Enzymes apyrase

Any excess nucleotide dNTP and any excess ATP are degraded by the nucleotide degrading enzyme apyrase to their respective mono-and diphosphates (Fig. 5.29). When degradation is complete, the next dNTP can be added. 

Fig. 5.29. The nucleotide degrading enzyme apyrase destroys any remaining dNTP and ATP.

The levels of extracellular adenosine could increase step-wise up to micromolar levels as the outcome of the transport and/or diffusion of intracellular adenosine, formed from the large pools of intracellular ATP in hypoxic conditions (Sitkovsky et al. 2005,2008). Hypoxia can upregulate an adenine nucleotide-metabolizing ecto-enzyme cascade comprising ecto-ATP apyrase (CD39) and CD73 (Synnestvedt et al. 2002). 

Pyrosequencing is a method to determine the nucleic acid sequence of short segments without the use of electrophoresis. A sequencing primer is hybridized to a single-stranded template that is usually generated by PCR. Four enzymes, a DNA polymerase, ATP sulfurylase, luciferase and apyrase, and two substrates— adenosine 5 phosphosulfate and luciferin— are included in the reaction mixture (Figure 37-17). One of the four dNTPs is added to the reaction (dATPaS is substituted for dATP because it is incorporated by the polymerase but is not a luciferase substrate). If the base is complementary to the template strand, DNA polymerase catalyzes its incorporation. Each incorporation event is accompanied by release of a pyrophosphate (PPi) so that the quantity of PPi produced is equimolar to the... 

The problem of a poor detection limit was caused by high background ATP and by the low sensitivity of the luciferin-luciferase (L-L) reagent. We have already developed an ATP elimination system using two ATP degrading enzymes (adenosine phosphate deaminase and apyrase) and a surfactant tolerant luciferase that was a mutated Luciola lateralis firefly luciferase. We optimized this elimination system, and investigated its suitability as a detection system. 

Fig. 2.7. Effect of adding increasing amounts of purified phosphofructokinase (PFK) on glycolytic oscillations in muscle extracts. The quantities of PFK added are (a) 0 (b) 0.2 unit (c) 0.4 unit (d) 0.6 unit (e) 0. The addition of apyrase each time induces oscillations, because of the transformation by this enzyme of ATP into the activator ADP (Frenkel, 1968).

Three additional enzymes are required for this process, i.t.,ATP sulfurylase, luciferase, and apyrase, and the substrates adenosine 5 phosphosulfate (APS) and luciferin. 

First, a single stranded DNA template, usually PCR amplified and purified by chromatography, gel filtration or electrophoretic techniques, is immobilised onto a surface. A suitable primer is then hybridised to this single strand (Fig. 5.26). This ensemble is incubated with four enzymes (DNA polymerase, ATP sulfyrase, luciferase and apyrase) and two substrates (adenosine 5 phosphosulfate (APS) and luciferin). 

Nucleoside di- and triphosphates are dephosphorylated by enzymes of different specificities than those which act on the monophosphates. Nucleoside triphosphates, first of all, may be dephosphorylated both to di-and monophosphates by the many enzymes which use triphosphates functionally as substrates in intermediary metabolism. In addition, they are also acted upon by several enzymes whose physiological role is not clear. For example, inorganic pyrophosphatase in the presence of Zn +, and apyrase (ATP diphosphohydrolase) degrade both ATP and ADP to adenylate. 

Glucose may be assayed in the following manner. An aliquot of the extract is treated with apyrase and boiled (10 minutes or more) a portion of this is added to the firefly enzyme and hexokinase is added (1,17). The glucose is titrated with known concentrations of ATP and the excess ATP is determined as in Section 11,3,C,4 (see Fig. 5). Fructose and mannose are also acted upon by hexokinase. 

Apyrase can be measured by plotting the decrease in luminescence as a function of time, when the preparation containing the apyrase is added to the firefly enzyme system (see Fig. 6). 

Fig. 6. Time course ci luminescence of firefly enzyme plus different concentrations of potato apyrase in the presence of a constant added amount of ATP. Apyrase AI(OH) treated ATP — 140 pg./0.8 ml.

Further phosphates of thiamine, thiamine monophosphate (TMP), and thiamine triphosphate (TTP) have also been found as natural products 19, 80). Kiessling has shown that potato apyrase, ch cleaves Y-phosphates, hydrolyzes TTP in the same manner as it does ATP, suggesting that the two compounds have their phosphate groups arranged in a similar manner 81). Yeast incorporates P K)4 to a greater extent into the phosphate of TTP than into any other thiamine phosphate 88). Neither TMP nor TTP possess cocarboxylase activity 83). TTP can, however, transfer its terminal phosphate enzymically to glucose, just as does ATP 84). 

Pardee and co-workers (225,225a,227,227a) have investigated the activity of 9 bacterial enzymes after infection of E. coli with various T phages, i.e., apyrase, ribonuclease, deoxyribonuclease, alkaline and acid proteases, pyruvic oxidase, formic dehydrogenase, serine deaminase, and catalase. The difficulties of assay of labile enzymes in the broken cell systems are indicated by the early report of an increase in apyrase activity (225) which later was attributed to a technical defect in the... 

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