Adenosine monophosphate assay

Guerrant RL, Brunton LL, Schnaitman RC, Rebhun LI, Gilman AG Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology A rapid, sensitive in vitro assay for the entertoxins of Vibrio cholerae and Escherichia coli. Infect Immun 1974 10 320-327. 

Brydon and Roberts- added hemolyzed blood to unhemolyzed plasma, analyzed the specimens for a variety of constituents and then compared the values with those in the unhemolyzed plasma (B28). The following procedures were considered unaffected by hemolysis (up to 1 g/100 ml hemoglobin) urea (diacetyl monoxime) carbon dioxide content (phe-nolphthalein complex) iron binding capacity cholesterol (ferric chloride) creatinine (alkaline picrate) uric acid (phosphotungstate reduction) alkaline phosphatase (4-nitrophenyl phosphate) 5 -nucleotidase (adenosine monophosphate-nickel) and tartrate-labile acid phosphatase (phenyl phosphate). In Table 2 are shown those assays where increases were observed. The hemolysis used in these studies was equivalent to that produced by the breakdown of about 15 X 10 erythrocytes. In the bromocresol green albumin method it has been reported that for every 100 mg of hemoglobin/100 ml serum, the apparent albumin concentration is increased by 100 mg/100 ml (D12). Hemolysis releases some amino acids, such as histidine, into the plasma (Alb). 

Pennington (PI) and Uberti (U1) first introduced the technique of liquid chromatographic enzyme assays by using the ion-exchange mode of HPLC in their analyses of 3, 5 -cyclic adenosine monophosphate phosphodiesterase and adenosine deaminase, respectively. Since that time, a number of liquid chromatographic enzyme assays have been developed. 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

The measurement of TSH was originally based on bioassays such as the stimulation of colloid droplet formation in the guinea pig thyroid gland and the release of labeled thyroidal iodide into mouse blood. These early in vivo bioassays, however, were of limited sensitivity and precision and were not applicable to the measurement of TSH in unfractionated serum. Most TSH bioassays have involved the in vitro stimulation of thyroid cyclic adenosine monophosphate (cAMP) or adenylate cyclase activity. The rat FRTL-5 thyroid cell line is an example of a particularly convenient and precise assay system. Unfortunately, such methods require purification and concentration of TSH from serum before assay. Sensitive detection of TSH in unfractionated serum is possible using a cytochemical bioassay, but this procedure is technically difficult and time-consuming. At present, immunoassay is the procedure of choice for the measurement of serum TSH in the clinical laboratory. 

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.

The cyclic adenosine monophosphate (cAMP) assay, in which the ability to inhibit forskolin-induced cAMP production is measured. 

OTHER TECHNIQUES A number of other techniques have been used to determine specific organic phosphorus compounds in the environment, including a firefly luciferase assay for adenosine di- and triphosphate (Kaplan and Bott, 1985), a protein assay for cyclic adenosine monophosphate (Francko and Wetzel, 1982) and cetryltrimethylammonium bromide precipitation of dissolved RNA and DNA in marine and freshwaters (Karl and Bailiff, 1989). However, none of these are regularly used in studies of organic phosphorus dynamics in aquatic ecosystems. 

A polyacrylamide-boronate affinity gel, Affi-gel 601 , has been used for the separation of cyclic adenosine monophosphate, cyclic guanosine monophosphate, and cyclic cytidine monophosphate from their corresponding 5-nucleotides and nucleosides. A simple direct assay of 3 5 -cyclic nucleotide phosphodiesterase activity has been developed, based on the use of the gel. 

Cyclic 3 ,5 -adenosine monophosphate phosphodiesterase (EC 3.1.4.17) activity, assayed in rat pineal organ cultures at 1 p,M substrate concentration, was significantly increased 4 h after choleragen exposure and was maximally activated (170 percent of control) after 6 h, thereafter declining to basal levels (Minneman and Iversen 1976). 

Conditions for the assay of liver phosphorylases a and b have been reviewed, and procedures were suggested for the estimation of these enzymes. For example, both forms can be estimated at pH 6.5 in the presence of 1% glycogen, 0.05M-D-glucose 1-phosphate, and 0.15M-sodium fluoride. This assay can be made specific for phosphorylase a by the addition of either caffeine or maleate. The total activity is best measured in the presence of ImM-adenosine monophosphate and 0.5M-sulphate. 

Wandelt et al. [87] have reported the application of a FILA for nucleotide monitoring. The assays were based on the fluorescence quenching of 3-diphenyl-6-vinyl-1 //-pyrazole 3.4-/ quinoline (PAQ), by guanosine 30,50-cyclic monophosphate (cGMP), adenosine 30,50-cyclic monophosphate (cAMP), and cytidine 30,50-cyclic monophosphate (cCMP). Although no analytical characterization of the polymers was reported, the authors carried out a rebinding study to ascertain the applicability of the MIPs for such application. 

Bressan, R. A. Ross, C. W. Attempts to detect cyclic adenosine 3 5 -monophosphate in higher plants by three assay methods. Plant Physiol., 1976, 57, 29-37. 

One such assay is based on the synthesis of adenosine 3 ,5 -cydic monophosphate (cAMP) by adenylate cyclase [59]. The adenylate cyclase from B. pertussis can be split into two functionally complementary fragments, T18 and T25, allowing protein dimerization to be assayed based on dimerization of T18 and T25 and reconstitution of... 

In the assay described by Amici et al. (1994), a wide variety of pyrimidine and purine nucleoside 5 -monophosphates were separated from their nucleosides by chromatography on a Supelco LClg guard column (4.6 mm x 20 mm, 5 fim). The short column allows separations in less than a minute. The mobile phase was 0.1 M potassium phosphate buffer (pH 6.0) except in the case of adenosine and deoxyadenosine, when 5% methanol was also included. The flow rate was 2 mL/min. Compounds were detected by monitoring the effluent at 254 nm, although sensitivity could be improved in some cases by using a different wavelength. 

Enz5une sensors or assays exist for the determination of adenosine triphosphate (ATP) and its degradation products inosine 5 -monophosphate (IMP), inosine (HxR), hypoxanthine (Hx) and xanthine (X) as ATP is used as an indicator of the presence of microorganisms and the concentrations of its degradation products are used as indicators for fish and meat freshness in food industry [118]. 

Vitti P, Rotella CM, Valente WA, et al. Characterization of the optimal stimulatory effects of Graves monoclonal and serum immunoglobulin G on adenosine 3, 5 -monophosphate production in FRTL5 thyroid cells A potential clinical assay. J Clin Endocrinol Metab 1983 57 782. 

A more recent cell-based in vitro assay involves the use of the microphysiometer, a sensitive extracellular pH sensor, which has been used to measure luminal (or apical) secretion and basolateral release of OH- as well as liberation of acidic metabolites in rabbit gastric glands. Adenosine 3, 5 -cyclic monophosphate stimulation produced a biphasic change... 

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. 

The poly(ADP-ribose) glycohydrolase has been pmified from a number of sources 84, 171). It is an exoglycohydrolase, localized in the nucleus 31, 136), and is readily inhibited by NAD, ADP-ribose, adenosine 3, 5 -cyclic monophosphate, p-chloromercuribenzoate, and, in some cases, DNA 135, 136). The enzyme, in the presence of added nicotinamide, did not catalyze the synthesis of NAD it did not hydrolyze the ribose to ribose linkage of iso-ADP-ribose or the terminal ADP-ribose to histone bond 31,84,171). [An assay for the enzjrme has... 

Ultrasensitive enzymic radioimmunoassay (USERIA). The combination of RIA and ELISA has on occasion been used to create an assay for a given Ag (e. g. X) that is more sensitive by up to three orders of magnitude than either of these techniques used by themselves. For this assay three different Ab are required (i) an Ab to X (i.e. anti-X,), which is bonded to a solid support, (ii)a second Ab to X which binds to a different site on the X molecule (i.e. anti-Xj), and (iii) an alkaline phosphatase-labeled Ab that binds to the Fc region of anti-X2 (i.e. anti-[anti-X2]-alkaline phosphatase), along with pH]adenosine-S -monophosphate, a substrate of alka-... 

---