Anal. Chem. 2006, 78, 1731-1738
Accelerated Articles
Detection of Adenylyl Cyclase Activity Using a Fluorescent ATP Substrate and Capillary Electrophoresis Jennifer M. Cunliffe,† Roger K. Sunahara,‡ and Robert T. Kennedy*,†,‡
Department of Chemistry and Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109
A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CELIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Grs-GTPγS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Grs-GTPγS of 27 ( 6 µM and 317 ( 56 nM, respectively. These EC50 values compared well to those previously reported using [r-32P]ATP as substrate. When AC was concurrently activated with 2.5 µM forskolin and 25 nM Grs-GTPγS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2′-(or-3′)-O-(N-methylanthraniloyl) guanosine 5′triphosphate with an IC50 of 9 ( 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column * Corresponding author: (phone) 734-615-4363; (fax) 734-615-6462; (e-mail)
[email protected]. † Department of Chemistry. ‡ Department of Pharmacology. 10.1021/ac0521201 CCC: $33.50 Published on Web 02/08/2006
© 2006 American Chemical Society
chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.
Adenylyl cyclase (AC, EC 4.6.1.1) acts as an important enzyme in many signaling pathways by synthesizing the second messenger cyclic AMP (cAMP) from ATP. Once cAMP is produced, it participates in a variety of signaling events such as activation of protein kinase A, guanine nucleotide exchange factors for Raslike G proteins, or cyclic nucleotide channels.1-3 Nine isoforms of mammalian AC (ACI-ACIX) have been identified. While all isoforms share a primary structure of 12 transmembrane domains and 2 large cytosolic regions (C1 and C2), they are differentially expressed and regulated.3,4 AC is involved in a variety of physiological responses including learning, memory, drug dependence, and apoptosis.5-9 The important roles of AC, along with the potential for developing isoform-specific drugs for selective modulation, suggest AC as a possible therapeutic drug target.5-11 (1) Sunahara, R. K.; Dessauer, C. W.; Gilman, A. G. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 461-480. (2) Bos, J. L. Nat. Rev. Mol. Cell Biol. 2003, 4, 733-738. (3) Whorton, M. R.; Sunahara, R. K. In Handbook of Cell Signaling; Bradshaw, R. A., Dennis, E. A., Eds.; Elsevier Academic Press: San Diego, CA, 2003; Vol. 2, pp 419-426. (4) Sunahara, R. K.; Taussig, R. Mol. Interventions 2002, 2, 168-184. (5) Wong, S. T.; Athos, J.; Figueroa, X. A.; Pineda, V. V.; Schaefer, M. L.; Chavkin, C. C.; Muglia, L. J.; Storm, D. R. Neuron 1999, 23, 787798. (6) Storm, D. R.; Hansel, C.; Hacker, B.; Parent, A.; Linden, D. J. Neuron 1998, 20, 1199-1210. (7) Avidor-Reiss, T.; Nevo, I.; Saya, D.; Bayewitch, M.; Vogel, Z. J. Biol. Chem. 1997, 272, 5040-5047. (8) Nevo, I.; Avidor-Reiss, T.; Levy, R.; Bayewitch, M.; Heldman, E.; Vogel, Z. Mol. Pharmacol. 1998, 54, 419-426. (9) Lim, G.; Wang, S. C.; Lim, J.-A.; Mao, J. Neurosci. Lett. 2005, 389, 104108.
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For example, cardiac responses initiated by β-adrenergic receptor activation could be controlled by specifically inhibiting type 5 AC, causing a reduction in cardiac myocyte apoptosis while preserving cardiac myocyte contraction.10 Development of novel drugs, or chemical probes for further study of the role of AC in cell signaling, would be aided by high-throughput assays to monitor AC activity. In this work, we describe a novel approach to measure AC activity that is potentially scalable to high-throughput applications. AC activity is commonly measured by monitoring the conversion of [R-32P]ATP to [R-32P]cAMP using ion-exchange chromatography to separate the nucleotides.12-14 Although low limits of detection are achievable, this technique is laborious, sample intensive, and has a radioactivity requirement that results in added complications for safety and environmental disposal. A competitive radioimmunoassay for cAMP that uses a radioiodinated succinyl ester derivative of cAMP as the tracer may also be used for detection of AC activity, but again, the radioactivity requirement makes this technique less than ideal.15 More recently, enzyme immunoassays have been commercialized, but the cost and time required to perform the assay impede routine use. An alternative route to detect AC activity would be to monitor turnover of a fluorescent substrate. A potential difficulty with fluorescence assays for enzymes that utilize small-molecule substrates such as ATP is that the fluorophore may interfere with the enzymatic reaction. For example, the fluorescent ATP analogue 2′-(or-3′)-O-(N-methylanthraniloyl) adenosine 5′-triphosphate (MANT-ATP) inhibits AC, obviating its use in AC enzyme assays.16 A second issue is the method of detection of the reaction. While direct fluorescence readout is convenient for high-throughput assays, it requires a change in fluorescent properties upon reaction that is not always feasible to engineer into small-molecule substrates. For this reason, a separation method may be necessary to monitor the reaction. For example, when BODIPY ATPγS was used as a substrate for the tumor repressor protein Fhit, thinlayer chromatography with epi-UV illumination was used to separate and detect substrate and product.17 An alternative method for assaying enzyme activity with greater potential for drug screening is capillary electrophoresis (CE).18-25 (10) Iwatsubo, K.; Minamisawa, S.; Tsunematsu, T.; Nakagome, M.; Toya, Y.; Tomlinson, J. E.; Umemura, S.; Scarborough, R. M.; Levy, D. E.; Ishikawa, Y. J. Biol. Chem. 2004, 279, 40938-40945. (11) Johnson, R. A.; Desaubry, L.; Bianchi, G.; Shoshani, I.; Lyons, E., Jr.; Taussig, R.; Watson, P. A.; Cali, J. J.; Krupinski, J.; Pieroni, J. P.; Iyengar, R. J. Biol. Chem. 1997, 272, 8962-8966. (12) Salomon, Y.; Londos, C.; Rodbell, M. Anal. Biochem. 1974, 58, 541548. (13) Smigel, M. D. J. Biol. Chem. 1986, 261, 1976-1982. (14) Sunahara, R. K.; Dessauer, C. W.; Whisnant, R. E.; Kleuss, C.; Gilman, A. G. J. Biol. Chem. 1997, 272, 22265-22271. (15) Wu, G. C.; Lai, H. L.; Lin, Y. W.; Chu, Y. T.; Chern, Y. J. Biol. Chem. 2001, 276, 35450-35457. (16) Gille, A.; Lushington, G. H.; Mou, T. C.; Doughty, M. B.; Johnson, R. A.; Seifert, R. J. Biol. Chem. 2004, 279, 19955-19969. (17) Draganescu, A.; Hodawadekar, S. C.; Gee, K. R.; Brenner, C. J. Biol. Chem. 2000, 275, 4555-4560. (18) Zhao, D. S.; Gomez, F. A. Electrophoresis 1998, 19, 420-426. (19) Krylov, S. N.; Zhang, Z.; Chan, N. W.; Arriaga, E.; Palcic, M. M.; Dovichi, N. J. Cytometry 1999, 37, 14-20. (20) Lorieau, J.; Shoemaker, G. K.; Palcic, M. M. Anal. Chem. 2003, 75, 63516354. (21) Meredith, G. D.; Sims, C. E.; Soughayer, J. S.; Allbritton, N. L. Nat. Biotechnol. 2000, 18, 309-312. (22) Zarrine-Afsar, A.; Krylov, S. N. Anal. Chem. 2003, 75, 3720-3724.
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Figure 1. Structure of BATP and enzymatic product. The BODIPY FL label is attached to the 2′- or 3′-O-ribosyl position in BATP (A). When AC interacts with BATP, the enzymatic product 2′-BcAMP (B) is produced.
CE enzyme assays have been implemented both on- and off-line. In on-line assays, the enzyme and substrate are mixed in the column. Such assays are best for minimizing reagent usage and for rapid reactions; however, they tend to require more care in injection and sample loading. Off-line assays, in which enzyme and substrate are mixed and incubated and the resulting substrateproduct mixture separated by CE, tend to be more straightforward allowing well-controlled incubation times. They are also well-suited for cases where enzyme reactions are slow; however, they use more sample and can be more time-consuming. CE has been utilized for enzyme assays due to its high-resolution separations, high mass sensitivity, minimal sample preparation, and automation.26 With the advent of capillary array instruments,27-29 CE also has the potential for high-throughput assay development. In this work, we demonstrate that the fluorescent ATP analogue BODIPYATP (Figure 1A) is a good substrate for AC and that the reaction can be monitored by CE with laser-induced fluorescence (LIF) detection. (23) Shoemaker, G. K.; Lorieau, J.; Lau, L. H.; Gillmor, C. S.; Palcic, M. M. Anal. Chem. 2005, 77, 3132-3137. (24) Van Dyck, S.; Kaale, E.; Novakova, S.; Glatz, Z.; Hoogmartens, J.; Van Schepdael, A. Electrophoresis 2003, 24, 3868-3878. (25) Novakova, S.; Van Dyck, S.; Van Schepdael, A.; Hoogmartens, J.; Glatz, Z. J. Chromatogr., A 2004, 1032, 173-184. (26) Schultz, N. M.; Tao, L.; Rose, D. J. J.; Kennedy, R. T. In Handbook of Capillary Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton, 1997; pp 611-637. (27) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 21492154. (28) Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2256-2261. (29) Emrich, C. A.; Tian, H.; Medintz, I. L.; Mathies, R. A. Anal. Chem. 2002, 74, 5076-5083.
EXPERIMENTAL SECTION Chemicals. Tris-HCl, sodium phosphate monobasic monohydrate, MgCl2‚6H2O, and MnCl2‚4H2O were purchased from Fisher (Fair Lawn, NJ) and 10× Tris-glycine buffer was purchased from Bio-Rad Laboratories (Hercules, CA). Forskolin (from Coleus forskohlii) was purchased from Calbiochem (San Diego, CA). BODIPY FL 2′-(or-3′)-O-(N-(2- aminoethyl)urethane) ATP (BATP), BODIPY FL iodoacetamide, 2′-(or-3′)-O-(N-methylanthraniloyl) guanosine 5′-triphosphate (MANT-GTP), and Rhodamine 110 were from Molecular Probes (Eugene, OR). All other materials were purchased from Sigma (St. Louis, MO). Buffers were made in deionized water purified by E-Pure water systems (Barnstead International Co., Dubuque, IA). Protein Expression and Purification. Cytosolic AC domains VC1 (isoform V, first cytosolic domain) and ArgC-IIC2 (IIC2, isoform II, second cytosolic domain) were expressed and purified as previously described.14,30 Bovine GRs (short form) was expressed, purified, and activated with GTPγS.14,31 All proteins were stored at -80 °C until use. Sample Preparation for Adenylyl Cyclase Assays. For experiments studying basal AC production of BcAMP, samples contained 100 nM BATP, 250 nM VC1, and/or 3.8 µM IIC2. When indicated, AC was denatured by immersing sample in boiling water for 5 min prior to addition of BATP. Experiments measuring the rate of BODIPY cyclic AMP (BcAMP) formation used 1 µM BATP and 50-500 nM VC1 with a 30-fold excess of IIC2. (IIC2 was always in at least 30-fold excess over VC1 to ensure maximum IIC2/VC1 heterodimer formation; AC concentrations are reported as the concentration of the limiting cytosolic domain, VC1). For experiments examining the effect of sampling frequency on BcAMP formation, samples containing 100 nM AC and 1 µM BATP were serially injected every 7.5, 15, or 30 min. For experiments studying BcAMP synthesis with activated AC, samples contained 100 nM-4 µM BATP, 50-100 nM AC (50-100 nM VC1 and 1.9-3.8 µM IIC2), 0-1 mM forskolin, and/or 0-3.16 µM GRs-GTPγS. All samples were prepared in 10 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2, 5 mM MnCl2, and 1 mM dithiothreitol pH 7.7 (TEMMD) with a final volume of 50 µL and were incubated for 10 min at room temperature unless otherwise indicated. Experiments demonstrating the inhibition of BcAMP production used 50 nM AC, 4 µM BATP, 1 mM MnCl2, 100 µM forskolin, 50 nM GRs-GTPγS, and 0-1 µM MANT-GTP and were incubated for 5 min at room temperature before injection. Enzymatic reactions were quenched by injection onto the capillary because the enzyme and substrate were electrophoretically separated. The internal standard (IS), BODIPY FL thiophosphate (BSP), was synthesized as previously described.32 All samples contained 20 nM BSP or 50-200 nM fluorescein as internal standard with the exception of experiments measuring the rate of product formation, which used 27 nM Rhodamine 110. CE-LIF. AC assays were performed using a P/ACE MDQ capillary electrophoresis unit (Beckman Coulter Inc., Fullerton, (30) Tesmer, J. J.; Sunahara, R. K.; Gilman, A. G.; Sprang, S. R. Science 1997, 278, 1907-1916. (31) Lee, E.; Linder, M. E.; Gilman, A. G. Methods Enzymol. 1994, 237, 146164. (32) Jameson, E. E.; Roof, R. A.; Whorton, M. R.; Mosberg, H. I.; Sunahara, R. K.; Neubig, R. R.; Kennedy, R. T. J. Biol. Chem. 2005, 280, 77127719.
CA) with the separation cartridge temperature maintained at 15 °C and sample storage maintained at 25 °C. The LIF detector used 5 mW of 488-nm light from an Ar+ laser as previously described.33 Emission was detected after passing through a 488-nm notch filter and a 520 ( 10 nm band-pass filter. Data acquisition (16 Hz) and control were performed using P/ACE 32 Karat Software Version 5.0 (Beckman) for Windows 2000 on a 2.0-GHz IBM personal computer. Fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) had a total length of 30 cm, length to the detector of 20 cm, inner diameter of 50 µm, and outer diameter of 360 µm. To increase temporal resolution, the length to the detector was shortened to 10 cm (total length was maintained at 30 cm) for experiments measuring the rate of product formation. Electrophoresis buffer was 60:40 (v/v) 25 mM Tris, 192 mM glycine pH 8.5/100 mM sodium phosphate, pH 7.1. The electrophoresis buffer had a final pH of 7.8. Phosphate electrophoresis buffer has previously been used for nucleotide separations18,32,34,35 and was necessary to resolve the BATP isomers. At the beginning of each day, the capillary was rinsed with 0.1 M NaOH, H2O, and electrophoresis buffer for 5 min each. The capillary was rinsed with 0.1 M NaOH and electrophoresis buffer for 1 min each prior to injection. Samples were injected for 3 s at 0.5 psi, and separation was at 15 kV (500 V/cm) for 5.0 min. Enzyme Immunoassay. The presence of a cAMP derivative in the assay mixture was confirmed using the Direct cAMP enzyme immunoassay (EIA) kit from Assay Designs (Ann Arbor, MI) following manufacturer’s instructions. Samples containing 1 µM BATP, 100 nM AC (natured or denatured), and 100 µM forskolin were incubated for 30 min at room temperature prior to a 4× dilution with 0.1 M HCl. Simultaneous analysis by EIA and CE-LIF confirmed that formation of a cAMP-like product and the peak at 2.4 min coincided. Data Analysis. CE data were analyzed using software written in-house.36 A calibration curve for BATP was constructed summing the peak areas of 3′- and 2′-BATP and was corrected for injection variation with internal standard. BcAMP was quantified using the BATP calibration curve because BcAMP was not available in purified form. This was considered acceptable because the adenosine BODIPY analogues are not quenched17,37 and substrate and product have the same quantum yield (see BATP as an AC substrate section). The concentration of activator or inhibitor required to produce 50% of maximal response (EC50 or IC50, respectively) was determined by fitting dose-response data using GraphPad Prism (Version 3.0, GraphPad Software, San Diego, CA) to y ) bottom + ((top - bottom)/(1 + 10LogEC50-x)), where x is the logarithmic concentration of modulator (in M) and y is the BcAMP formed (in nM). For each modulator, experiments were performed in triplicate and similar EC50 or IC50 values were obtained. Error is reported as ( standard error. (33) Cunliffe, J. M.; Liu, Z.; Pawliszyn, J.; Kennedy, R. T. Electrophoresis 2004, 25, 2319-2325. (34) Tseng, H. C.; Dadoo, R.; Zare, R. N. Anal. Biochem. 1994, 222, 5558. (35) Pyo, M.; Maeder, G.; Kennedy, R. T.; Reynolds, J. R. J. Electroanal. Chem. 1994, 368, 329-332. (36) Shackman, J. G.; Watson, C. J.; Kennedy, R. T. J. Chromatogr., A 2004, 1040, 273-282. (37) Korlach, J.; Baird, D. W.; Heikal, A. A.; Gee, K. R.; Hoffman, G. R.; Webb, W. W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2800-2805.
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Figure 2. Reaction of only one isomer to yield a single enzymatic product when BATP interacts with AC. (A) Samples contained 100 nM BATP, 250 nM VC1, and 3.8 µM IIC2. Electropherograms of blank (trace 1), natured AC (trace 2), and denatured AC (trace 3) are vertically set off for clarity. (B) Sample contained 100 nM AC, 200 nM BATP, and 100 µM forskolin and the peak area of BcAMP, BATP isomers 1 and 2, and BcAMP + isomer 2 are monitored with time.
RESULTS AND DISCUSSION BATP as an AC Substrate. The overall goal of these experiments was to determine if BATP could serve as a substrate for AC and if CE could be used to monitor the reaction. BATP has two isomers with the BODIPY label attached to either the 2′or 3′- O-ribosyl position for 2′-BATP or 3′-BATP, respectively (Figure 1). The two isomers of BATP migrated at 3.1 and 3.2 min by CE as shown in trace 1 of Figure 2A. BSP, used as an internal standard to correct for injection volume variation, migrated at 4.0 min. Upon addition of AC, a new single peak was detected at 2.4 min (trace 2, Figure 2A). The area of the peak increased with time until it reached a plateau (data not shown). Once at the plateau, the peak area of product was constant with relative standard deviation (RSD) of
