotyrosine-containing peptides, respectively. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-molecule inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.

Topics: Drug Evaluation, Preclinical; Fluorescent Dyes; Hymecromone; Kinetics; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Sensitivity and Specificity

A number of aromatic substrates were evaluated for their ability to detect tyrosine phosphatase and serine/threonine phosphatase activity. Results demonstrated that the fluorinated coumarin DiFMUP is the most sensitive substrate for detecting LAR and PP-2A activity. Using this substrate, selective high-throughput screening assays for serine/threonine and tyrosine phosphatases were developed. Specific inhibitor cocktails were added to each assay to limit the activity of other phosphatases. LAR, CD-45, and PTP-1B all rapidly hydrolyze DiFMUP in the tyrosine phosphatase assay. The activity of non-tyrosine phosphatases is less than 6% of the LAR activity. PP-1 and PP-2A are highly active in the serine/threonine phosphatase assay. Inhibition of LAR and PP-2A in these assays is demonstrated using known inhibitors. Both of these assays are sensitive, robust, kinetic assays that can be used to quantify enzyme activity.

Topics: Animals; Enzyme Inhibitors; Humans; Hymecromone; Jurkat Cells; Kinetics; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Sensitivity and Specificity; Spectrometry, Fluorescence

Protein phosphatase inhibition assays currently used for the detection of cyanobacterial peptide hepatotoxins in drinking water require an enrichment step using C18 cartridges to achieve lower the detection limit. This paper describes a colorimetric and fluorometric protein phosphatase inhibition method for the direct detection of microcystin-LR (MCYST-LR) in drinking water without complex clean-up steps and preconcentration procedures. In this assay three different substrates, p-nitrophenyl phosphate (p-NPP) and two fluorogenic compounds, 4-methylumbelliferyl phosphate (MUP) and 6,8-difluoro-4-methylumbelliferyl phosphate DiFMUP), were tested. The detection limits of the assay are 0.25 and 0.1 microg/l using colorimetric and fluorometric methods, respectively. These levels are well below the provisional guideline value for MCYST-LR of 1 microg/l of drinking water. The detection limit of the fluorometric method is comparable to that of the classical ELISA test. Although both the latter tests allow the detection of MCYST-LR in drinking water directly without pretreatment, the protein phosphatase inhibition assay remain less expensive and therefore more attractive for use in the routine assessment of drinking water contamination by microcystins.

Topics: Bacterial Toxins; Colorimetry; Cyanobacteria; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fluorometry; Hymecromone; Marine Toxins; Microcystins; Nitrophenols; Organophosphorus Compounds; Peptides, Cyclic; Phosphoprotein Phosphatases; Reproducibility of Results; Sensitivity and Specificity; Water

A fluorogenic assay for human T-cell phosphatase (TCPTP) was conducted on an etched glass microchip using pressure driven flow. The TCPTP enzyme catalyzes the removal of a phosphate group from 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP) to produce the fluorogenic product 6,8-difluoro-4-methylumbelliferone (DiFMU). Enzyme assays with real-time on-chip dilution were performed in both low-viscosity (1 cP) buffer and an enzyme solution containing 50% glycerol (6 cP). Single side channels connect a series of reagent wells to a main channel where the fluorescent product of the enzyme reaction passes the detector region. Flow regulation of mixed viscosity fluids requires a pressure control on each arm of the chip contributing to the overall flow. An 8-channel pressure controller was built to regulate the air pressure above all wells feeding channels of the chip, thereby controlling the dilution ratios of buffer, substrate and enzyme. Well pressures maintained a constant concentration of enzyme in the detector channel while adjusting the flow contribution of substrate and buffer. The substrate concentration was stepped over two orders of magnitude while verifying fluid dilutions using marker dyes. The kinetic parameters, Km, Vmax, and Kcat, showed good agreement with the values determined using a standard well plate and fluorometer.

Topics: Chromogenic Compounds; Computer Systems; Equipment Design; Fluorometry; Humans; Hymecromone; Kinetics; Microchemistry; Osmosis; Pressure; Protein Tyrosine Phosphatase, Non-Receptor Type 2; Protein Tyrosine Phosphatases; Rheology; T-Lymphocytes; Viscosity

A fluorescent enzyme inhibition assay for microcystin-LR was developed using a new fluorescent substrate of protein phosphatases 1 (PP1) and 2A (PP2A), 6,8-difluoro-4-methylumbelliferyl phosphate. The PP1 and PP2A inhibition assay for microcystin-LR was performed in a microtiter plate and the fluorescence yielded by the enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The concentration of microcystin-LR causing 50% inhibition of PP1 and PP2A activity (IC50) was 0.01 nM for PP1 and 0.08 nM for PP2A. The measurable range of microcystin-LR was 800 to 0.08 pg/well for both enzymes. The described assay is fast and very sensitive for the detection of microcystin-LR. Furthermore, this assay can be successfully applied to the study of toxins that inhibit PP1 or PP2A.

Topics: Cyanobacteria; Fluorescent Dyes; Hymecromone; Kinetics; Marine Toxins; Microcystins; Peptides, Cyclic; Phosphoprotein Phosphatases; Sensitivity and Specificity; Spectrometry, Fluorescence; Substrate Specificity

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pK(a) approximately 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pK(a) values lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A beta-galactosidase substrate was also prepared based on 6, 8-difluoro-4-methylumbelliferone. This substrate, 6, 8-difluoro-4-methylumbelliferyl beta-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl beta-d-galactopyranoside (MUG), for the detection of beta-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and beta-galactosidase activity, respectively, at neutral and acid pH.

Topics: Acid Phosphatase; Alkaline Phosphatase; beta-Galactosidase; Fluorescent Dyes; Hydrogen-Ion Concentration; Hymecromone; Models, Chemical; Phosphoric Monoester Hydrolases; Spectrometry, Fluorescence