Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3

The cascade of cellular events in response to an internal signal or environmental stimulus requires a diversity of molecular participants, ranging from ions to enzymes. Signal transduction pathways frequently activate specific protein kinases, leading to the phosphorylation of particular cellular proteins and subsequent initiation of a multitude of cellular responses. Binding and hydrolysis of nucleotides plays a major role in these activities, and our nucleotide analogs and assays for phosphate-producing enzymes are important tools for signal transduction research and high-throughput screening of compounds that affect signal transduction.

We offer a selection of native and modified biomolecules to aid the researcher in dissecting this highly complex branch of the signal transduction process. In addition to the probes below, we have developed the P_iPer and EnzChek assay kits for quantitation of inorganic phosphate and pyrophosphate that are extremely useful for following hydrolysis of nucleotides by various enzymes and of phosphate esters by protein phosphatases. These kits and other kits to measure ATP by chemiluminescence and protein phosphatase activity are described in Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3.

Protein kinase C (PKC) is a key player in many transmembrane signal transduction systems. This Ca²⁺-dependent serine/threonine protein kinase is activated in the presence of certain membrane-derived lipids, such as diacylglycerols and phosphatidyl serines, and phosphorylates a wide variety of substrates, including ion-channel proteins and cytoskeletal proteins.

Fim-1: A Fluorescent Bisindolylmaleimide

Bisindolylmaleimides selectively inhibit PKC by binding to the enzyme's catalytic domain. To monitor PKC activation and translocation from the cytoplasm to membranes, Chen and Poenie have developed fluorescent derivatives of bisindolylmaleimides. We offer the membrane-permeant fim-1 diacetate (F7453, ), a fluorescein-conjugated bisindolylmaleimide.

Fim-1 has been shown to be an effective inhibitor of PKC and to exhibit 16-fold selectivity for PKC over PKA. Enzyme kinetic analysis has demonstrated that fim-1 inhibits PKC by competing with ATP and not with phosphatidylserine or diacylglycerol, which is consistent with its binding at the catalytic domain rather than the regulatory domain. Using fixed and permeabilized cells, Chen and Poenie have shown that the pattern of staining produced by fim-1 is very similar to that of an anti-PKC antibody, except that the fluorescent inhibitor also appeared to stain mitochondria; this mitochondrial staining is not yet understood. Fim-1 has significant potential for monitoring PKC activity in live cells.

Fluorescent Polymyxin B Analogs

Polymyxin B is a cyclic polycationic peptide antibiotic that binds to anionic lipids. It is also a selective inhibitor of protein kinase C (IC₅₀ ~35 µM). Other direct and indirect biological effects of polymyxin B include induction of apoptosis, blocking the effect of endotoxins, modulation of K⁺-ATP channels, inhibition of the effect of phorbol ester–stimulated superoxide release, inhibition of sarcoplasmic reticulum Ca²⁺-ATPase and inhibition of insulin secretion. Polymyxin B is also reported to be a potent inhibitor of calmodulin (C23693), with an IC₅₀ of 80 nM in the presence of 500 µM Ca²⁺.

Our fluorescent polymyxin B analogs include those of the green-fluorescent BODIPY FL and Oregon Green 514 fluorophores (P13235, ; P13236), as well as the previously known dansyl polymyxin (P13238).

Hypericin

Hypericin (H7476, ), a natural pigment isolated from plants of the genus Hypericum, is a potent, selective inhibitor of PKC (IC₅₀ = 1.7 µg/mL = 3.4 µM) that should be useful for probing and manipulating PKC in live cells. When compared with other PKC probes, this aromatic polycyclic dione offers several advantages, including bright red fluorescence emission and exceptional photostability. Furthermore, hypericin exhibits light-induced inhibition of PKC, thus providing the researcher with another level of control in manipulating and evaluating PKC activity and distribution. Hypericin has a variety of pharmacological properties, from antibacterial and antineoplastic activities to antiviral activities and induction of apoptosis. Hypericin is also a potent photosensitizer, with a quantum yield of 0.75 for the generation of singlet oxygen.

Antibody against the CaM Kinase II Substrate Synapsin I

Ca²⁺/calmodulin-dependent kinase II (CaM kinase II) is a multifunctional kinase found in most, if not all, tissues. It regulates a number of cellular functions, including neurotransmitter synthesis and release, gene expression, carbohydrate metabolism, cytoskeletal function and Ca²⁺ homeostasis. CaM kinase II, which phosphorylates the Ins 1,4,5-P₃ receptor, is reported to be essential for the cADP-ribose–mediated Ca²⁺ mobilization that is required for insulin secretion by pancreatic β-islet cells.

Synapsin I is a synaptic vesicle–associated protein that inhibits neurotransmitter release, a function that is abolished upon its phosphorylation by CaM kinase II. Calmodulin also influences synapsin's function indirectly by activating CaM kinases and directly by binding to synapsin and potentiating the effects of phosphorylation. Unphosphorylated synapsin I is able to promote actin polymerization and bundling of actin filaments in the presence of synaptic vesicles. Upon membrane polarization and activation of protein kinases, synapsin becomes phosphorylated, which reduces its affinity for actin and disrupts the actin network that has been constraining the synaptic vesicles. These synaptic vesicles then migrate to the presynaptic membrane, where they undergo membrane fusion and release the neurotransmitter.

Synapsin is localized exclusively to synaptic vesicles and thus serves as an excellent marker for synapses in brain and other neuronal tissue. Antibodies directed against synapsin I have proven valuable in molecular and neurobiology research, for example to estimate synaptic density and to follow synaptogenesis. We offer a rabbit polyclonal anti–bovine synapsin I as an affinity-purified IgG fraction (A6442). This antibody was isolated from rabbits immunized against bovine brain synapsin I, but it is also active against human, rat and mouse forms of the antigen; it has little or no activity against synapsin II. Affinity-purified anti–synapsin I antibody is suitable for immunohistochemistry (), Western blots, enzyme-linked immunosorbent assays (ELISAs) and immunoprecipitation. Zenon Rabbit IgG Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling—Section 7.3, Molecular Probes' Zenon Labeling Kits—Table 7.14) can be used to prepare fluorescent dye– or enzyme-labeled complexes of this antibody.

Antibody Beacon Tyrosine Kinase Assay Kit: Real-Time Activity Measurements

Tyrosine kinases are critical players in signal transduction pathways. The fluorometric assay of all kinases, however, has been difficult because ATP-dependent phosphorylation of a fluorescent peptide target does not lead to appreciable changes in the fluorescence of the product. Previous attempts to develop fluorometric assays for kinases have typically utilized synthetic peptides labeled with an environment-sensitive fluorescent dye, which, after considerable trial and error in substrate selection, may exhibit an adventitious but small fluorescence change (either up or down) upon phosphorylation. We have discovered a unique and general method for the continuous assay of most or all tyrosine kinases that promises to be very useful for both routine laboratory use and high-throughput screening applications. Moreover, this method utilizes the natural, unlabeled target peptide or protein, yielding results that may be more relevant to the researcher.

The Antibody Beacon Tyrosine Kinase Assay Kit (A35725) provides a simple yet robust solution assay for measuring the activity of tyrosine kinases (Figure 10.47) and the effectiveness of potential inhibitors and modulators (Figure 10.48). The key to this tyrosine kinase assay is a small-molecule tracer ligand labeled with our bright green-fluorescent Oregon Green 488 dye. When an anti-phosphotyrosine antibody binds this tracer ligand to form the Antibody Beacon detection complex, the fluorescence of the Oregon Green 488 dye is efficiently quenched. In the presence of a phosphotyrosine-containing peptide, however, this Antibody Beacon detection complex is rapidly disrupted, releasing the tracer ligand and relieving its antibody-induced quenching. Upon its displacement by a phosphotyrosine residue, the Oregon Green 488 dye–labeled tracer ligand exhibits an approximately fourfold fluorescence enhancement, enabling the detection of as little as 50 nM phosphotyrosine-containing peptide with excellent signal-to-background discrimination (Figure 10.49). Key benefits of the Antibody Beacon Tyrosine Kinase Assay Kit include:

• Real-time measurements. Unlike many other commercially available tyrosine kinase assays, the Antibody Beacon Tyrosine Kinase Assay Kit permits real-time monitoring of kinase activity (Figure 10.50). Not only is the Antibody Beacon detection complex rapidly dissociated in the presence of phosphotyrosine residues, but the assay components have been designed to be simultaneously combined, eliminating any delay in the measurements.
• Simple detection protocol. Tyrosine kinase activity is measured by a simple increase in fluorescence intensity; no special equipment, additional reagents, or extra steps are required. The absorption and emission spectra of the Oregon Green 488 dye perfectly match those of fluorescein, making this assay readily compatible with any fluorescence microplate reader.
• Use of natural substrates. The Antibody Beacon tyrosine kinase assay utilizes unlabeled peptides or proteins (provided by the user) and is applicable to the assay of a wide variety of kinases.
• Compatibility. The anti-phosphotyrosine antibody provided in the Antibody Beacon Tyrosine Kinase Assay Kit is specific for phosphotyrosine residues; assay components such as ATP (up to 1 mM) and reducing agents such as dithiothreitol (DTT, up to 1 mM) do not interfere with this assay. This anti-phosphotyrosine antibody was selected from among several clones to produce the greatest fluorescence enhancement by the kinase-phosphorylated product.
• Reliability. This tyrosine kinase assay has a broad signal window, indicated by a Z' factor of >0.85.

We have optimized each component of this kit in order to provide minimal background fluorescence, maximal displacement of the tracer ligand and a large fluorescence enhancement upon the ligand's release. The Antibody Beacon Tyrosine Kinase Assay Kit comes with all the reagents needed to perform this assay right out of the box, including:

• Oregon Green 488 dye–labeled tracer ligand
• Anti-phosphotyrosine antibody
• Concentrated tyrosine kinase reaction buffer
• Two generic tyrosine kinase substrate solutions: a poly(Glu:Tyr) solution and a poly(Glu:Ala:Tyr) solution
• Dithiothreitol (DTT)
• Adenosine triphosphate (ATP)
• Phosphotyrosine-containing peptide, phospho-pp60 c-src (521–533), for use as a reference
• Detailed protocols (Antibody Beacon Tyrosine Kinase Assay Kit)

Each kit provides sufficient reagents to perform ~400 assays using a 50 µL assay volume in a fluorescence microplate reader.

Figure 10.48 Detection of the src kinase by staurosporine using the Antibody Beacon Tyrosine Kinase Assay Kit (A35725). Varying concentrations of staurosporine were incubated with src kinase (25 U/mL in reaction buffer) for 20 minutes at 37°C. The Antibody Beacon tyrosine kinase detection complex, kinase substrate (poly(Glu:Tyr), 4:1) and ATP were then added to each well, and the reactions were incubated at 37°C. After 1 hour, fluorescence was measured in a fluorescence microplate reader using excitation at 485 nm and emission at 535 nm.

Figure 10.49 Fluorescence response of the Antibody Beacon detection complex in the presence of a phosphotyrosine-containing peptide. The fluorescence of the free Oregon Green 488 peptide ligand (solid line), Antibody Beacon detection complex of the Oregon Green 488 peptide ligand (dashed line) and Antibody Beacon detection complex plus phosphorylated Abl substrate peptide (EAlpYAAPFAKKK; dot-dash line) was measured in tyrosine kinase assay buffer using the Antibody Beacon Tyrosine Kinase Assay Kit (A35725). In the presence of the phosphopeptide, the Oregon Green 488 peptide ligand was displaced from the Antibody Beacon complex and exhibited a fourfold enhancement over the fluorescence of the Antibody Beacon complex in buffer alone.

Figure 10.50 Real-time detection capability of the Antibody Beacon Tyrosine Kinase Assay Kit (A35725). Fluorescence of the Antibody Beacon detection complex in tyrosine kinase assay buffer was monitored over time. After ~15 seconds, an excess of phosphotyrosine-containing peptide was added to the Antibody Beacon detection complex and the off-rate was calculated.

RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kits

Protein tyrosine phosphatases (PTP) represent a large family of enzymes that play a very important role in intra- and intercellular signaling. PTPs work antagonistically with protein tyrosine kinases to regulate signal transduction pathways in response to a variety of signals, including hormones and mitogens. Our RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R22067) provide researchers with a sensitive and convenient method to monitor PTP and screen PTP inhibitors in a variety of research areas (Figure 10.43), including:

• Insulin regulation
• Cell proliferation and differentiation
• Axonal outgrowth
• Immune response and inflammation
• Angiogenesis

The EnzChek tyrosine phosphatase assay is based on 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, D6567, D22065; Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3), an acid and alkaline phosphatase substrate whose hydrolysis product (6,8-difluoro-7-hydroxy-4-methylcoumarin, DiFMU; D6566, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, ) exhibits excitation/emission maxima of ~358/455 nm, a low pK_a (~4.9) and a high quantum yield (~0.89). Unlike other end-point tyrosine phosphatase assay kits, the EnzChek tyrosine phosphatase assay is continuous, allowing researchers to easily measure fluorescence at various time points in order to follow the kinetics of the reaction. Furthermore, the assay is not affected by free phosphate and is compatible with most nonionic detergents, resulting in minimal sample processing before analysis. Most importantly, each assay well contains inhibitors to ensure that the assay is selective for tyrosine phosphatases; other phosphatases, including serine/threonine phosphatases, will not hydrolyze DiFMUP under our assay conditions (Figure 10.44). Unlike phosphopeptide-based assays, this DiFMUP-based assay can be used to monitor a variety of tyrosine phosphatases, including PTP-1B and CD-45 (Figure 10.44). Tyrosine phosphatase inhibitors can be evaluated quantitatively in the assay for their effect on tyrosine phosphatase activity.

Each RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit (R22067) includes:

• One RediPlate 96 EnzChek tyrosine phosphatase assay 96-well microplate
• Reaction buffer
• Detailed assay protocols (RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit)

To ensure the integrity of the fluorogenic components, the 96-well microplate is contained in a resealable foil packet and consists of twelve removable strips, each with eight wells (Figure 8.60). The removable strips allow researchers to perform only as many assays as required for the experiment. The first row of wells in the microplate contains a dilution series of the 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) reference standard for generating a standard curve, and the remaining wells are preloaded with the DiFMUP substrate. RediPlate Assay Kits—Table 10.3 summarizes our other RediPlate 96 Assay Kits for serine/threonine phosphatase activity (see below), RNA quantitation (Nucleic Acid Quantitation in Solution—Section 8.3) and protease activity (Detecting Peptidases and Proteases—Section 10.4).

RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit

The majority of protein phosphorylation occurs on serine and threonine residues, with <0.01–0.05% on tyrosine residues. Serine/threonine phosphatases represent a large family of enzymes that have been implicated in the regulation of metabolism, transcription, translation, differentiation, cell cycle, cytoskeletal dynamics, oncogenesis and signal transduction. The RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit (R33700) provides a fast, simple and direct fluorescence-based assay for detecting serine/threonine phosphatases and their corresponding modulators and inhibitors (Figure 10.43).

As with the RediPlate 96 EnzChek Tyrosine Phosphatase Kit, the substrate incorporated in the RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit is DiFMUP, which upon hydrolysis generates DiFMU (D6566, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, ) with excitation/emission maxima of 358/452 nm, a low pK_a (~4.9) and a high quantum yield (~0.89). Inhibitors are included in each assay well to ensure that the assay is selective for serine/threonine phosphatases; under the prescribed assay conditions, other phosphatases, including tyrosine phosphatases, do not significantly react with the substrate (Figure 10.45). Furthermore, unlike phosphopeptide-based assays, this DiFMUP-based assay can be used to monitor a variety of serine/threonine phosphatases including PP-1, PP-2A and PP-2B (Figure 10.45). Serine/threonine phosphatase inhibitors can be evaluated quantitatively in the assay for their effect on serine/threonine phosphatase activity (Figure 10.46). Additional advantages of this RediPlate assay include compatibility with nonionic detergents and insensitivity to free phosphate, resulting in minimal sample processing before analysis.

Each RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit includes:

• One RediPlate 96 EnzChek serine/threonine phosphatase assay 96-well microplate
• Concentrated reaction buffer
• NiCl₂
• MnCl₂
• Dithiothreitol
• Detailed assay protocols (RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit)

To ensure the integrity of the fluorogenic components, the 96-well microplate is contained in a resealable foil packet and consists of twelve removable strips, each with eight wells (Figure 8.60). Eleven of the strips (88 wells) are preloaded with the fluorogenic substrate DiFMUP; the remaining strip, marked with black tabs, contains a dilution series of the DiFMU reference standard for generating a standard curve. We also offer RediPlate 96 Assay Kits for tyrosine phosphatase activity (see above), RNA quantitation (Nucleic Acid Quantitation in Solution—Section 8.3) and protease activity (Detecting Peptidases and Proteases—Section 10.4), which are summarized in RediPlate Assay Kits—Table 10.3.

Figure 10.45 Specificity of the RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit (R33700) for serine/threonine phosphatases. The phosphatases listed in the tables were applied at the indicated concentrations to a RediPlate 96 EnzChek serine/threonine phosphatase assay microplate. Reactions were incubated at 37°C. After 1 hour, fluorescence was measured in a fluorescence microplate reader using excitation at 355 ± 20 nm and emission at 460 ± 12.5 nm.

Figure 10.46 Detection of PP-2A inhibition by okadaic acid using the RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit (R33700). Each reaction contained 50 µM DiFMUP, 10 mU/mL PP-2A and the indicated concentration (log scale) of okadaic acid in reaction buffer containing 50 mM Tris-HCl, 0.1 mM CaCl2, 1 mM NiCl2, 125 µg/mL bovine serum albumin (BSA) and 0.05% Tween 20. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 355 ± 20 nm and emission at 460 ± 12.5 nm.

Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit

The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit (P33706) provides a method for selectively staining phosphoproteins or phosphopeptides on microarrays without the use of antibodies or radioactivity. This kit permits direct detection of phosphate groups attached to tyrosine, serine or threonine residues in a microarray environment and has been optimized for microarrays with acrylamide gel surfaces. Each Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit provides:

• Pro-Q Diamond phosphoprotein/phosphopeptide microarray stain
• Pro-Q Diamond microarray destain solution
• Microarray staining gasket with seal tabs, 10 chambers
• Slide holder tube, 20 tubes
• Detailed protocols (Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit)

The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit is ideal for identifying kinase targets in signal transduction pathways and for phosphoproteomics studies.

3',5'-Cyclic AMP (cAMP) is an important second messenger in many signal transduction pathways, linking activation of cell-surface membrane receptors to intracellular responses, and ultimately, to changes in gene expression. cAMP is synthesized by plasma membrane–bound adenylate cyclase, which is coupled to transmembrane receptors for hormones, neurotransmitters and other signaling molecules by heterotrimeric G-proteins. Upon ligand binding, the intracellular receptor domain of a G-protein–coupled receptor (GPCR) interacts with a G-protein, which then dissociates and activates adenylate cyclase, resulting in an increase in the concentration of intracellular cAMP. Subsequently, cAMP activates cAMP-dependent protein kinases (protein kinase A), which phosphorylate specific substrate proteins, including enzymes, structural proteins, transcription factors and ion channels.

Adenylate Cyclase Probe: Fluorescent Forskolin

Forskolin, isolated from Coleus forskohlii, is a potent activator of adenylate cyclase, the enzyme that catalyzes the formation of cAMP from ATP. Cervical cells stained with our BODIPY FL forskolin (B7469, ) exhibit a staining pattern identical to that seen with a fluorescent anti–adenylate cyclase antibody (Daisy McCann, McCann Associates, Inc., personal communication). BODIPY FL forskolin was successfully used to assess the localization of adenylate cyclase in both live tissues and cultured neurons.

cAMP Chemiluminescent Immunoassay Kit

The cAMP Chemiluminescent Immunoassay Kit enables ultrasensitive determination of 3',5'-cyclic AMP (cAMP) levels in cell lysates, providing the highest sensitivity of any commercially available cAMP assay. As few as 60 femtomoles of cAMP can be detected. Furthermore, this assay has a wide dynamic range, detecting from 0.06 to 6000 picomoles without the need for sample dilution or manipulations such as acetylation. This extensive dynamic range is especially important in cell-based assays designed to measure Gs- or Gi-coupled agonist stimulation or inhibition. Intra-assay precision for duplicate samples is typically 5% or less.

This competitive immunoassay is formatted with maximum flexibility to permit either manual assay or automated high-throughput screening. The cAMP immunoassay is based on the highly sensitive CSPD alkaline phosphate substrate, a chemiluminescent 1,2-dioxetane, with Sapphire-II luminescence enhancer. The ready-to-use substrate⁄enhancer reagent generates sustained glow light emission that is measured 30 minutes after addition. Once the substrate⁄ enhancer reaches the glow signal, the plate can be read for hours with little or no degradation of the signal, facilitating screening protocols in which several plates are compared to each other. In addition, the assay exhibits exceptionally low crossreactivity with other adenosine containing or cyclic nucleotides.

• Alkaline phosphate conjugate of cAMP
• Anti-cAMP antibody
• cAMP standard
• CSPD substrate and Sapphire-II luminescence enhancer
• Assay and lysis buffer
• Conjugate dilution buffer
• Wash buffer
• Pre-coated microplates
• Detailed protocols (cAMP Chemiluminescent Immunoassay Kit)

The assay follows a simple protocol. Cells are seeded into plates, cultured and treated with test compounds as desired. Cell lysates are prepared in either the presence or absence of culture media. Lysates are incubated with the alkaline phosphate conjugate of cAMP and anti-cAMP antibody in a coated microplate; the resulting immune complexes are captured in the plate. In samples without cAMP, all of the alkaline phosphate cAMP is captured on the coated surface, resulting in a high substrate turnover and thus high signal. In the presence of cAMP, the amount of alkaline phosphate cAMP captured on the coated surface decreases as a result of competition for binding with unlabeled cAMP, causing a reduced signal; signal reduction is proportional to the amount of cAMP present in the cell lysate. After washing to remove unbound alkaline phosphate cAMP, the chemiluminescent substrate/enhancer is added, and the resulting glow signal is measured in a luminometer. When measured 30 minutes after substrate addition, the light signal intensity is inversely proportional to the cAMP level in the sample or standard preparation. The simple assay format and glow light emission kinetics achieved with the cAMP Chemiluminescent Immunoassay Kit provide an ideal assay system for automated high-throughput screening applications.

The cAMP Chemiluminescent Immunoassay Kit is designed for quantitating cellular cAMP for functional assays of receptor activation. It has been used with established cell lines for functional measurements with endogenous receptors, with cell lines containing exogenously expressed ligand receptors, with primary cells and with tissues in response to treatment with the appropriate ligands. It has also been used for receptor characterization, orphan receptor ligand identification and the characterization of novel chimeric receptors. In addition, this assay can be used for high-throughput screening assays of compounds that stimulate or interfere with these signal transduction pathways.

Nucleotide analogs that serve as substrates or inhibitors of enzymes, as well as nucleotide derivatives that selectively bind to regulatory sites of nucleotide-binding proteins, have been used as structural and mechanistic probes for isolated proteins, reconstituted membrane-bound enzymes, organelles such as mitochondria, and tissues such as skinned muscle fibers. More recently, however, these analogs have also been employed to study the effects of nucleotides on signal transduction and to screen for compounds that may affect signal transduction, such as G protein inhibitors and activators (G proteins and GTP Analogs for Binding Studies—Note 17.1).

We prepare a variety of nucleotide analogs, including:

• Alexa Fluor derivatives of cAMP for use as probes of type I cAMP-dependent protein kinases (PKA I) and Alexa Fluor 647 ATP (A22362)
• BODIPY dye–labeled nucleotides for use as enzyme substrates and as long-wavelength probes of nucleotide-binding sites
• Environment-sensitive, blue-fluorescent N-methylanthraniloyl (MANT) nucleotides
• Blue-fluorescent ethenoadenosine triphosphate (ε-ATP, E23691)
• Environment-sensitive trinitrophenyl (TNP) nucleotides
• Caged nucleotides, which are important probes for studying the kinetics and mechanism of nucleotide-binding proteins because they allow spatial and temporal control of the release of active nucleotide
• Photoaffinity nucleotides for site-selective covalent labeling
• Fluorescent ChromaTide nucleotides and aha-dUTP nucleotides (Characteristics of ChromaTide UTP nucleotides—Table 8.6, Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides—Table 8.7), which are primarily used for biosynthetic incorporation into DNA or RNA (Labeling Oligonucleotides and Nucleic Acids—Section 8.2)

Alexa Fluor cAMP and Alexa Fluor ATP

Our Alexa Fluor cAMP analogs are 8-(6-aminohexyl)amino derivatives; similar analogs have been shown to exhibit a marked preference for binding to type I cAMP-dependent protein kinases (PKA I). We offer the green-fluorescent Alexa Fluor 488 cAMP (A35775, ) and far-red–fluorescent Alexa Fluor 647 cAMP (A35777). Alexa Fluor 488 cAMP was loaded into cells by electroporation and then used to measure intercellular diffusion of cAMP from regulatory to responder T cells via gap junctions.

The Alexa Fluor 647 conjugate of ATP (A22362) comprises the long-wavelength Alexa Fluor 647 fluorophore linked to the ribose of ATP by a urethane bridge, as in the BODIPY ATP conjugates. We have not fully characterized the use of this probe in the study of nucleotide-binding proteins.

ATP γ-AmNS

ATP γ-AmNS (A12412), in which the aminonaphthalenesulfonate (AmNS) is attached to the terminal phosphate of ATP (), is a useful blue-fluorescent probe (absorpton/emission maxima ~330/463 nm) for studying ATP-binding sites of Escherichia coli RNA polymerase and the mitochondrial adenine-nucleotide carrier. This nucleotide analog can also be used as a fluorescence resonance energy transfer (FRET) donor or acceptor (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).

BODIPY Ribonucleotide Di- and Triphosphates

Our selection of BODIPY dye–modified ribonucleotides includes:

• BODIPY FL adenosine 5'-triphosphate (BODIPY FL ATP, A12410)
• BODIPY TR adenosine 5'-triphosphate (BODIPY TR ATP, A22352)
• BODIPY TR adenosine 5'-diphosphate (BODIPY TR ADP, A22359)
• BODIPY FL guanosine 5'-triphosphate (BODIPY FL GTP, G12411)
• BODIPY TR guanosine 5'-triphosphate (BODIPY TR GTP, G22351)
• BODIPY FL guanosine 5'-diphosphate (BODIPY FL GDP, G22360)

These mixed-isomer analogs comprise a BODIPY fluorophore attached to the 2' or 3' position of the ribose ring via an aminoethylcarbamoyl linker (). Interactions between the fluorophore and the purine base are evident from the spectroscopic properties of these nucleotide analogs. The fluorescence quantum yield of BODIPY FL GTP and BODIPY TR GTP is significantly quenched in solution (Figure 17.16) and increases upon binding to at least some GTP-binding proteins. Similar nucleotide analogs incorporating fluorophores such as fluorescein, tetramethylrhodamine and Cy3 have been primarily used for biophysical studies of nucleotide-binding proteins. The BODIPY dye–labeled nucleotides may be particularly useful for fluorescence polarization–based assays of ATP- or GTP-binding proteins.

Figure 17.16 Fluorescence emission spectra of (1) free BODIPY FL dye in phosphate-buffered saline, pH 7.2; (2) BODIPY FL ATP (A12410); and (3) BODIPY FL GTP (G12411). Samples were prepared with equal absorbance at the excitation wavelength (488 nm). The areas under the curves are therefore proportional to the relative fluorescence quantum yields, clearly showing the quenching effect caused by interaction of the BODIPY FL fluorophore with the guanine base of GTP.

Nonhydrolyzable BODIPY ATP and GTP Analogs

Among the most useful fluorescent nucleotides for protein-binding studies are those that stoichiometrically bind to ATP- or GTP-binding sites but are not metabolized. We offer the following nonhydrolyzable BODIPY nucleotides:

• BODIPY FL AMPPNP (B22356)
• BODIPY FL ATP-γ-S (A22184, )
• BODIPY FL GTP-γ-S (G22183)
• BODIPY 515/530 GTP-γ-S (G35779)
• BODIPY TR GTP-γ-S (G35780)
• BODIPY FL GTP-γ-NH amide (G35778)

The fluorescence of the BODIPY GTP-γ-S thioesters is quenched ~90% relative to that of the free dye but is recovered upon protein binding to at least some G-proteins. The green-fluorescent BODIPY FL GTP-γ-S has been used to detect GTP-binding proteins separated by capillary electrophoresis. As compared with BODIPY FL GTP-γ-S thioester, the green-fluorescent BODIPY 515/530 GTP-γ-S thioester has a greater fluorescence increase upon protein binding, . The BODIPY TR GTP-γ-S thioester is a red-fluorescent analog with spectral properties similar to the Texas Red dye.

Although BODIPY FL GTP-γ-NH amide exhibits less fluorescence enhancement upon protein binding, it is reportedly the best of the three green-fluorescent GTP-γ analogs for directly monitoring nucleotide exchange. The different linker lengths of the green-fluorescent GTP-γ analogs (six-carbon for BODIPY FL GTP-γ-NH amide, four-carbon for BODIPY FL GTP-γ-S and one-carbon for BODIPY 515/530 GTP-γ-S) may be useful for understanding protein active-site geometries.

In addition to their potential use for binding studies, BODIPY FL ATP-γ-S and BODIPY FL GTP-γ-S thioesters are important substrates for Fhit (Figure 17.18), a member of the histidine triad superfamily of nucleotide-binding proteins that bind and cleave diadenosine polyphosphates. Fhit, one of the most frequently inactivated proteins in lung cancer, functions as a tumor suppressor by inducing apoptosis. These BODIPY nucleotides should be especially useful for screening potential Fhit inhibitors and activators.

N-Methylanthraniloyl (MANT) Nucleotides

The blue-fluorescent MANT nucleotide analogs of ATP (), AMPPNP, GTP, GMPPNP, ADP and GDP are modified on the ribose moiety, making these probes particularly useful for studying nucleotide-binding proteins that are sensitive to modifications of the purine base. The compact nature of the MANT fluorophore and its attachment position on the ribose ring results in nucleotide analogs that induce minimal perturbation of nucleotide–protein interactions, as confirmed by X-ray crystal structures of MANT nucleotides bound to myosin and H^-ras p21. Furthermore, because MANT fluorescence is sensitive to the environment of the fluorophore, nucleotide–protein interactions may be directly detectable. These properties (Spectroscopic properties of MANT-ATP in aqueous solution (pH 8)—Table 17.1) make MANT nucleotides valuable probes of the structure and enzymatic activity of nucleotide-binding proteins.

Applications for MANT-ATP (M12417), MANT-ADP (M12416) and MANT-AMPPNP (M22354) include analysis of:

• ATPase kinetics of kinesin and other microtubule motor-proteins using stopped-flow fluorescence measurements
• Conformation of the myosin subfragment-1 nucleotide-binding site, as indicated by fluorescence quencher accessibility
• Interaction of P-glycoprotein ATP-binding sites with drug efflux–modulating steroids
• Myosin ATPase activity in rabbit skeletal muscle
• Structural characteristics of the nucleotide-binding site of Escherichia coli DnaB helicase

Applications for MANT-GTP (M12415), MANT-GDP (M12414) and MANT-GMPPNP (M22353) include analysis of:

• Activation of protein kinases by Rho subfamily GTP-binding proteins
• Conformational changes during activation of heterotrimeric G-proteins
• Effects of nucleotide structural modifications on binding to H^-ras p21
• Nucleotide hydrolysis and dissociation kinetics of H^-ras p21 and other low molecular weight GTP-binding proteins
• GTP-binding proteins Rab5 and Rab7, Raf-1, Rho and Rac, as well as Ras-related proteins

Ethenoadenosine Nucleotide

The ethenoadenosine nucleotides—developed in 1972 by Leonard and collaborators —bind like endogenous nucleotides to several proteins. The properties and applications of ethenoadenosine nucleotides have been comprehensively reviewed by Leonard. The etheno ATP analog (ε-ATP, E23691; ) can often mimic ATP in both binding and function. This probe has been used to replace ATP in actin polymerization reactions and is frequently incorporated in place of the tightly bound actin nucleotide. It also supports contraction of actomyosin, facilitates the measurement of nucleotide-exchange kinetics in actin and carbamoyl-phosphate synthetase and serves as a substrate for myosin, which converts it to ε-ADP. ε-ATP is not a substrate for firefly luciferase; however, when added to a reaction mixture containing luciferase, luciferin and ATP, ε-ATP enhances light production, probably by increasing the rate at which the enzyme releases the inhibitory product, oxyluciferin.

The ethenoadenosine nucleotides usually bind to nucleotide-binding sites with high specificity. Because their blue fluorescence is not very environment sensitive, however, protein binding is typically detected by fluorescence anisotropy (Fluorescence Polarization (FP)—Note 1.5) or circular dichroism. The radiative lifetime of the ethenoadenosine nucleotides is unusually long (>30 nanoseconds). Binding of ethenoadenosine nucleotides to proteins is frequently accompanied by energy transfer from the protein's tryptophan residues to the nucleotide. These nucleotides are also often used as FRET donors to longer-wavelength dyes as a means of establishing distances within and between cytoskeletal and other proteins (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2).

Trinitrophenyl (TNP) Nucleotides

Unlike the etheno derivatives, the free trinitrophenyl (TNP) nucleotides are essentially nonfluorescent in water. The TNP nucleotides undergo an equilibrium transition to a semiquinoid structure that has relatively long-wavelength spectral properties; this form is only fluorescent when bound to the nucleotide-binding site of some proteins. The TNP derivative of ATP frequently exhibits a spectral shift and fluorescence enhancement upon protein binding and actually binds with higher affinity than ATP to several proteins. The broad, long-wavelength absorption of TNP nucleotides makes them useful for FRET studies (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). The TNP derivatives of ATP (TNP-ATP, T7602; ), ADP (TNP-ADP, T7601) and AMP (TNP-AMP, T7624) have been used as structural probes for a wide variety of proteins, including:

• Myosin
• Tubulin
• Na⁺/K⁺-ATPase
• Gastric H⁺/K⁺-ATPase
• Erythrocyte Ca²⁺/Mg²⁺-ATPase
• F₁-ATPase
• Sarcoplasmic reticulum ATPase
• Adenylate kinase
• Aspartokinase
• Phosphoglycerate kinase

We also prepare the TNP analog of GTP (T7600). This analog has been used as a spectroscopic probe for the GTP inhibitory site of liver glutamate dehydrogenase and as an inhibitor of adenylate cyclase.

We have found that chromatographically purified TNP nucleotides are unstable during lyophilization. Consequently, these derivatives are sold in aqueous solution and should be frozen immediately upon arrival.

Caged Nucleotides

Caged nucleotides are nucleotide analogs in which the terminal phosphate is esterified with a blocking group, rendering the molecule biologically inactive. Photolytic removal of the caging group by UV illumination results in a pulse of the nucleotide—often on a microsecond to millisecond time scale—at the site of illumination. Because photolysis ("uncaging") can be temporally controlled and confined to the area of illumination, the popularity of this technique is growing. We are supporting this development by synthesizing a variety of caged nucleotides, ligands for biological receptors, probes involved in signal transduction and fluorescent dyes. Our current selection of caged nucleotides includes:

• NPE-caged ATP (A1048)
• DMNPE-caged ATP (A1049)
• NPE-caged ADP (A7056)
• DMNB-caged c-AMP (D1037)

Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3 discusses our selection of caged probes and the properties of the different caging groups that we use (Properties of six different caging groups—Table 5.2).

Researchers investigating the cytoskeleton have benefited greatly from advances in caging technology, primarily originating from the work of Trentham, Kaplan and their colleagues. NPE-caged ADP (A7056) is a useful probe for studying the effect of photolytic release of ADP in muscle fibers and isolated sarcoplasmic reticulum. Although it is sometimes difficult to properly abstract papers that describe experiments with caged ATP because they could be referring to either NPE-caged ATP (A1048), DMNPE-caged ATP (A1049) or earlier caged versions of this nucleotide, most researchers have used NPE-caged ATP. Caged ATP has been employed in a variety of experimental systems, including tissues, cells and isolated proteins:

• Isolated myosin subfragment-1
• F-actin
• Skinned cardiac and skeletal muscle fibers
• ATP-regulated K⁺ channels of pancreatic β-cells and rat heart cells
• Sarcoplasmic reticulum Ca²⁺-ATPase
• Submitochondrial particles

Because the caged nucleotides may be added to an experimental system at relatively high concentrations, use of the enzyme apyrase was recommended by Sleep and Burton to eliminate any traces of ATP that may be present in the caged ATP probes. Once the caged ATP solutions have been preincubated with apyrase, the enzyme can be removed by centrifugal filtration.

These caged nucleotides are generally cell impermeant and must be microinjected into cells or loaded by other techniques (Techniques for loading molecules into the cytoplasm—Table 14.1). Permeabilization of cells with staphylococcal α-toxin or the saponin ester β-escin is reported to make the membrane of smooth muscle cells permeable to low molecular weight (<1000 daltons) molecules, while retaining high molecular weight compounds. α-Toxin permeabilization has permitted the introduction of caged nucleotides, including caged ATP (A1048) and caged GTP-γ-S, as well as of caged inositol 1,4,5-triphosphate (NPE-caged Ins 1,4,5-P₃; I23580; Calcium Regulation—Section 17.2) into smooth muscle cells. Caged inositol 1,4,5-triphosphate has also been successfully loaded in ECV304 cells using electroporation.

BzBzATP

Functional ion channels can be assembled from both homomeric and heteromeric combinations of the seven P2X receptor subunits so far identified (P2X_1–7). Due to the lack of specific agonists or antagonists for P2X receptors, it is difficult to determine which receptor subtypes mediate particular cellular responses. We offer one of the most potent and widely used P2X receptor agonists, BzBzATP (2'-(or 3'-)O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, B22358). BzBzATP has more general applications for site-directed irreversible modification of nucleotide-binding proteins via photoaffinity labeling.