Nucleotides and Nucleotide-Coenzymes

Nucleotides contain both acid and basic groups mixtures of these substances may therefore be separated by anion- or cation exchange chromatography [12, 85]. Column chromatography on Dowexion- 

Randerath was the first to describe fractionations of nucleotides on ECTEOLA- [58, 59] and DEAE-cellulose layers [64, 66]. He found that TLC on ion exchangers renders possible better and faster separations than column chromatography and is also considerably more sensitive than PC. In 1962 Randerath described the preparation of PEI-cellulose and the application of this new anion exchanger in thin-layer chromatography of nucleotide mixtures [63, 65]. 

The chromatographic behaviour of the various nucleotides can be forecast with fair certainty from physical data [64]. This represents a marked advantage of ion exchange TLC over partition chromatographic procedures. Ion exchange TLC on modified celluloses may be used also for separations on the micro-preparative scale [64]. 

Plates coated with ion exchangers are best used as soon as possible after preparation. PEI-cellulose layers become unusable within a few 

Saturated ammonium sulphate-M sodium citrate-isopropanol (80 + 18 + 2), a solvent developed for PC of mononucleotides [45], can be employed also for separating isomeric purine-mononucleotide on cellulose layers [60] it migrates about 10 cm in 90 min. Excellent separations are obtained on cellulose layers with the solvent tert, amyl alcohol-formic acid-water (30 + 20+ 10), likewise applied in paper chromatographic separation of mononucleotides [48] the time of run is about 120 min. It is superior to all other solvent systems described here (Table 194). 

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The amino acids not only function as energy metabolites but also used as precursors of many physiologically important compounds such as heme, bioactive amines, small peptides, nucleotides and nucleotide coenzymes. In... 

Todd was named president of the Royal Academy in 1975. He was also a member of the National Academy of Sciences (USA), the American Academy of Arts and Sciences, and several prestigious European societies. Todd served as the chairman of the British government s advisory committee on scientific policy from 1952 until 1964. He was awarded the Nobel Prize in chemistry in 1957 for his work on nucleotides and nucleotide coenzyme studies. He was knighted in 1954 and died in 1997. see also Nucleotide. 

A.R. Todd. The Nobel Prize for Chemistry in 1957 was awarded for his work on nucleotides and nucleotide coenzymes . 

First DNA polymerase discovered, which copied DNA strands Nobel Prize for work on nucleotides and nucleotide coenzymes Calvin cycle of photosynthesis published First diphosphane metal complex reported Used DNA polymerase for laboratory synthesis of DNA from nucleotides... 

Pantothenic acid, sometimes called vitamin B3, is a vitamin that makes up one part of a complex coenzyme called coenzyme A (CoA) (Figure 18.23). Pantothenic acid is also a constituent of acyl carrier proteins. Coenzyme A consists of 3, 5 -adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage. Phosphopantetheine in turn consists of three parts /3-mercaptoethylamine linked to /3-alanine, which makes an amide bond with a branched-chain dihydroxy acid. As was the case for the nicotinamide and flavin coenzymes, the adenine nucleotide moiety of CoA acts as a recognition site, increasing the affinity and specificity of CoA binding to its enzymes. 

Silver ions will oxidatively desulphurize nucleoside and other phos-phorothioates to generate a phosphorylating agent. This reaction has been utilized for the preparation of nucleotide coenzymes and has the advantage that it can be carried out on a large scale without the formation of contaminating symmetrical pyrophosphates. 

Synthesis of nucleotides and nucleosides, nucleotide coenzymes, polynucleotides, and histidine... 

The natural substrates which are most frequently used are the nucleotide coenzymes NAD+ and NADP+, which are reversibly reduced by many enzymes ... 

Fluorimetric methods are useful for monitoring reactions involving the nucleotide coenzymes. The natural fluorescence of the reduced forms in the region of 460 nm can be used in kinetic assays. However, this fluorescence is destroyed at pH values below 2.0, whereas any oxidized forms of the coenzymes present are stable. If the pH of the solution is then raised above 10.5 and heated, the oxidized forms are themselves converted to fluorescent derivatives. This latter procedure lends itself to fixed time assays such as is illustrated in Procedure 8.6. 

J. London and M. Knight, Concentrations of nicotinamide nucleotide coenzymes in micro-organisms, J. Gen. Microbiol. 44, 241-254 (1966). 

The pyridine nucleotides NAD and NADP always function in unbound form. The oxidized forms contain an aromatic nicotinamide ring in which the positive charge is delocalized. The right-hand example of the two resonance structures shown contains an electron-poor, positively charged C atom at the para position to nitrogen. If a hydride ion is added at this point (see above), the reduced forms NADH or NADPH arise. No radical intermediate steps occur. Because a proton is released at the same time, the reduced pyridine nucleotide coenzymes are correctly expressed as NAD(P)H+HT... 

The best-known aldopentose (1), D-ribose, is a component of RNA and of nucleotide coenzymes and is widely distributed. In these compounds, ribose always exists in the fura-nose form (see p. 34). Like ribose, D-xylose and L-arabinose are rarely found in free form. However, large amounts of both sugars are found as constituents of polysaccharides in the walls of plant cells (see p.42). 

We conclude this chapter by describing some chemical features of nucleotide coenzymes and some of the enzymes (dehydrogenases and flavoproteins) that use them. The oxidation-reduction chemistry of quinones, iron-sulfur proteins, and cytochromes is discussed in Chapter 19. 

Dolphin, D., Avramovic, O., Poulson, R (eds) (1987) Pyridine Nucleotide Coenzymes Chemical, Biochemical, and Medical Aspects, John Wiley Sons, Inc., New ork. 

NADPH formed in the oxidative phase is used to reduce glutathione, GSSG (see Box 14-3) and to support reductive biosynthesis. The other product of the oxidative phase is ribose 5-phosphate, which serves as precursor for nucleotides, coenzymes, and nucleic acids. In cells that are not using ribose 5-phosphate for biosynthesis, the nonoxidative phase recycles six molecules of the pentose into five molecules of the hexose glucose 6-phosphate, allowing continued production of NADPH and converting glucose 6-phosphate (in six cycles) to C02. 

Phosphorus -31. NMR spectroscopy using 31P, the ordinary isotope of phosphorus, also has many uses.466 Application of 31P NMR to living tissues has been extraordinarily informative467 and is dealt with in Chapter 6. The many phosphorus nuclei in nucleotides, coenzymes, and phosphorylated metabolites and proteins are all suitable objects of investigation by NMR techniques. 

Three facts account for the need of cells for both the flavin and pyridine nucleotide coenzymes (1) Flavins are usually stronger oxidizing agents than is NAD+. This property fits them for a role in the electron transport chains of mitochondria where a sequence of increasingly more powerful oxidants is needed and makes them ideal oxidants in a variety of other dehydrogenations. (2) Flavins can be reduced either by one- or two-electron processes. This enables them to participate in oxidation reactions involving free radicals and in reactions with metal ions. (3) Reduced flavins... 

Eklund, H., and Branden, C.-I. (1987) in Pyridine Nucleotide Coenzyme Chemical, Biochemical and Medical Aspects, Vol. 2,... 

Since NADPH is continuously used in biosynthetic reactions, and is thereby reconverted to NADP+, the cycle of Eq. 17-46 must operate continuously. As in Eq. 17-42, a true equilibrium does not exist but steps b and c are both essentially at equilibrium. These equilibria, together with those of Eq. 17-42 for the NAD system, ensure the correct redox potential of both pyridine nucleotide coenzymes in the cytoplasm. 

Keilin soon realized that three of the absorption bands, those at 604,564, and 550 nm (a, b, and c), represented different pigments, while the one at 521 nm was common to all three. Keilin proposed the names cytochromes a, b, and c. The idea of an electron transport or respiratory chain followed6 quickly as the flavin and pyridine nucleotide coenzymes were recognized to play their role at the dehydrogenase level. Hydrogen removed from substrates by these carriers could be used to oxidize reduced cytochromes. The latter would be oxidized by oxygen under the influence of cytochrome oxidase. 

An alternative pathway for synthesis of quinoli-nate from aspartate and a triose phosphate exists in bacteria and in plants and provides the major route of nicotinic acid synthesis in nature. In E. coli the reaction is catalyzed by two enzymes, one an FAD-containing L-aspartate oxidase which oxidizes aspartate to a-iminoaspartate.228 The latter condenses with dihydroxyacetone-P to form quinolinate (Eq. 25-13).229 There are at least two other pathways for synthesis of quinolinic acid as well as five or more salvage pathways for resynthesis of degraded pyridine nucleotide coenzymes.224/230/231... 

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