The metabolism of adenine derivatives in different parts of the brain of the rat,and their release from hypothalamic preparations on excitation

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [¹⁴C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [¹⁴C]adenine, the hypothalamic tissues released about 0.2% of their ¹⁴C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 μUM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned, and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

The metabolism of adenine derivatives in different parts of the brain of the rat, and their release from hypothalamic preparations on excitation.

Five enzymes concerned with the metabolism of adenine derivatives were assayed in seven regions of the rat brain. A region which included the hypothalamus had the highest AMP deaminase and adenosine deaminase activities, while its 5'-nucleotidase activities were relatively low. The enzymes named and also the uptake of [14C]adenine by incubated tissue samples were more active with hypothalamic than with neocortical tissues. On superfusion with glucose-bicarbonate saline after assimilating [14C]adenine, the hypothalamic tissues released about 0.2 per cent of their 14C content per minute. This release was increased fourfold with electrical excitation but the presence of 0.25 muM tetrodotoxin prevented most of this increase. The compounds released during superfusion and electrical stimulation were preponderantly hypoxanthine, inosine, and adenosine, with only small amounts of adenine nucleotides. The output of all these compounds increased during the period of stimulation and also the proportion of adenine nucleotides increased when stimulation was carried out in the presence of tetrodotoxin. The output of the nucleotides and adenosine increased more promptly when stimulated than did that of the other compounds named. The results are discussed in terms of the metabolic roles of the enzymes concerned. and in relation to whether the enzymes are acting on intracellular or extracellular substrates.     

Metabolism of [14C]adenine and derivatives by cerebral tissues, superfused and electrically stimulated

1. Uptake of [(14)C]adenine and [(14)C]adenosine from surrounding fluids to guinea-pig cerebral tissues was measured during incubation in vitro. Output of (14)C-labelled compounds from the loaded tissues to superfusion fluids occurred on continued incubation, at about 0.2% of the tissue's content/min, and this rate was increased about fourfold by electrical excitation of the tissue. 2. The compounds released from the tissue to superfusion fluids included adenine, adenosine, inosine and hypoxanthine with small amounts of nucleotides. Output of all these compounds, except adenine, increased on excitation. Media depleted of oxygen or glucose also increased the output of (14)C-labelled derivatives from [(14)C]adenine-loaded tissues, and this augmented output was further increased by electrical stimulation. 3. [(14)C]Adenosine was found as the main product from [(14)C]ATP when this was added at low concentrations to fluids superfusing cerebral tissue. Metabolic and neurohumoural explanations of the liberation and action of adenosine derivatives in the tissue are discussed.     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

Adenosine in cerebral homeostatic role: appraisal through actions of homocysteine, colchicine, and dipyridamole

Mammalian neocortical tissues were incubated in [14C]adenine-containing fluids and their newly-synthesized adenine derivatives examined after periods of superfusion. Increased [K+] released adenine derivatives from the tissues, a release diminished by homocysteine. Homocysteine acted also to diminish the tissue content of adenosine plus its metabolites hypoxanthine and inosine, while increasing that of S-adenosylhomocysteine. Hypoxia also increased the tissue content and the output of adenosine plus its metabolites, and again homocysteine augmented the S-adenosylhomocysteine. Glutamic acid also increased tissue content and output of adenosine and derivatives, an action diminished by homocysteine and associated with augmented S-adenosylhomocysteine. Colchicine or dipyridamole did not prevent augmentation of S-adenosylhomocysteine by the reagents described; the sequence from adenosine phosphates to S-adenosylhomocysteine is concluded to be intracellular and not to involve extracellular formation of precursor adenosine. Adenosine displayed properties consistent with its being involved in two distinct categories of homeostasis, and also with its exerting an inhibitory tone in normal cerebral systems.     

Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

ACTIONS OF NEUROHUMORAL AGENTS AND CEREBRAL METABOLITES ON OUTPUT OF ADENINE DERIVATIVES FROM SUPERFUSED TISSUES OF THE BRAIN

—Adenine nucleotides of guinea-pig neocortical tissues were labelled by prior incubation with [¹⁴C]adenine and excess of adenine was then removed by superfusion with precursor-free media. During continued superfusion labelled adenine derivatives were released at a stable rate of about 0·05 per cent of the tissue ¹⁴C/min and this rate was increased about five-fold by electrical stimulation. Various compounds, including some known to increase the cyclic AMP content of cerebral tissues, were examined for action on the release of [¹⁴C]adenine derivatives from the tissue and also on the rates of lactate production by the tissue, both before and during electrical excitation. The tissue content of adenine nucleotides following exposure of the tissue to these compounds was also determined. Noradrenaline, γ-aminobutyrate and acetylcholine together with carbamoylcholine at the concentrations examined were without effect on the release of ¹⁴C compounds from the tissue. Also, noradrenaline and γ-aminobutyrate caused no alteration in lactate production but brought about some decrease in the adenylate energy charge of the tissue. Histamine, 100 μm , brought about a small but consistent increase (35 per cent) both in release of ¹⁴C-compounds and lactate output, while reducing the adenylate energy charge of the tissues. l -Glutamate at 5 mm decreased the tissue adenylate energy charge to a greater extent than did histamine; it increased the release of ¹⁴C-compounds seven to eight-fold and similarly increased the tissues' rates of lactate production. Lower concentrations of glutamate had smaller effects. In those cerebral tissues whose cyclic AMP content is increased by l -glutamate, the increase is probably brought about by intermediation of released adenosine.     

Purine reutilization in phytohemagglutinin-stimulated human T-lymphocytes.

Incubation of human peripheral blood T-lymphocytes with phytohemagglutinin (PHA) resulted in increased rates of metabolism of the purine bases adenine, hypoxanthine, and guanine. The respective rates decreased to unmeasurable levels in cells incubated without PHA. [¹⁴C]Adenine was converted predominantly into adenine nucleotides, with slight catabolism to hypoxanthine and very low conversion into guanine nucleotides. [¹⁴C]Guanine labeled predominantly the guanine nucleotide pool, but some adenine nucleotide formation also took place. From [¹⁴C]hypoxanthine, adenine nucleotides in the soluble pool were more heavily labeled than the guanine nucleotides, whereas in the nucleic acid fraction the latter contained more radioactivity. Adenosine at low concentrations was mainly phosphorylated to adenine nucleotides, but at higher concentrations this process leveled off, while deamination continued to increase linearly. PHA-stimulation resulted in an increased rate of adenosine metabolism but no qualitative differences in comparison to unstimulated cells were observed. Enzyme assays indicated that after PHA-stimulation the activities of adenine and hypoxanthine phosphoribosyltransferases, and those of adenosine deaminase and kinase, increased with a peak at 48 h, when expressed on a per cell basis, but not at all when expressed per mg of protein. We conclude that stimulation of human T-lymphocytes with PHA increases the capacity of the cells for purine nucleotide synthesis from all the directly re-utilizable catabolic products, namely the purine bases and adenosine.     

Release of14C-adenine derivatives from cerebral cortical slices: Effect of phenytoin and phenobarbital

Both ouabain, 0.1 mM, and veratridine, 0.05 mM, increased the release of¹⁴C-labeled compounds from rat cortical slices prelabeled with¹⁴C-adenine and incubated in vitro. The increment in radioactivity released by both depolarizing agents was almost entirely a result of increases in adenosine, inosine, and hypoxanthine. However, the distribution of these three compounds in the ouabain-induced efflux (adenosine, 12%; inosine, 51%; hypoxanthine, 36%) contrasted with that evoked by veratridine (adenosine, 42%; inosine, 15%; hypoxanthine, 38%). Phenytoin significantly reduced the efflux of¹⁴C-labeled compounds produced by both ouabain and veratridine, but phenobarbital had no effect. The intracortical injection of adenosine, inosine, and hypoxanthine has been shown to induce epileptiform discharges in rats, and it is suggested that the inhibitory effect of phenytoin on the release of adenine derivatives may play a role in its antiepileptic action.     

5'-adenine mononucleotides in preparations from guinea pig cerebral cortex. Accumulation, translocation and release.

Isolated nerve terminals (synaptosome beds) were prepared from the neocortex of guinea pig and their ability to accumulate and release adenine nucleotides was studied. Synaptosome beds prelabelled with [14C]adenosine released newly synthesized [14C] adenine derivatives on superfusion. Electrical stimulation and K+ depolarization gave augmented output of both [14C] adenine derivatives and lactate from the preparations. Action of metabolic inhibitors on this output was examined. During incubation and superfusion, the synaptosomes displayed glycolysis and synthesis of ATP. Supply of adenine derivatives to the nerve terminals also occurred by translocation from other parts of the tissue.     

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.     

Metabolism of Adenine and Hypoxanthine in a Hormone Autonomous Genetic Tumour Line of Tobacco

Genetic tumour tissues of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grow on auxin and cytokinin-free medium, were incubated with [14C]-/[3H]-adenine or [3H]-hypoxanthine to investigate cytokinin biosynthesis. Adenine was supplied to tissues of two different ages (2- and 3.5-week-old) for 8, 24 or 30 h. The uptake was over 91.0 % (of "supplied radioactivity") by 2-week-old tissues as compared to around 50.0 % uptake by 3.5-week-old tissues. Incorporation into cytokinins could not be detected. While unmetabolized adenine accounted for only about 24.0 and 13.4 % of "extracted radioactivity" (following 8 and 30 h incubation, respectively) in 2-week-old tissues, relatively higher levels, i.e. 36.0 and 34.5 % (following 8 and 24 h incubation, respectively) were present in 3.5-week-old tissues. The metabolites formed were adenosine and its nucleotides (4.5 - 16.5 % and 37.4 - 60.2 % of the extracted radioactivity, respectively). Hypoxanthine was supplied to 3.5-week-old tissues for 8 and 24 h. While the uptake was low (<28.0 % of supplied radioactivity), the major proportion of extracted radioactivity was due to unmetabolized hypoxanthine (79.8 % and 85.9 % after 8 and 24 h incubation periods, respectively); the minor metabolites were inosine and adenosine (both <0.5 %) and their nucleotides (< 3.5 %). Radioactivity incorporation into cytokinins from hypoxanthine was not detected. Thus in the present investigations precursor incorporation from either adenine or hypoxanthine into cytokinins could not be demonstrated. It is possible that this may be due to slow rate of cytokinin turnover in these tissues.     

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.     

Combined effect of adenosine and papaverine on the metabolism of adenine nucleotides in rat thymocytes

It is shown that in [14C]adenine-labelled thymocytes adenosine increases the content of adenine nucleotides and simultaneously accelerates their catabolism. Papaverine induces acceleration of splitting and a decrease of the specific ATP radioactivity but increases the AMP content and its specific radioactivity. The both effectors intensify considerably the outlet of total radioactive label from cells. If the papaverine effect in the extracellular medium results in accumulation mainly of hypoxanthine in the extracellular medium then the adenosine presence causes accumulation of inosine and hypoxanthine approximately in equal amounts. The release of labelled adenosine from thymocytes in all cases is an insignificant part of extracellular radioactivity. A conclusion is drawn that under conditions of the combined action of the substances under study papaverine removes the adenosine effect caused by its under study papaverine removes the adenosine effect caused by its phosphorylation with the formation of ATP and exerts the dose-depended action on adenine nucleotide metabolism in thymocytes.     

Circadian variations of adenosine level in blood and liver and its possible physiological significance

The role of adenosine as a possible physiological modulator was explored by measuring its concentration in different tissues during a 24-hour period. Initially the circadian variations of adenosine and other purine compounds such as inosine, hypoxanthine, uric acid and adenine nucleotides were studied in the rat blood. A daily cyclic response was observed, with low levels of adenosine from 08.00 - 20.00 h, followed by an increase from this time on. Inosine and hypoxanthine levels were elevated during the day and low at night. The uric acid changes observed indicate that the decrease in purine catabolism coincides with a decrease in inosine and hypoxanthine levels and an increase in adenosine. The blood adenine nucleotides, energy charge and phosphorylation potential remained constant during the day and showed oscillatory changes during the night. Similar studies were made in the liver, a primary source of circulating purines. Liver adenosine was high during the night while inosine and hypoxanthine remained low along the 24 hours. The results suggest that liver purine metabolism might participate in the maintenance and renewal of the blood purine pool and in the energy state of erythrocytes in vivo.     

Characteristics of adenosine activity on adenine nucleotide metabolism of rat thymocytes

The effects of adenosine on adenine nucleotide metabolism in [14C]adenine-labeled rat thymocytes were studied. It was shown that adenosine increases the intracellular pool of adenine nucleotides, predominantly ATP, which is accompanied by marked acceleration of their catabolism and a release of labeled products (especially inosine, hypoxanthine and adenosine) from the thymocytes. The effect of adenosine depends on its concentration and manifests itself already at 10(-6) M. 2-Deoxycoformycin partly relieves the effect of adenosine on adenine nucleotide metabolism. Exogenous deoxyadenosine, inosine, hypoxanthine and adenine, unlike adenosine, do not significantly affect the adenine nucleotide catabolism and the label release from the cells. All the effectors under study strongly increase inosine transport from the thymocytes, and inhibit, with the exception of adenosine, the hypoxanthine release from the cells.     

Stimulation-evoked release of purines from the rabbit retina

The evoked release of purines from rabbit retinae preloaded with [³H]adenosine was studied in vitro. Potassium (8.6–43.6 mM) and ouabain (1 or 10 μM) increased the release of radioactivity in a concentration-dependent manner. The K⁺-evoked release was significantly reduced when the superfusion was carried out at 2–4°C. The effect of K⁺ (8.6, 13.6 and 23.6 mM) and of ouabain (1 μM) were completely abolished when the retinae were superfused with a Ca²⁺-free medium containing 0.1 mM EGTA. Calcium removal only partially reduced the effect of higher K⁺ and ouabain concentrations (43.6 mM and 10 μM, respectively). Further, the effect of K⁺ was found to be independent of extracellular Ca²⁺ when retinae were pretreated with ouabain for 30 min. Stimulation of the retina with light flashes induced a small, persistent increase in the release of radioactivity observable for several minutes after the end of stimulation.The superfusate contained mainly hypoxanthine and inosine. There were no significant changes in the relative proportions of the different purine compounds released before or in response to either K⁺ (23.6 mM) or ouabain (10 μM) stimulation. Potassium stimulation significantly increased the release of adenosine, inosine and hypoxanthine. Addition of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), significantly increased the relative proportions of released endogenous adenosine and inosine.The results indicate that K⁺ stimulation induces the release of purines from the rabbit retina by a Ca²⁺- and energy-dependent process. Light flashes also induce a purine release. The results suggest an active role for adenosine in retinal neurotransmission.     

Output of [14C]adenine nucleotides and their derivatives from cerebral tissues. Tetrodotoxine-resistant and calcium ion-requiring components

1. Neocortical tissues, exposed briefly to [(14)C]adenine and containing over 98% of their (14)C as adenine nucleotides, when superfused with glucose-bicarbonate salines released about 0.1% of their (14)C content/min to the superfusate. 2. Addition of unlabelled adenosine to the superfusing fluid increased the (14)C output three- to four-fold; half-maximal increase was given by about 40mum-adenosine, and reasons are adduced for considering the activity of adenosine kinase to be a major factor in conditioning the (14)C output. Adenosine similarly increased the enhanced (14)C output caused by electrical excitation of the superfused tissue; it brought about only a small increase in tissue glycolysis. 3. Output of (14)C from the [(14)C]adenine-labelled tissues was increased when Ca(2+) was omitted from the superfusing fluids, but electrical stimulation did not then liberate more (14)C. Nevertheless, such tissues still responded to electrical stimulation by increased glycolysis, and their (14)C output again became susceptible to increase by electrical stimulation when Ca(2+) was restored. 4. The six-fold increase in tissue glycolysis caused by electrical excitation was almost completely inhibited by tetrodotoxin at 0.1mum and above, but this was associated with about 50% inhibition only in the output of (14)C from tissues preincubated with [(14)C]adenine. The (14)C-labelled compounds of which output was most inhibited by tetrodotoxin were adenosine, inosine and hypoxanthine whereas output in a nucleotide fraction was little affected.     

Metabolism of adenosine, AMP and ATP in rats

Metabolism of [14C]adenosine in a dose of 100 mg per 1 kg of mass and [14C]ATP in the equimolar quantity was studied in rats after intraperitoneal administration. Adenosine is shown to enter tissues of the liver, spleen, thymus, heart and erythrocytes where it phosphorylates into adenine nucleotides (mainly ATP) and deaminates into inosine. The content of adenosine increases for a short period in the above tissues, except for erythrocytes and plasma. The latter accumulates a considerable amount of inosine and hypoxanthine, but only traces of uric acid, xanthine and adenine nucleotides. ATP administered to rats catabolizes through the adenosine formation. The exogenic adenosine and ATP replace in tissues and erythrocytes only a slight part (1-12%) of their total adenine nucleotide pool. The content of these metabolites and ADP in the blood plasma does not change essentially under the effect of adenosine, ATP and AMP. It is shown on rats whose adenine nucleotide pool of cells is marked by the previous administration of [14C]adenine that injections of adenosine, ATP and inosine do not accelerate catabolism of adenine nucleotides in tissues and erythrocytes as well as do not increase the level of catabolism products in the blood plasma. Adenosine enhances and ATP lowers the content of cAMP in spleen and myocardium, respectively.     

Pentatrichomonas hominis: purine salvage pathway

1. Pentatrichomonas hominis was found incapable of de novo synthesis of purines. 2. Pentatrichomonas hominis can salvage adenine, guanine, hypoxanthine, adenosine, guanosine and inosine, but not xanthine for the synthesis of nucleotides. 3. HPLC tracing of radiolabelled purines or purine nucleosides revealed that adenine, adenosine and hypoxanthine are incorporated into adenine nucleotides and IMP through a similar channel while guanine and guanosine are salvaged into guanine nucleotides via another route. There appears to be no direct interconversion between adenine and guanine nucleotides. Interconversion between AMP and IMP was observed. 4. Assays of purine salvage enzymes revealed that P. hominis possess adenosine kinase; adenosine, guanosine and inosine phosphotransferases; adenosine, guanosine and inosine phosphorylases and AMP deaminase.