Nucleic acids synthesis, salvage

Salvage pathway. A family of reactions that permits nucleosides or purine and pyrimidine bases resulting from the partial breakdown of nucleic acids, to be reutilized in nucleic acid synthesis. 

Once the fusion has taken place, it is necessary to eliminate any unfused myeloma cells and to select only hybrid cells secreting antibody. This is primarily achieved by the use of hypoxanthine aminopterin thymidine (HAT) media and cells that are deficient in the enzyme responsible for incorporation of hypoxanthine into DNA. Figure 3 illustrates this process. The unfused splenocytes are not immortal and naturally die off in culture. The elimination of the unfused myeloma cells is carried out by the initial use of mutant myeloma cells selected for a deficiency in the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK), rendering them unable to use the salvage pathway for nucleic acid synthesis. The myelomas will die off... 

Purine nucleotides are required by the rapidly proliferating malaria parasite primarily for nucleic acid synthesis and energy metabolism. The malaria parasite cannot synthesize purines novo and depends for Its Intraerythrocytlc (IE) growth and development on salvage of purine bases from the host RBC and extracellular environment (2). We have shown with falciparum. In vitro, that hypoxanthlne is an essential purine base precursor for parasite Synthesis of adenosine and guanoslne nucleotides (3). Whetiier hypoxanthlne Is the malaria parasites preferred substrate vivo Is not known. 

The purine salvage pathway enzyme HGPRT phosphoribosylates hypoxanthine and guanine, which allows the free bases to be reused as precursors for nucleic acid synthesis. The purine analogues, 8-azaguanine (8AG) and 6-thioguanine (6TG), are also substrates for HGPRT. After further phosphorylation, 8AG and 6TG ribonucleotides become substrates for nucleic... 

Since there has been no evidence presented to support the hypothesis that free adenine can be formed de novo in biological systems from small molecule precursors, and furthermore, since purines have never been reported to have been essential dietary additions, the formation of nucleotides from free purines may be looked upon as a minor biosynthetic pathway. Undoubtedly, there is some utilization of free purines which are derived from the intestinal tract as well as from catabolic events within the cell. The term salvage pathway has been aptly applied to the reactions utilizing free bases for nucleic acid synthesis (206). 

A. Salvage pathways allow synthesis of nucleotides from free purines or pyrimidines that arise from nucleic acid degradation or dietary sources, which is more economical for the cell than de novo synthesis. 

Two types of pathways lead to nucleotides the de novo pathways and the salvage pathways. De novo synthesis of nucleotides begins with their metabolic precursors amino acids, ribose 5-phosphate, C02, and NH3. Salvage pathways recycle the free bases and nucleosides released from nucleic acid breakdown. Both types of... 

In addition to the pathways for synthesis de novo, mammalian cells and microorganisms can readily form mononucleotides from purine bases and their nucleosides and to a lesser extent from pyrimidine bases and their nucleosides. In this way bases and nucleosides formed by constant breakdown of mRNA and other nucleic acids can be reconverted (or salvaged ) to useful nucleotides, and the energy expended by the cell in synthesizing the bases is retained. 

Adenine phosphoribosyltransferase catalyzes the conversion of adenine to AMP in many tissues, by a reaction similar to that of hypoxanthine-guanine phosphoribosyltransferase, but is quite distinct from the latter. It plays a minor role in purine salvage since adenine is not a significant product of purine nucleotide catabolism (see below). The function of this enzyme seems to be to scavenge small amounts of adenine that are produced during intestinal digestion of nucleic acids or in the metabolism of 5 -deoxy-5 -methylthioadenosine, a product of polyamine synthesis. 

The synthesis of purine and pyrimidine nucleotides needed for the biosynthesis of nucleic acids, and in turn the processes of transcription and translation provide a number of suitable targets for therapeutic intervention. These have been subject of much interest for the discovery of new antiprotozoal drugs as well. Developments in two areas, viz. the purine salvage pathway and the pyrimidine biosynthesis have yielded useful drugs and are discussed. 

It has been indicated above that for purine requirements the protozoans depend on salvage of preformed purines. Contrary to this, the protozoal parasites have capability for the de novo biosynthesis of pyrimidines, which are equally essential for nucleic acid and protein synthesis. The pyrimidine biosynthesis in protozoans is very similar to the pathway mapped for eukaryotes [1,20,21]. 

In the purine salvage pathway, purine bases obtained from the normal turnover of cellular nucleic acids or (to a lesser extent) from the diet are reconverted into nucleotides. Because the de novo synthesis of nucleotides is metabolically expensive (i.e., relatively large amounts of phosphoryl bond energy are used), many cells have mechanisms to retrieve purine bases. Hypoxanthine-guaninephos-phoribosyltransferase (HGPRT) catalyzes nucleotide synthesis using PRPP and either hypoxanthine or guanine. The hydrolysis of pyrophosphate makes these reactions irreversible. 

Eimeria tenella. It appears that the sporulated oocysts of E. tenella are capable of de novo pyrimidine synthesis since sporozoites incorporate " C-labeled aspartate and orotate into pyrimidine nucleotides (103). Besides de novo synthesis, E. tenella is also capable of pyrimidine salvage. E. tenella will incorporate uracil, cytidine and uridine but not thymidine into its nucleic acids (104). This parasite is dependent on UMP for the synthesis of TMP and thymidylate synthase activity is present in extracts of E. tenella (105). As with both Plasmodium and the kinetoplastids, the thymidylate synthase of E. tenella exists as a bifunctional protein (91). 

Dietary uptake of purine and pyrimidine bases is minimal. The diet contains nucleic acids and the exocrine pancreas secretes deoxyribonuclease and ribonucle-ase, along with the proteolytic and lipolytic enzymes. This enables digested nucleic acids to be converted to nucleotides. The intestinal epithelial cells contain alkaline phosphatase activity, which will convert nucleotides to nucleosides. Other enzymes within the epithelial cells tend to metabolize the nucleosides to uric acid, or to salvage them for their own needs. Approximately 5% of ingested nucleotides will make it into the circulation, either as the free base or as a nucleoside. Because of the minimal dietary uptake of these important molecules, de novo synthesis of purines and pyrimidines is required. 

The salvage pathway utilizes preformed pyrimidines and purines for the synthesis of nucleic acids and is highly active in various types of cells. Uridine kinase plays a key role in the pyrimidine salvage pathway and its concentration is considered to reflect the relative efficiency of the system in utilizing preformed pyrimidines [74]. Adenosine kinase plays a similar role in making use of preformed purines [75]. It should be noted, however, that uridine and adenosine kinases are not the only enzymes involved in the salvage pathway and other deoxynucleoside kinases, phosphorylases, and phosphoribosyltransferases [76] also have important roles. 

Since synthesis of pyrimidine and purine nucleotides de novo (anew) is energetically demanding, it may occur using heterocyclic bases from dietary sources or from those released by the turnover of nucleic acids. Such reactions, called salvage pathways (Figure 16.7) since they enable the reutilization of existing bases, facilitate considerable savings in ATP. 

For the past decade this laboratory has devoted much of its attention to an examination of various facets of purine metabolism in human erythrocytes. These cells do not have the complete pathway for the novo synthesis of purines and do not make nucleic acids. On the other hand, they have an active nucleotide metabolism and contain the salvage enzymes, hypoxan-thine-guanine phosphoribosyl transferase (HGPRTase), adenine phosphoribosyl transferase (APRTase) and adenosine kinase. In view of the fact that the activities of certain enzymes of purine metabolism are quite high (e.g., purine nucleoside phos-phorylase occurs at a level of about 15 umolar units/ml of erythrocytes) and the total mass of erythrocytes in the adult human being is in excess of two liters, it appears that these cells play an important and perhaps not yet fully appreciated role in the whole body economy of purines in man. Therefore, we believe that the human erythrocyte provides a very useful model system for the examination of purine metabolism in man as well as for investigations of the action of certain purine and purine nucleoside antimetabolites, many of which are important in medicine. 

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