Methods Using Nonlabeled ATP

Enzymatic Fluorimetric Assay In order to eliminate risks and costs associated with the use of radioactive compounds, Weign et al. [23] and Sugiyama et al. [24, 25] introduced a novel enzymatic fluorimetric assay to assess adenylyl cyclase activity. This method, firstly applied on ventricular membrane preparations and later extended also to tissues, is based upon previous observations by Lowry and Passonneau [26]. These authors developed a number of sensitive assays which can measure small amounts of biological compounds based on the fluorescence of reduced pyridine nucleotides. These methods employ one or more of a series of enzymatic reactions which ultimately lead to the production of either P-nicotinamide-adenine dinucleotide phosphate (NADP+) or P-nicotinamide-adenine dinucleotide (NAD+) or the reduced forms, NADPH and NADH. Indeed NADPH is a fluorescent molecule (340 nm excitation wavelength/460 emission wavelength) which can be easily detected using a fluorescence spectrophotometer. The enzymatic fluorimetric assay consists of two parts, namely: the production of cAMP by the adenylyl cyclase in samples (membranes or tissues), and the measurement of NADPH which is generated in proportion to newly formed cAMP. The second part of the procedure comprises several steps:

1. Enzymatic destruction of the adenine nucleotides other than cAMP in the samples with a mixture of apyrase, 5' nucleotidase and adenosine deaminase;

2. Conversion of cAMP to AMP with phosphodiesterase;

3. Quantification of the resulting AMP (Fig. 9.3). This can be used to stimulate the activity of added glycogen phosphorylase A, which converts added glycogen and inorganic phosphate into glucose-1-

5' nucleotidase ATP + H20 ->- adenosine + Pi adenosine

deaminase

phosphodiesterase

glycogen phosphorilase A

phosphoglucomutase Glucose-1-phosphate ->- glucose-6-phosphate glucose-6-phosphate + dehydrogenase + Glucose-6-phosphate + NADP+ ->- 6-phosphogluconolactone + NADPH + H+

Figure 9.3 Enzymatic fluorimetric assay of cAMP. Schematic representation of the steps required for detection of cAMP using coupled enzymatic reactions to glycogen Phosphorylase A.

phosphate. Ultimately, glucose - 1-phosphate is enzymatically converted into 6-phosphogluconolactone, NADPH and H+. The NADPH concentration is then determined fluorimetrically and can be correlated with the concentration of adenylyl cyclase, cAMP or AMP in the sample.

Alternatively, AMP can be converted to ADP by combining it with ATP in the presence of myokinase (Fig. 9.4, panel a). The resulting ADP is then converted to ATP and pyruvate by combining it with 2-phospho(enol)pyruvate (PEP) and pyruvate kinase. ATP is finally involved in a reaction with fructose and esokinase to produce 6-phosphogluconolactone and NADPH.

This enzymatic assay has a number of potential advantages over the classical radioactive methods: (1) the risks and costs associated with radioactivity are eliminated; (2) no overtime incubation or counting of radioactivity is necessary, so the time required for the assay is significantly shorter; and (3) finally, the standard curve is linear over a wide range of cAMP concentration.

Bioluminescent Enzymatic Assay The bioluminescent assay of adenylyl cyclase activity shares benefits of previously described fluorimetric assays including high degree of sensitivity, less expense, more versatility, and no radioactivity. In addition, it has two additional advantages: (1) it can be performed in fewer steps and, under similar experimental conditions, (2) it is more

myokinase

ADP + phospho(enol)pyruvate pyruvate kinase

ATP + fructose esokinase

> fructose-6-phosphate + ADP

Fructose-6-phosphate phosphoglucose isomerase

■> glucose-6-phosphate

Glucose-6-phosphate + NADP+

glucose-6-phosphate dehydrogenase

Luciferin + ATP + O2

luciferase

Figure 9.4 Enzymatic fluorimetric and bioluminescent assays of cAMP. (a) Schematic representation of the steps required for fluorimetric detection of cAMP using coupled enzymatic reactions to myokinase; (b) schematic representation of cAMP detection using luciferase.

sensitive than the fluorimetric techniques. The first steps are identical to those previously described: there is a cleaning reaction to degrade adenine nucleotides other than cAMP, the conversion of cAMP to AMP with phosphodiesterase, and the conversion of AMP to ATP using myokinase and pyruvate kinase. What is different is just the final reaction to measure ATP. In this case, luciferin is used, which is enzymatically converted to dehydroluciferin by luciferase in an ATP-consuming reaction and production of light (Fig. 9.4 , panel b). Resulting luminescence is proportional to initial cAMP concentration [27].

Another assay employing bioluminescence, also feasible for wide compound library screening (HTS) has been recently introduced by Kumar et al. [28]. The assay is based on the principle that cAMP modulates PKA holoen-zyme activity, decreasing available ATP and leading to decreased light production in a coupled luciferase reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both PKA activity and cAMP intracellular concentration is observed. Thus, the functional activity of agents that modulate Gs- or Gi-coupled GPCRs can be measured by changes in the amount of RLU readout.

Immunoassays: RIA and EIA The immunoassays' approach for determining cAMP concentration relies on the highly specific antigen-antibody interaction. The RIA, introduced by Steiner et al. [29] uses an antibody generated against 2- -O-monosuccinyl cAMP and 125I labeled cAMP. The concept behind this method is competition between radiolabeled cAMP and cAMP from samples and standards, for binding to the antibody. Whereas the first RIA protocol was sensitive to 1-2pmoles of cAMP, the method has been subsequently improved by means of 2'-O-acetylation of cAMP in samples and standards. This step allows to reach a higher sensitivity to readily detect fmole amounts of cAMP in tissue extracts [30, 31] . RIA has also been adapted to investigate adenylyl cyclase activity in cell membranes with several advantages over the conventional method utilizing [a-32P]ATP. First, rather than using up to a million cpm of 32P to perform a single assay, only about 10,000 cpm of 125I are required, thus increasing safety. Second, no column chromatography or purification of any kind are necessary. Moreover, even if the protocol is easier in comparison with the one elaborated by Salomon's methods [19-21], sensitivity is comparable [31].

Immunoassays, both RIA and EIA, require separation of antibody-bound cAMP fraction from free cAMP; the efficiency with which this is done is crucial to the overall assay performance and to the simplicity and convenience of the protocol. The most common methods for separation are adsorption and precipitation. The first one utilizes a suspension of particles that could absorb free cAMP onto their surface (e.g., charcoal); following centrifugation, an aliquot of the supernatant is transferred to a scintillation vial to enable determination of the antibody-bound fraction. Alternatively, addition of a precipitating agent (a second antibody, or, less selective, ammonium sulfate or polyethylene glycol) can be useful to isolate immunoglobulins from the reaction mixture. After incubation with the precipitating agent, the antibody-bound cAMP is separated from the unbound fraction by centrifugation. Supernatant is decanted away and the activity (radioactivity or enzyme activity) is determined in the pellet.

RIA protocol has been adapted also to a solid-phase procedure in order to simplify bound and unbound cAMP separation. This assay, useful for tissues, body fluids, and cultured cells, includes microtiter wells or strips coated with polyclonal anti- cAMP antibody. In this solid-phase procedure, separation is achieved by pouring the content of the wells away or washing strips, respectively, leaving the bound fraction physically attached [32].

RIA has been subsequently improved in order to completely skip the separation step, thus obtaining a new simpler protocol called scintillation proximity assay (SPA). The Flashplate technology, introduced by Perkin Elmer, uses SPA method: microtiter plates coated with scintillant enable the detection of specific binding of radiolabeled molecules. These plates are coated with an anticAMP antibody and the assay uses 1 25I-cAMP as a tracer. In the absence of cellular cAMP, the antibody sequesters 125I-cAMP, bringing it in close enough proximity to the scintillant on the plate, such that light is produced. In the presence of cellular cAMP, the unlabelled cAMP competes off the iodinated molecule and thereby reduces the signal [18]. Since any unbound radioligand remains too distant to activate scintillant, the need for physical separation process is eliminated.

Even if RIA is surely a highly sensitive and precise method to detect cAMP in different biological samples, given the safety and environmental concerns, the use of radioactive materials should be avoided. On these bases, the introduction of EIA has been very useful. In this case, cAMP is not radioiodinated but it is labeled with an enzyme, for instance P-D-galactosidase [33] or acetyl-cholinesterase [34]. The assessment of cAMP concentration is determined evaluating enzymatic activity usually by means of a colorimetric or a fluori-metric assay. Like RIA, EIA too needs a separation step of bound from unbound cAMP. Therefore, starting from the early 1990s, several solid-phase strategies have been introduced [35, 36]. At present, there are many commercially available kits for solid -phase EIA like the one distributed by Sigma-Aldrich (St. Louis, MO). The kit uses a polyclonal antibody to cAMP to bind, in a competitive manner, the cAMP in the sample or an alkaline phosphatase molecule that has cAMP covalently bound. Samples or standard, alkaline phosphatase conjugate, and antibody are simultaneously incubated in a secondary antibody- coated multiwell plate. After a short incubation time with alkaline phosphatase substrate, the reaction is stopped and the yellow color generated is read on a multiwell plate reader. The intensity of the yellow color is inversely proportional to the concentration of cAMP in either the standards or the samples. Even if these kits, either for RIA or EIA, are very simple to use, they are usually expensive.

HTS Methods The concept behind immunoassays, that is competition between labeled or nonlabeled cAMP for binding immunoglobulins, has been widely extended and modified, especially to develop new assays suitable for HTS campaigns aimed at seeking novel receptor modulators. Both fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) technologies have been applied to measurement of cAMP for HTS. A description of these and other methods for HTS is not in the scope of this chapter, but it can be found in some recent reviews [18].

Protein Binding Assay This protocol was firstly described by Gilman [37] and Brown et al. [38] in the early 1970s. According to their works, the concentration of cAMP can be assessed by means of competition between the cAMP present in samples or standards and [3H]cAMP for association with a cAMP-dependent protein kinase. In this case, a separation step is also required to separate bound from free cAMP. The original method has been improved with the introduction of faster separation techniques such as ammonium sulfate precipitation [39], or filtration by polyethylenimine-treated glass filters [40] . This protocol, used firstly to determine cAMP concentration in tissues, was adapted also to cell culture supernatants and body fluid [41]. This procedure can be easily performed for samples containing >3 nM concentration of cAMP [22]. However, the efficacy of protein binding assay is surely lower than immu-noassays (RIA and EIA) since antibodies have clear advantages over cAMP binding proteins in terms of affinity and stability.

Protein Kinase Activation Together with the introduction of RIA by Steiner et al. [29], another method was suggested to assess tissue cAMP concentration: the protein kinase activation assay. This method is based upon the ability of the cyclic nucleotide to activate cAMP-dependent protein kinase, which in turn phosphorylates a substrate (such as caseine), using ATP. Thus, by means of exposure of tissue extracts to y32P-ATP, a certain amount of phos-phorylated casein is obtained in relation with cAMP tissue concentration. The phosphorylated product can be isolated through filtration [42, 43] . However, this technique has the disadvantage of requiring radiolabeled ATP.

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