ATP ADP, AMP

The commonly accepted method for the analysis of ATP is based on enzymatic determination employing the lantern firefly extract luciferin— luciferase a detailed review has been presented by Deluca (1977). Methods for the analysis of ATP, ADP and AMP may be found in the reports by Skjoldal and Bamstedt (1977), Karl and Holm-Hansen (1978), Witzel (1979) and Graf and Lagaly (1980). In the majority of investigations, the seawater particulates and the microporous filters used for collection were extracted with boiling Tris buffer (0.02 M, pH 7.8) for periods between 1 and 5 min. To avoid stress on the cells, the volume filtered should be kept to a minimum (50—200 ml). The extracts obtained may be stored deep frozen for several months prior to analysis back in the laboratory. 

Hodson and Azam (1977) have reported that up to 20% of the total ATP in seawater occurs in the free form. It is, however, difficult to say whether this finding was a product of sample manipulation. At least in bacterial cultures, it has been shown that nucleotides may be released into the medium (Chapmann et al., 1971). On the other hand, it is questionable whether ATP is stable enough to exist in solution over longer periods owing to its low stability constant or the possibility of complexation with divalent cations and fulvic acids (Tetas and Lowenstein, 1963 Hulett, 1970 Bulleid, 1978). 

The results of numerous analyses show that ATP concentrations occur within a wide range from 10 to 2000 ng r for seawater with almost an order of magnitude higher values for the particulates from interstitial waters. These levels lie well above the absolute detection limit of the luciferin— 

---

By structural complementarity, dicationic l,4-diazabicyclo[2.2.2]octane (VII) provides an appropriate recognition site for phosphate ions and two stearyl side chains attached to the amines add lipophilic properties 59,60). Such a carrier model can selectively extract nucleotides from aqueous solution to chloroform solution via lipophilic salt formation. The order of nucleotide affinity is ATP > ADP > AMP. The selectivity ratios were 45 for ADP/AMP and 7500 for ATP/AMP at pH 3. The relative transport rate was ATP > ADP > AMP. The ratios were 60 for ATP/AMP and 51 for ADP/AMP. The modes of interaction of ADP and ATP are proposed to be as shown in Fig. 6. 

Examples of coenzymes vitamin-derived nucleotides for example adenosine phosphates ATP, ADP, AMP nicotinamide derivatives NAD+, NADH, NADP+, NADPH flavin derivatives FAD, FADH2 coenzyme A (abbreviated to CoA, CoASH or CoA-SH). 

Concerning human studies, only three reports have been reported (for review see Burnstock et al., 2000). Bleehen and Keele (1977) reported observations on the algogenic actions of adenosine compounds on blister base preparations. Coutts et al. (1981) injected ATP, ADP, AMP, adenosine, adenine and inosine intradermally. The area of erythema induced by the injection was delineated at 30 s. and again after a further 4.5 min when the size of the response was maximal. ATP, ADP and AMP evoked weal and flare responses in the skin in a dose-dependent manner. The rank order of potency was ATP > ADP > AMP other metabolites were apparently inactive. Injections of ATP and high doses of ADP produced a sensation of persistent pain. 

Inhibition of the initial step of a biosynthetic pathway by an end product of the pathway is a recurrent theme in metabolic regulation. In addition, many key enzymes are regulated by ATP, adenosine diphosphate (ADP), AMP, or inorganic phosphate ion (Pi). The concentrations of these materials provide a cell with an index of whether energy is abundant or in short supply. Because ATP, ADP, AMP, or P often are chemically unrelated to the substrate of the enzyme that must be regulated, they usually bind to an allosteric site rather than to the active site. 

Three membrane-bound adenosine triphosphatase enzymes have been characterized using Mn(II) and Gd(III) electron paramagnetic resonance (EPR) and a variety of NMR techniques. Mn(II) EPR studies of both native and partially delipidated (Na+ + K+)-ATPase from sheep kidney indicate that the enzyme binds Mn2+ at a single, catalytic site with Kq = 0.21 x 10- M. The X-band EPR spectrum of the binary Mn(II)-ATPase complex exhibits a powder line shape consisting of a broad transition with partial resolution of the 55 n nuclear hyperfine structure, as well as a broad component to the low field side of the spectrum. ATP, ADP, AMP-PNP and Pj all broaden the spectrum, whereas AMP induces a substantial narrowing of the hyperfine lines of the spectrum. 

The most generally interesting intracellular metabolites are the adenine nucleotide phosphates (ATP/ADP/AMP) and the nicotinic acid/adenine dinucleotide phosphates (NAD(P)H/NAD(P)). The pool concentrations and ratios of these can serve as markers for the physiological status of a culture. 

Nonequilibrium reactions can be detected by determining metabolite concentrations in the tissue of interest. Conventionally, a tissue sample is rapidly frozen by compression between metal plates that have been cooled to 77 K by immersion in liquid nitrogen (freeze-clamping). This procedure rapidly halts any enzymatic processes that might alter the metabolite concentrations the concentrations can then be determined by enzymatic or chemical assays. Recently, 31P-NMR spectroscopy has shown considerable value in measuring the concentrations of such metabolites as ATP, ADP, AMP, phosphate, and phosphocreatine in living cells or tissues. 

Likewise, biochemical parameters of heart tissue and coronary flow (ATP, ADP, AMP, catabolites of adenine nucleotides, ascorbate and urate), following 18, 35 and 60 min ischemia, clearly indicated an analogous myocardial protection (data not shown). 

P2Y, (P2Y1) receptors have been cloned from human and several other mammalian and non-mammalian species, and show an apparent functionai order of potency ATP = ADP > AMP, and seiective agonists include 2-methylthio-ATP which is more potent than ADP also ADPpp, ADPPS and 2-hexylthloATP. These receptors are found in a number of tissues inciuding intestinal smooth muscle, endothelial cells and hepatocytes. Normally they couple to the InsPs/DAG system. Recently, expression studies have provided evidence that this receptor type may also account for at least some of... 

Before launching wound study, the ability of ATP-vesicles to penetrate the tissue was tested in skin penetration. The rat skin, which is known to have similar permeability characteristics as those of humans (19), was mounted in the FDC-6 Franz Diffusion Cells (26-28). ATP-vesicles or free ATP solution was placed in the donor dome and the receiving chamber was filled with neutral buffer. ATP, ADP, AMP and their metabolites were measured by HPLC using a modified technique described previously (29-31) in the two chambers at 2, 4, and 24 h and the contents were compared to obtain the permeability ratio. The result indicated that the ATP-vesicles dramatically increased nucleotide penetration through the skin (dermis and epidermis) by 10-20-fold (N=9, Fig. 5). 

---