Nucleotides terminal phosphate, reactions

In this experiment the Pstl fragment was first digested with DNAase II in sodium acetate buffer, pH 4.7 at room temperature and the reaction halted by chilling and extraction with phenol. After precipitation the DNA was electrophoresed on an 8% polyacrylamide gel and a slice of gel, corresponding to fragments of chain length 150-250 nucleotides cut out and eluted. The 3 -terminal phosphates were removed by treatment with alkaline phosphatase and, after denaturation and removal of the phosphatase with phenol, the DNA was reprecipitated and dissolved in a small volume of water. 

Of course the whole process of DNA synthesis requires a considerable input of energy. We have already said that the substrates for the polymerase are the nucleotide triphosphates, but it is the monophosphates that are inserted into the chain. So once again the splitting off of the two terminal phosphate groups from the nucleotides provides the driving force for a biosynthetic reaction. 

ITP is the only nucleotide that is known to substitute for GTP. The reaction was thought for many years to use ATP, but the apparent utilization of ATP was the result of the presence of other nucleotides in ATP preparations and the presence of nucleoside diphosphate kinase in the enzyme preparations. Thus the active nucleotide reacted in catalytic quantities and the nucleoside diphosphate produced accepted the terminal phosphate of ATP to give the appearance of participation of ATP in the decarboxylation of oxalacetate. Another complication in the analysis of this reaction is the secondary reaction of phosphopyruvate with nucleoside diphosphates. Phosphopyruvate kinase, which is present in crude extracts, transfers phosphate from the product of decarboxylation to the nucleotide also formed in the reaction, so that no phosphate transfer is seen, and the reaction appears to be a simple decarboxylation. It... 

These reactions are catalyzed by kinases, some of viiich have already been discussed in this chapter. The reaction may be virtually irreversible as in phosphate ester formation. Here the phosphate acceptor may be a hydroi l group of a carbohydrate (glucose, ycerol, fructose, nucleotides, etc.), (Moline, or pantetheine. The terminal phosphate may also be transferred to an acceptor without loss of high chemical potential, such as to nucleoside mono- or diphosphate or to a nitro n atom. These reactions are freely reversible. Nucleotide diphosphate may also donate its terminal phosphate as in the nucleotide monophosphate kinase reaction. 

Alkyl-S-esters of phosphoric acid are cleaved on oxidation with iodine [16] or periodate, or on hydrolysis under acid conditions, but are stable to sdkali and at neutral pH. These properties have been used to protect terminal phosphate groups in nucleotides [17], and allow specific removal of a number of other protecting groups in other parts of the molecule without the S-dkyl ester being affected. Alkyl-S-phosphates arc obtained via reaction of thiophosphoric acid with the corresponding alkyl hsdides. The S- ilkyl group is then introduced with... 

In the preceding sections the conversion of purines and purine nucleosides to purine nucleoside monophosphates has been discussed. The monophosphates of adenosine and guanosine must be converted to their di- and triphosphates for polymerization to RNA, for reduction to 2 -deoxyribonucleoside diphosphates, and for the many other reactions in which they take part. Adenosine triphosphate is produced by oxidative phosphorylation and by transfer of phosphate from 1,3-diphosphoglycerate and phosphopyruvate to adenosine diphosphate. A series of transphosphorylations distributes phosphate from adenosine triphosphate to all of the other nucleotides. Two classes of enzymes, termed nucleoside mono-phosphokinases and nucleoside diphosphokinases, catalyse the formation of the nucleoside di- and triphosphates by the transfer of the terminal phosphoryl group from adenosine triphosphate. Muscle adenylate kinase (myokinase)... 

We first describe the synthesis of unsymmetrical a.y-dinucleoside triphosphates involving 7-methylguanosine. For the construction of the terminal cap structure, there might be two possible reaction modes where an activatable protecting group (X) was introduced into a nucleotide by direct displacement with phosphate hydroxyl group (Method A) and by pyrophosphorylation between Xp and pN (Method B). 

Fig. 2.8. Nearest neighbour analysis and quantitative depurination analysis of a defined product from a primed synthesis reaction. When radioactive dATP (or dGTP) is used in the primed synthesis, depurination analysis will yield pyrimidine tracts each of which terminate in a radioactive 3 -phosphate. Thus only those depurination products which lie 5 -adjacent to the labelled nucleotide will be labelled. Each depurination product will be labelled to the same specific activity thus greatly simplifying the quantitation. Digestion of the labelled product with a mixture of micrococcal nuclease and bovine spleen phosphodiesterase yields the nucleoside 3 -monophosphates. Identification of the labelled products (by paper electrophoresis at pH 3.S) gives the nearest neighbours to the labelled substrate.

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