Adenylate kinase assay

Erwinia herbicola NCIMB 12126 was obtained from the National Collection of Industrial and Marine Bacteria (Aberdeen, UK), HEPES buffer sachets and magnesium acetate were obtained from Sigma (Poole, UK), adenylate kinase assay kits were obtained from Acolyte Biomedica (Salisbury, UK), sterile tissue culture grade distilled water was obtained from Gibco (Paisley, UK), L-broth and tryptone soya agar plates were obtained from Oxoid (Basingstoke, UK). 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

Figure 9.111 Adenylate kinase activity measured by the HPLC assay method. The assay mixture contained 200 fiM ATP, 20 fiM pH]AMP (approximately 120,000 cpm), 50 mM Tris-HCl (pH 7.4), and 32 fig of protein of the enzyme from Dictyostelium discoideum. Samples (20 juL) were injected and the radioactivity monitored continuously with a Berthold LB 503 detector using PICO-Fluor 30 scintillant. Absorbance was measured at 254 nm. Three representative time points were shown (A) 1 minute (B) 30 minutes, and (C) 60 minutes after initiation of the reaction. (From Rosso-mando, 1987.)... 

The following method has been described for the assay of cyclic AMP [144, 145]. Cyclic AMP is converted to 5 -AMP with the aid of phosphodiesterase and then to ATP with myokinase (EC 2.V.4.3 ATP AMP phosphotransferase adenylate kinase) and pyruvate kinase (EC 2.7.1.40 ATP pyruvate phosphotransferase). ATP is measured by determining the orthophosphate which accumulates during incubation of ATP with a cycling system containing myosin, pyruvate kinase, and phosphoenol pyruvate. Alternately, the ATP is determined by its luminescent reaction with firefly luciferin and luciferase [145-147]. With a sensitivity to about 1 pmol/tube of cyclic AMP, this assay is almost as sensitive as the phosphorylase method, but with a linearity over three orders of magnitude, it is linear over a much wider range than the phosphorylase method. 

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. ... 

The development of automated luminometers is focused primarily on devices that achieve high sample throughput rates, typically through the use of 96 and 384 well microtitre plates. Whilst suited for many applications, these systems require a skilled operator and often do not provide rapid results when the time taken to prepare the microtitre plate is included in the assay time. An instrument that can perform on demand, automated, near real time analysis using a variety of luminescent assay protocols has been developed. The instrument has been designed to automate various luminescent assays including adenylate kinase (AK) assays and those that use magnetic separation steps in conjunction with bioluminescence. ... 

Squirrell DJ, Murphy MJ. Adenylate kinase as cell marker in bioluminescent assays. In Campbell AK, Kricka LJ, Stanley PE, Eds. Bioluminescence and Chemiluminescence Fundamentals and Applied Aspects. Chichester John Wiley and Sons, 1994 468-9... 

Previously, we tested a manual method using antibiotic-mediated lysis of nontarget cells followed by immuno-magnetic separation with an adenylate kinase (AK) bioluminescence endpoint determination. This 4 h assay for patient swabs was carried out on a limited number of samples. The work reported here was to introduce automation into the assay and to carry out tests on a larger number of samples in a hospital setting. Initial assay development was done in a non-clinical laboratory using spiked samples. The assay was then adapted to fit in with the standard hospital test. Modifications were introduced as testing in the hospital laboratory proceeded. 

Strain specific lysis by bacteriophages can be used to release intracellular ATP or adenylate kinase (AK). At the 13 ISBC a paper is presented on identification of bacteria using bacteriophages. There are also several papers on bioluminescent realtime detection of nucleic acid amplification, which may be used for identification of bacteria. In these assays either pyrophosphate or AMP is converted to ATP as a measure of the amplification reaction. 

The use of adenylate kinase (AK) rather than ATP for biomass estimations has been advocated/ AK is a very stable enzyme and the assay does not differentiate between living and dead cells. However, AK may be used in hygiene control. The ATP level in ADP, i.e. the AK substrate, sets the detection limit of the assay. This is a problem since ADP easily disproportionate to ATP and AMP. 

Jansson V, Jansson K. An enzymatic cycling assay for AMP using adenylate kinase, nucleoside-diphosphate kinase and firefly luciferase. Anal Biochem. 2003 321 263-5. 

Brovko Y, Romanova NA, Ugarova NN. Bioluminescent assay of bacterial intracellular AMP, ADP and ATP with the use of coimmobilised three-enzyme reagent (adenylate kinase, pyruvate kinase, firefly luciferase). Anal Biochem. 1994 220 410-4. 

Assays for enzymes (adenylate kinase, ATPase, creatine kinase, creatine kinase isoenzyme MB, lactate dehydrogenase, proteases)... 

The most versatile system for the synthetase assay in homogenates has been developed by Hurlbert (25). The method utilizes endogenous or added lyase and adenylate kinase to trap the adenylosuccinate into ADP and ATP. Phosphoenolpyruvate and pyruvate kinase are added in excess to serve as a regenerating system for GTP, and to convert ADP... 

Table 4-II shows the adenylate kinase activities of unfractionated soluble extracts of several rat tissues (5). The assay used was ADP synthesis from ATP and adenylate these data indicate that rat tissues differ widely in their adenylate kinase activities. The adenylate kinase activity of liver cells is also present in mitochondria as well as in the soluble phase but is absent from nuclei and microsomes about 90% of the total activity in rat liver is found in the cytosol. The mitochondrial enzyme appears not to be a contaminant from the cjrtosol.

FcR-mediated cytotoxicity assays are variable and difficult to perform, especially the ADCC assay, which uses primary cells from peripheral blood. However, recently these assays have been simplified and used as a bioactivity assays for characterization of therapeutic proteins and as a routine potency assays. Some laboratories in industry are able to use cloned NK cells as effector cells and the established cell lines or transfected cells as target cells in simplified ADCC assays. The CDC assay is used as a potency release assay for several therapeutic proteins. The use of cell lines instead of the primary cells in ADCC assays and nonradioactive detection methods, adenylate kinase (AK) or ATP with luminescent readouts, for example, have a potential to improve the performance of these assays and make them more suitable for the routine use. 

Assay of Adenylate Kinase Activity and other Analytical Methods were performed according to Muller et al. (1982) and Neufang et al. (1983). 

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

Fig. 11. Response of inactivation of acetyl-CoA carboxylase to the adenylate energy charge. Acetyl-CoA carboxylase-protein kinase preparation was preincubated as described in Fig. 1 at 37°C for 30 minutes except without AMP. Following this preincubation, inactivation and phosphorylation of the carboxylase were measured by determining the carboxylase activity remaining after incubation for 4 minutes in the presence of different adenylate energy charge systems containing ATP and AMP, or ATP alone as indicated. Carboxylase was then assayed in the presence of a final concentration of 10...

Polynucleotide phosphorylase can be assayed in several ways. The liberation of inorganic phosphate can be used to follow the reaction. Both disappearance of acid-soluble nucleotides and formation of acid-insoluble nucleotides have been measured. The reverse reaction in the presence of P Mabeled phosphate allows an exchange reaction, the incorporation of P into nucleotides, to be used as an assay. The exchange reaction was the first reaction of this enzyme to be discovered, and studies on the responsible enzyme led to the finding of polynucleotide synthesis. Another assay based on the reverse reaction has been devised for rapid spectrophoto-metric determinations. A polymer of adenylic acid is incubated with the phosphorylase in the presence of phosphate, phosphopyruvate, pyruvate kinase, DPNH, and lactic dehydrogenase. The formation of ADP is thus coupled with the formation of pyruvate which reacts stoichiometrically with PPNH, so that the entire reaction can be followed at 340 m/a. 

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