ATP analysis

Adenosine triphosphate is utilized in portions of the cell other than the mitochondria and chloroplasts therefore, the utilization as well as the production of ATP is of importance to total adenylate status. As a result, it became important to consider total ATP content of plants. When detached pinto bean leaves were exposed to 1,0 yl/1 ozone for 30 min total ATP content of the leaf decreased (12), Since ozone altered leaf ATP content it could also alter the leaf s adenylate status we wished to determine if a correlation existed between alteration in adenylates and the change previously reported in photosynthesis and respiration. Since ATP is readily broken down by adenosine triphosphatases, a reliable method of extraction and quantitative method of ATP analysis was designed for the study (8),... 

Figure 4.7 Effect of increasing amounts of Ca2+ in a mixture of this cation and ATP. Analysis by HPLC as described in Figure 4.6. Chromatograms are of 20 /jlL samples containing 40 nmol ATP and CaCk at (A) 60 nmol, (B) 100 nmol, (C) 120 nmol, (D) 160 nmol, ( ) 200 nmol, and (F) 400 nmol. (From Jahngen and Rossomando, 1983.)...

A more rapid test method, developed by the British Textile Technology Group in the late 1980s, is based on adenosine triphosphate (ATP) luminescence. The growth of microorganisms is assessed by firefly bioluminescent detection and ATP analysis. ... 

Table I lists the various methods of ATP analysis and an evaluation of the ease and sensitivity as well as advantages or disadvantages of each method.

Some compounds and enzymes which are conjugate in their action with ATP have been assayed with the same equipment required for ATP analysis. 

ATP analysis is used in quality testing in somatic cells for stockbreeding, such as in the test for mastitis in milking cows [38], Similar tests are applied for in vitro sperm estimations and cryoprotection in farming [39]. 

In a recent study [69] myocardial tissue metabolism has been measured by calorimetry in experimental hyperthyroidism in rats. It is known that hyperthyroidism is associated with increased myocardial aerobic metabolism and accelerated heart function, but it is unknown whether there is also an increase of anaerobic metabolism. Myocardial heat production, oxygen consumption and ATP content have been measured in a group of rats treated for 2 weeks with triiodothyronine. The results were compared with the corresponding values obtained from control rats who were administered saline. Tissue slices, about 5 mg, from the apical region of the heart were prepared immediately for measurement of heat production and oxygen consumption, whereas separate specimen were frozen in liquid nitrogen for ATP analysis. 

Firefly. Firefly luciferase (EC 1.13.12.7) is a homodimeric enzyme (62 kDa subunit) that has binding sites for firefly luciferin and Mg ATP . Amino acid sequence analysis has iadicated that beetle luciferases evolved from coen2yme A synthetase (206). Firefly bioluminescence is the most efficient bioluminescent reaction known, with Qc reported to be 88% (5), and at 562 nm (56). At low pH and ia the presence of certain metal ions (eg, Pb ", ... 

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

So far, as in Equation (3.33), the hydrolyses of ATP and other high-energy phosphates have been portrayed as simple processes. The situation in a real biological system is far more complex, owing to the operation of several ionic equilibria. First, ATP, ADP, and the other species in Table 3.3 can exist in several different ionization states that must be accounted for in any quantitative analysis. Second, phosphate compounds bind a variety of divalent and monovalent cations with substantial affinity, and the various metal complexes must also be considered in such analyses. Consideration of these special cases makes the quantitative analysis far more realistic. The importance of these multiple equilibria in group transfer reactions is illustrated for the hydrolysis of ATP, but the principles and methods presented are general and can be applied to any similar hydrolysis reaction. 

What molecular architecture couples the absorption of light energy to rapid electron-transfer events, in turn coupling these e transfers to proton translocations so that ATP synthesis is possible Part of the answer to this question lies in the membrane-associated nature of the photosystems. Membrane proteins have been difficult to study due to their insolubility in the usual aqueous solvents employed in protein biochemistry. A major breakthrough occurred in 1984 when Johann Deisenhofer, Hartmut Michel, and Robert Huber reported the first X-ray crystallographic analysis of a membrane protein. To the great benefit of photosynthesis research, this protein was the reaction center from the photosynthetic purple bacterium Rhodopseudomonas viridis. This research earned these three scientists the 1984 Nobel Prize in chemistry. 

Eu(III) complexes of quinolinecarboxylic acids, including 5, were studied (00MI31). Those of complexes of 5 with Eu(III) and Tb(III) ion were studied, and they were applied for analysis of 5 in medicinal preparations (00UKZ115). Stability of lanthanide complexes with 5 was studied (00MI67). The fluorescence spectra of 5 complexed with Co(II) and ATP was measured (01SA(A)1317). 

An amine-terminated poly ether (ATPE) is prepared as follows. Charge poly(tetramethylene oxide) diol (PolyTHF 1000, BASF, 75.96 g, 0.0759 m) to a 500-mL three-neck round-bottom flask fitted with a thermocouple, a mechanical stirrer, and a vacuum port. Add tert-butylacetoacetate (24.04 g, 0.1582 m) and apply vacuum. Heat at 175° C for 4 h, Fourier transform infrared (FTIR) analysis should indicate complete loss of the polyol OH absorption at 3300 cm. The room temperature viscosity of the product should be about 520 mPa-s. React this acetoacetylated product (85.5 g, 0.0649 m) with cyclohexylamine (14.5 g, 0.1465 m) at 110° C under vacuum for several hours. Cool the resultant cyclohexylaminocrotonate poly ether product to room temperature (1790 mPa-s at room temperature). 

The second of these steps, the rate of hydrolysis at 12 °C, measured by analysis of the ADP content in fibers rapidly frozen at different times after ATP release from caged-ATP, is 40-60 s (Ferenczi, 1986) which is similar to the rate of active force increase at 20°C once the difference in temperature has been accounted for. This rate is similar to that measured in solution however, and is not rate limiting in solution. Therefore, force generation in the caged-ATP experiments could be limited by hydrolysis, or more likely, by a step following hydrolysis such as Pj release. The idea that release of phosphate is linked to force production in muscle... 

Two strains of Sphingomonas sp. that could degrade pentachlorophenol maintained their levels of ATP even in the presence of high concentrations of pentachlorophenol. Analysis of the lipids using P NMR showed that this could be attributed to the increased levels of cardiolipin (Lohmeier-Vogel et al. 2001). 

The reversibility of QM adducts also creates numerous challenges. For example, measuring the full burden of DNA alkylation by a QM can be obscured by the loss of its labile products during or before chemical identification can be completed. Results from a deoxynucleotide model system indicated that only a small fraction of the possible adducts could be measured after the interval required for analysis of DNA. Perhaps the kinetic products of QMs also contribute to the cellular activity of these intermediates although this has yet to be explored. QM equivalents can be envisioned to migrate from one reversible nucleophile such as the N1 of adenine in such cofactors as ATP to another until quenched by a compound such as glutathione that is present in cells as a defense against undesirable electrophiles. 

Liquid Chromatography. - Diasteriomeric phosphonodipeptides have been separated by ion exchange column chromatography.267 H.p.l.c. has been used for the analysis of a variety of biologically active phosphorus compounds, such as aminoacid phosphate esters,26 phosphinothrycin,269 inositol triphosphate,270 fructose diphosphate,271 pyridoxal phosphate,272 and ATP.273... 

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