Purine into nucleotide pools

INCORPORATION OF PURINE ANALOGS INTO NUCLEOTIDE POOLS... 

Evidence is accumulating from radioactive labelling and other experiments that pyrimidine nucleosides supplied exogenously are not incorporated into parasite nucleic acids in contrast to purine nucleosides [58, 346, 348-351, 353-355]. In particular, the parasite in vitro utilises a variety of preformed purines, especially adenosine, and there is now evidence that it depends upon the host for supplies of purines in vivo, such material coming from the adenosine nucleotide pool of the red cells [356]. The plasmodial necessity for de novo synthesis of pyrimidines is apparently due both to a lack of penetration of pyrimidines through the red cell membrane and to a similar transport deficiency through the parasite membrane, rather than to an inability to polymerise them into nucleic acids [349]. Five possible points of attack have therefore been suggested for an antimalarial to exert its effects, (a) com-... 

The importance of blood and, in particular, of erythrocytes as a vehicle for transport of purines is well known >. Considerable quantities of purines enter and leave the nucleotide pools of red cells which take up adenine, guanine, hypoxanthine, and xanthine and convert them into nucleotides. No matter what purine is taken up by erythrocytes, hypoxanthine appears to be the main purine released in vivo. Human erythrocytes cannot synthesize purines de novo and are unable to convert hypoxanthine or guanine into adenine. Hypoxanthine release is mediated by prior conversion of the various purine nucleotides to IMP. In the present paper, some of the mechanisms which regulate the catabolic paths of this nucleotide are studied. 

The present report summarizes some of our recent investigations into the incorporation of natural purine bases and nucleosides, as well as related analogs, into the nucleotide pools of human erythrocytes. A number of these studies were greatly facilitated by the recent development of high pressure liquid chromatography which makes it possible to examine intracellular... 

INCORPORATION OF NATURAL PURINE RIBONUCLEOSIDES AND THEIR ANALOGS INTO THE NUCLEOTIDE POOLS OF HUMAN ERYTHROCYTES... 

The enzyme studies described above are also compatible with a number of experiments in which incorporation of ribonucleotides into DNA has been shown to be more efficient than incorporation of deoxyribonucleotides Functional compartmentation of DNA precursors is also observed in the utilization of deoxyuridine or thymidine for DNA which in E. coli or in eukaryotic cells occurs without prior equilibration with the free dTTP pool On the other hand, in thymocytes purine deoxyribonucleosides are converted to nucleotides but are not utilized for DNA replication however here they allosterically inhibit ribonucleotide reduction . All these data agree with the existence of two different deoxyribonucleotide pools, one associated with the replitase complex and another independent pool of free deoxyribonucleotides. 

Formate serves as a precursor of carbons 2 and 8 of the purine ring and of the 5-methyl group of thymine. Thus one may obtain information on the dynamic state of nucleic acids in tissues by measuring the rate of incorporation of injected formate-C into nucleic acids. It must be emphasized that the specific activities of isolated nucleic acids taken at early time intervals after injection of formate-C may reflect changes in pool size and specific activities of the precursors, either formate or the nucleotides, which are the immediate precursors of the nucleic acids, in addition to reflecting changes in the rate of nucleic acid synthesis. 

Aminobenzamide (SAB), an inhibitor of ADP-ribosyl transferase, also affects de novo purine biosynthesis, demonstrated by an inhibition of incorporation of radiolabel from glucose, methionine and formate into the DNA (1-3). The step(s) affected have not been identified. Mutant ceU lines, deficient in salvage nucleotide synthesis, are no more sensitive to SAB than wild-type (4). Also, there is no effect of SAB on dNTP pool sizes (2, 5). These data indicate that the effect of SAB on de novo purine biosynthesis is not normally rate-limiting. Other effects of SAB include inhibition of cell growth and inhibition of adenosine transport (5). 

Table 1 shows the effect of various purine inhibitors on incorporation of ( H) hypoxanthine into purine nucleotides of PRBC. Control cultures and inhibitor treated cultures were all prepared from a common pooled source of malaria infected RBC and, therefore, have the same initial parasitemia (4.8% PRBC) and hematocrit (12%). 

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