Adenosine kinase assay

In addition, unlike many other discontinuous assays that focus on only one of the components of the reaction, the HPLC assay offers the potential to monitor several. For example, consider adenosine kinase, the enzyme that uses two substrates and forms two products according to the reaction Ado + ATP —> AMP + ADP. Since HPLC can readily separate all four compounds (see Fig. 1.4), and all four compounds can be detected at 2S4 nm, it is apparent that with the HPLC method, the level of each component can be monitored during the course of the reaction, providing a complete analysis of each time point. ... 

Analogs can be used in another way. Consider the development of an assay procedure for adenosine kinase, the enzyme that catalyzes the primary reaction Ado + ATP —> AMP + ADP. Problems will arise during the assay of this activity in crude extracts, since other enzymes may be present that can form AMP directly from ATP. 

Alternatively, the same reaction can be assayed if adenosine is replaced by formycin A (FoA) (Fig. 4.14), a fluorescent analog. With this substrate, one product of the adenosine kinase reaction would be FoMP, the fluorescent analog of AMP, while AMP formed directly from ATP would not be fluorescent. Therefore, by monitoring both the fluorescence and the ultraviolet absorbance, using equipment arranged as shown in Figure 4.15, the analyst could follow both the kinase reaction and any secondary reactions. 

Adenosine kinase catalyzes the transfer of phosphate from ATP to adenosine (Ado) to form AMP and ADP. The separation of the reactants, Ado and ATP, from the products, AMP and ADP, can be accomplished by reversed-phase HPLC (Ci8) with isocratic elution with a mobile phase of 0.1 M potassium phosphate (pH 5.5) and 10% methanol. Detection depends on the substrate. In this assay, it is useful to replace the substrate adenosine with the fluorescent analog formycin A (FoA) and to monitor the column eluent with a fluorescence detector. Thus, ATP and any of its hydrolytic products will not be detected. 

In order to determine whether the released adenosine could serve as a precursor of human erythrocyte adenine nucleotides, a rabbit liver was labeled by perfusion with Hj -hypoxanthine as described. Washout perfusion with an isotonic balanced salt solution removed residual extracellular label. The labeled liver was then perfused by recirculating a 400ml washed human erythrocyte suspension for one hour. Erythrocytes were then collected and washed, and the liver was excised. Extracts were prepared and the purine nucleotides were assayed for distribution of radioactivity (Table II). Within the liver, the radioactivity was approximately evenly distributed between the adenine nucleotides and the hypoxanthine plus xanthine nucleotides, indicating again that extensive conversion of hypoxanthine to IMP to AMP can occur in the liver cell. Within the human erythrocyte, over 80 percent of the label appeared in the adenine nucleotides. Since IMP is not converted to AMP in that cell, the labeled adenosine formed in the liver from the perfused hypoxanthine must have been taken up by the human erythrocyte and converted to AMP by the adenosine kinase. Since free adenine, the only other possible precursor of human erythrocyte adenine nucleotides, was not detected in hypoxanthine perfused liver or in hepatic venous effluent from hypoxanthine perfused liver, a possible role is unlikely. 

In order to enhance affinity and selectivity for Brc-Abl, we modified the inhibitor methylating at positions I and II (Fig. 7.5d). The synthesis of the wrapping prototype recapitulates imatinib synthesis [38], as described in [39], To test whether the specificity and affinity for Brc-Abl improved, we conducted a spectrophotometric kinetic assay to measure the phosphorylation rate of peptide substrates in the presence of the kinase inhibitor at different concentrations. This assay couples production of adenosine diphosphate (ADP), the byproduct of downstream phosphorylation, with the concurrent detectable oxidation of reduced nicotinamide adenosine dinucleotide (NADH). The oxidation results upon transfer of phosphate from PEP (phospho-enolpyruvate) to ADP followed by the NADH-mediated reduction of PEP to lactate. Thus, phosphorylation activity is monitored by the decrease in 340 nm absorbance due to the oxidative conversion NADH->-NAD+ [34, 39]. 

The ATP produced is measured by hexokinase (HK)/ glucose-6-phosphate dehydrogenase (G6PD) coupled reactions that ultimately convert NADP to NADPH, which is monitored spectrophotometricaUy. Oliver first reported this method that RosaUd also described with the improvement of adding AMP to inhibit adenylate kinase (AK) and cysteine to activate CK. Subsequently, Szasz and colleagues optimized the assay by adding N-acetylcysteine to activate CK, EDTA to bind Ca and to increase the stability of the reaction mixture, and adenosine pentaphosphate (ApsA) in addition to AMP to inhibit AK. A reference method based on this previous experience was developed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) it was modified recently to produce a reference procedure for the measurement of CK at 37 °C. ... 

Fig. 4 Assays for G-protein-coupled receptors. The two main ciasses are binding and functional assays. Binding assays detect compounds that are ligands of the receptor. Functional assays probe the signaling of the receptor within the cell. Gs/i and Gq/i, G-proteins PLC, phospholipase C AC, adenylyl cyclase DAG, diacylglycerol cAMP, cyclic adenosine monophosphate PKC, protein kinase C PKA, protein kinase A (PKA) lns(l,4,5)P3, inositol phosphates P-CREB, phosphorylated cAMP response element binding protein CRE, cAMP regulatory element.

Fig. 15.2-15 Development of kinase-biased screening libraries during an NMR-based fragment approach. The K, values of the fragments are obtained by quantification of the STD NMR resonances of an adenosine derivative, which is used as the reporter ligand in a competition assay, (a) As a proof of principle, published high-affinity ligands are fragmented into their components. The NMR method is applied to this validation set, which reveals the typical affinities of a particular kinase toward the standard kinase fragments. For example, fragments usually exhibit higher affinities to kinase A...

P.B., and Chau, V. (1981) Fluoromet-ric assay for adenosine 3, 5 -cyclic monophosphate-dependent protein kinase and phosphoprotein phosphatase activities. Proc. Nad. Acad. Sci. U.S.A., 78, 6048-6050. 

Of the second class, a mutant, unable to take up adenosine, and a mutant, unable to take up guanosine and inosine were obtained. Then tested by assay on cell extracts these mutants showed normal kinase activities for all three nucleosides. 

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