Jennifer Allsop

Purine de novo synthesis in liver and developing rat brain, and the effect of some inhibitors of purine nucleotide interconversion.

The rate of purine de novo synthesis from sodium formate in developing rat brain falls in the late gestational stages to birth, rises again in the 1st week of life and then decreases rapidly to the 3rd week, and continues declining up to 8 weeks of life (adulthood). The changes in the overall purine biosynthetic rate with respect to time are similar to those in the activity of the rate-limiting enzyme [amidophosphoribosyltransferase (phosphoribosyl diphosphate amidotransferase; EC 2.4.2.14)]. Azaserine [O-diazoacetyl-L-serine], a known inhibitor of glutamine requiring metabolic steps, inhibits purine de novo synthesis by more than 90%. This confirms that the method used to assess purine de novo synthesis in fact does so. The effects of virazole [1-beta-ribofuranosyl-1-H,1,2,4-triazole-3-carboxamide], an inhibitor of IMP dehydrogenase (EC 1.2.1.14), and of alanosine [L-2-amino-3-(hydroxynitrosamino)propanoic acid] an inhibitor of adenylosuccinate synthetase (EC 6.3.4.4), on the rate of purine de novo synthesis were investigated in liver and brain tissue. The effect of the xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] was also investigated in liver tissue. The biosynthesis of the purines which were extruded into the incubation medium as well as those which remained in the tissue was studied. Only inhibitory effects were observed, and these were confined to the purines remaining in the tissue. Allopurinol was completely inert from this viewpoint. The results are compared with those of other workers using lymphoid cells, and emphasize the differences in the control of de novo purine synthesis in different tissues and under different conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Purine Synthesis and Salvage in Brain and Liver

Previous work in which we measured the specific activities of amidophosphoribosyltransferase (PRPP-At; EC 2.4.2.14) and hypo-xanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) in the brains of rats at different ages suggested that the HPRT-catalysed purine salvage pathway became more important relative to the purine de novo synthesis pathway after the main bursts of neuroblast and neuroglial proliferation.1 This interpretation involves the assumption that changes in the specific activity of PRPP-At, which catalyses the first and presumably ratelimiting reaction on the purine de novo synthesis pathway, are a valid measure of changes in the flow of metabolites along the whole pathway. The evidence2 that the cyclic nucleotides (cAMP and cGMP) have important second messenger functions in the central nervous system, some of which are related to post-synaptic neurotransmission, and which presumably become more important as neuronal function increases, suggested to us that HPRT might have a specific function in maintaining the supply of a neuropharmacologically important low molecular weight purine derivative which could be cGMP. We also suggested that a failure of this mechanism might be responsible for the functional disorders of the Lesch-Nyhan syndrome3.

The effect of gold salts on the biosynthesis of uridine nucleotides in human granulocytes

Abstract The action of sodium aurothiomalate on the activity of the enzymes which catalyse the synthesis of the uridine 5′-phosphates has been studied in vitro using normal human granulocytes. The following enzymes were markedly inhibited in disrupted cell preparations: carbamoyl phosphate: l -aspartate transferase (EC 2.1.3.2), orotidine-5′-phosphate-pyrophosphate phosphoribosyltransferase (EC 2.4.2.10) and uridine monoand diphosphokinases [ATP: nucleosidemonophosphate phosphotransferase (EC 2.7.4.4) and ATP:nucleosidediphosphate phosphotransferase (EC 2.7.4.6)]. ATP: uridine 5′-phosphotransferase (EC 2.7.1 f) was also inhibited although to a lesser extent than the other enzymes. The activities of l -4,5-dihydroorotate aminohydrolase (EC 3.5.2.3), l -4,5-dihydroorotate: O2 oxidoreductase (EC 1.3.3.1) and orotidine-5′-phosphate carboxylase (EC 4.1.1.23) were not affected under the conditions used. The inhibitory effects could be prevented by adding sulphydryl group-protecting reagents (2-mercaptoethanol, l -cysteine, dithiothreitol and reduced glutathione), the inhibition was reduced slightly by adding serum to the system. There was no inhibition when intact cells were used. Some preliminary observations on the possible relationship of the gold-sensitive sulphydryl groups to the active sites of enzymes have also been made. The possible implications of these findings in connection with gold induced neutropoenia are discussed.

Activities of Amidophosphoribosyltransferase and Purine Phosphoribosyltransferases in Developing Rat Brain

The availability of purine nucleotides at their sites of physiological action depends upon the balance between purine synthesis de novo and the purine phosphoribosyltransferase catalysed purcine salvage reactions. It was reported that purine synthesis de novo was considerably less active in brain tissue than in liver1.

Purine De Novo Synthesis and Salvage During Testicular Development in the Rat

We have encountered several patients with the Lesch-Nyhan syndrome in whom, although the testes were clinically normal in early childhood, they failed to develop at puberty and in one they could not be found at autopsy when he was 18 years old. This patient showed no evidence of pre-pubertal development although the plasma testosterone and gonadotrophin levels were consistent with approaching puberty and with his bone age of 13.2 years1. The testes of another clinically prepubertal Lesch-Nyhan syndrome patient who died at age 14 years showed tubules lined by simple germinal epithelium with no evidence of spermatogenesis. The interstitial tissue contained: numerous fibroblastic cells but no interstitial cells (Leydig cells) were seen.

PURINE DE NOVO SYNTHESIS AND SALVAGE DURING TESTICULAR DEVELOPMENT IN THE RAT: 3

Purine de novo synthesis and salvage occur in the testis. Cell division accelerates at puberty. We have correlated changes in the activity of purine de novo synthesis and purine salvage with the histology of the developing testis and with serum hormone levels. Purine de novo synthesis peaked at 17 days and HPRT activity at 35 days. APRT activity decreased at 17 days and remained very low. The 17 day peak of purine de novo synthesis coincided exactly with the appearance of spermatids and prominent spermatocytic meiosis. The 35 day HPRT peak coincided with the first appearance of active spermatogenesis, full thickness of precursor cells and fully fledged spermatozoa. These findings indicate that the full development of reproductive function in the male requires HPRT as well as purine de novo synthesis.

Phosphoribosylpyrophosphate (PRPP) Amidotransferase (EC 2.4.2.14) Activity in Unstimulated and Stimulated Human Lymphocytes

The mechanisms whereby new DNA synthesis is initiated after the exposure of lymphocytes to mitogens are poorly understood. It has been suggested that an influx of calcium ions, and increased levels of cyclic guanosine 3′, 5′- monophosphate (cGMP) in the cells may be involved. Hovi, Allison & Allsop (1975) reported that the phosphoribosylpyrophosphate (PRPP) content of phytohaemagglutinin (PHA) stimulated lymphocytes increases transiently during the first hour of PHA-stimulation. They suggested either that this is a step in a chain of events which increases purine biosynthesis de novo and makes purine ribonucleotides available for DNA synthesis, or that a pulse of PRPP and purine biosynthesis is in some way needed to push the lymphocytes from the G0 to the G1 phase of the cell cycle.