Purine metabolism in cultured human fibroblasts derived from patients deficient in hypoxanthine phosphoribosyltransferase, purine nucleoside phosphorylase, or adenosine deaminase.

Abstract

ABS14RACT Rates of purine synthesis de novo, as measured by the incorporation of [4C]formate into newly synthesized purines, have been determined in cultured human fibroblasts derived from normal individuals and from patients deficient in adenosine deaminase, purine nucleoside phosporylase, or hypoxanthine phosphoribosyltransferase, three consecutive enzymes of the purnne salvage pathway. All four types of cell lines are capable of incorporating [14C]formate into purines at approximately the same rate when the assays are conducted in purine-free medium. The purine overproduction that is characteristic of a deficiency in either the transferase or the phosphorylase and that results from a block in purine reutilization can be demonstrated by the resistance of [14Clformate incorporation into purines to inhibition by hypoxanthine in the case of hypoxanthine phosphoribosyltransferase-deficient fibroblasts and by resistance to inhibition by inosine in the case of purine nucleoside phosphorylast-deficient fibroblasts. Human cells in culture utilize purines derived from two metabolic routes-either by synthesis de nowo beginning with 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) and glutamine or by "salvage" from PP-ribose-P and purines present in the medium or arising from the intracellular degradation of nucleic acids. Individuals who lack purine nucleoside phosphorylase (PNP) or hypoxanthine phosphoribosyltnferase (HPRT), two consecutive enzymes of the purine salvage pathway, synthesize purines de novo at an accelerated rate (1-3). Although several cell culture systems have been devised to demonstrate the purine overproduction characteristic of patients with a deficiency of HPRT (4-8), none has been devised to show the comparable overproduction in PNP deficiency. The mechanism by which the rate of purine synthesis de novo is controlled has been the subject of intensive study by numerous investigators. It is believed to involve the regulation of PP-ribose-P synthetase or PP-ribose-P amidotransferase, the enzymes which catalyze the first two reactions of this pathway. In Ehrlich ascites tumor cells, the rate of synthesis of PP-ribose-P has been shown to be inversely related to the concentration of adenine and guanine nucleotides (9). Partially purified human PP-ribose-P synthetase has been shown to be inhibited by ADP and AMP (10). The activity of PP-ribose-P amidotransferase and of the de novo pathway as a whole may be controlled by the intracellular levels of PP-ribose-P and of adenine and guanine nucleotides (4,(11)(12)(13)(14)(15)(16). PP-ribose-P is normally present at concentrations well below the Km for the enzyme. It also converts the human enzyme to its more active, low molecular weight form. Adenine and guanine nucleotides act as inhibitors of the partially purified enzyme and also convert the human enzyme to its less active, high molecular weight form. Thus, either an increase in the concentration of PP-ribose-P or a decrease in the levels of inhibitory nucleotides could potentially accelerate the rate of pur...

Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets.

“…Thus, our studies overall are quite compatible with data from other cells, which suggest that the cellular content of NTP is modulated by two interactive variables-the negative feedback control of nucleotides upon their own synthesis (at several enzymatic levels; references 11, 24,31,[55][56][57][58] and positive regulatory control by PRPP (27,38,41,56). Possible site(s) ofaction ofglucose on nucleotide synthesis.…”

“…The rise in GTP (and in insulin secretion) induced by guanine, and the fall in both parameters induced by MPA, were in general, inversely proportional to the starting content ofpurine nucleotides, which are feedback inhibitors of salvage ( 11 ) and de novo ( 1 1, 31, 55 ) synthetic pathways as well as ofthe synthesis of their common precursor PRPP (1 1,24,41,[55][56][57]. Thus, our studies overall are quite compatible with data from other cells, which suggest that the cellular content of NTP is modulated by two interactive variables-the negative feedback control of nucleotides upon their own synthesis (at several enzymatic levels; references 11, 24,31,[55][56][57][58] and positive regulatory control by PRPP (27,38,41,56).…”

The purine nucleoside phosphorylasepnp-1regulates epithelial cell resistance to infection inC. elegans

“…Obligate intracellular pathogens as diverse as viruses and the eukaryotic pathogens Plasmodium falciparum and microsporidia depend on their hosts for purines, with several types of transporters identified in eukaryotic pathogens used to steal purines from hosts [4–6, 44–46]. Extensive work on purine metabolism and its effects on human physiology have come from studies of so-called ‘inborn errors of metabolism’, which are often due to mutations in purine enzymes [21, 38, 47–50]. Interestingly, although mutations in enzymes of the purine de novo and salvage pathways cause of various disorders (e.g.…”

De novo purine synthesis, purine salvage, and DNA synthesis in normal and Lesch-Nyhan fibroblasts infected with Mycoplasma pneumoniae

“…In this latter case, any metabolic determinations reflect the total cell population and not just the target cells. Previous work indicated that there is a close correlation between host cell nucleic acid metabolism and the ability of M. pneumoniae to cause a cytopathic effect (CPE) in host cells (24). In this study, we used normal human lung fibroblasts (MRC-5 cells) and Lesch-Nyhan fibroblasts, which are deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8), to probe the effects of M. pneumoniae infection further (15,23,27). As the Lesch-Nyhan cells are HGPRT deficient, they lack the purine salvage pathway.…”