Developmental changes in purine phosphoribosyltransferases in human and rat tissues.

1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5'-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme.     

Inhibition by 6-mercaptopurine of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells that are resistant to this drug

1. A strain of Ehrlich ascites-tumour cells that showed little inhibition of growth in the presence of 6-mercaptopurine accumulated less than 5% as much 6-thioinosine 5′-phosphate in vivo, in the presence of 6-mercaptopurine, as did the sensitive strain from which it was derived. 2. Specific activities of the phosphoribosyltransferases that convert adenine, guanine, hypoxanthine and 6-mercaptopurine into AMP, GMP, IMP and 6-thioinosine 5′-phosphate were similar in extracts of the resistant and the sensitive cells. 3. As found previously with sensitive cells, 6-mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase from the resistant cells and does not inhibit the adenine phosphoribosyltransferase from these cells. Michaelis constants and inhibitor constants of the purine phosphoribosyltransferases from resistant cells did not differ significantly from those measured with the corresponding enzymes from sensitive cells. 4. Resistance to 6-mercaptopurine in this case is probably not due to qualitative or quantitative changes in these transferases.     

Analogues of ribose 5-phosphate and 5-phosphoribosyl pyrophosphate. The preparation and properties of ribose 5-phosphorothioate and 5-phosphoribosyl 1-methylenediphosphonate

1. 5-Phosphoribosyl 1-methylenediphosphonate was isolated after reaction of ribose 5-phosphate and O-adenylyl methylenediphosphonate with 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. 2. The analogue reacted with adenine phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase [K(m) (analogue)/K(m) (5-phosphoribosyl pyrophosphate) 0.17, 0.19 and 6.3 respectively; V(max.) (analogue)/V(max.) (5-phosphoribosyl pyrophosphate) 0.011, 0.26 and 1.1 respectively]. 3. The analogue was not a substrate for 5-phosphoribosyl pyrophosphate amidotransferase or orotate phosphoribosyltransferase. 4. Ribose 5-phosphorothioate was synthesized by allowing ribose to react with thiophosphoryl chloride in triethyl phosphate. The analogue was a substrate for 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites-tumour cells. When this reaction was coupled to either adenine phosphoribosyltransferase or hypoxanthine phosphoribosyltransferase, adenosine 5'-phosphorothioate or inosine 5'-phosphorothioate was formed respectively.     

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Adenine nucleotide metabolism in relation to purine enzymes in liver,erythrocytes and cultured fibroblasts

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hypoxanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5′-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5′-nucleotidase present in this tissue interferred with the formation of LaCl₃ salts. The lanthanum and the thin-layer chromatographic methods agreed with-in 10%.Liver cell sap had the highest activities of all purine enzymes except for 5′-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erythrocytes were devoid of 5′-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue.Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5′-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control of adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte > liver > fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Inhibition of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells by purine nucleotides

1. The purine bases adenine, hypoxanthine and guanine were rapidly incorporated into the nucleotide fraction of Ehrlich ascites-tumour cells in vivo. 2. The reaction of 5'-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase from ascites-tumour cells (K(m) 6.5-11.9mum) was competitively inhibited by AMP, ADP, ATP and GMP (K(i) 7.5, 21.9, 395 and 118mum respectively). Similarly the reactions of 5'-phosphoribosyl pyrophosphate with both hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase (K(m) 18.4-31 and 37.6-44.2mum respectively) were competitively inhibited by IMP (K(i) 52 and 63.5mum) and by GMP (K(i) 36.5 and 5.9mum). 3. The nucleotides tested as inhibitors did not appreciably compete with the purine bases in the phosphoribosyltransferase reactions. 4. It was postulated that the purine phosphoribosyltransferases of Ehrlich ascites-tumour cells may be effectively separated from the adenine nucleotide pool of these cells.     

Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.     

Purine metabolism in germinating wheat embryos

1. Both the acid-soluble fraction and the nucleic acid fraction of wheat embryos were extensively labelled after incubation for 6hr. in the presence of [8-(14)C]adenine. Subsequent incubation in the absence of labelled adenine resulted in no loss of radioactivity to the medium during a 48hr. period. Radioautography indicated that during this period there was a continuous increase in the radioactivity present in the acid-insoluble fractions of the root and leaf tissues relative to that present in the coleorhiza and coleoptile. 2. During incubation at 25 degrees there was a 26-fold increase in the activity of 3'-nucleotidase between 4hr. and 24hr.; the activities of enzymes hydrolysing AMP and IMP increased to a smaller extent. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase increased three- to five-fold during incubation at 25 degrees for 24hr. 3. Adenosine kinase, inosine phosphorylase and 5-phosphoribosyl pyrophosphate synthetase activities were high in extracts from dry embryos and did not increase during 48hr. at 25 degrees . 4. The increase in 3'-nucleotidase activity was prevented by cycloheximide, cryptopleurine or incubation at 4 degrees , but not by actinomycin D; these treatments did not depress the activity of the other enzymes measured. 5. The results are discussed in relation to RNA translocation within the wheat embryo during germination.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

The activities and kinetic properties of purine phosphoribosyltransferases in developing mouse liver

1. The total activity of adenine phosphoribosyltransferase/liver of mice remained constant from 1 to 16 days after birth despite a fourfold increase in liver weight. The total activity of this enzyme increased fivefold from 16 to 36 days and then remained relatively constant at least until 96 days after birth. Total hypoxanthine-phosphoribosyltransferase activity/liver steadily increased between 1 and 57 days after birth. 2. The mean K_m of 5-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase was 10·1μm between 3 and 11 days, at 64 days and at 96 days after birth. Between 17 and 51 days the mean K_m value was 3·0μm. The K_m of 5-phosphoribosyl pyrophosphate with hypoxanthine phosphoribosyltransferase remained constant at 28·2μm between 2 and 64 days. 3. Adenine-phosphoribosyltransferase activity was stimulated between 15 and 83% by 60μm-ATP when extracts were made between 3 and 11 days, at 64 days or at 96 days after birth. Between 17 and 51 days ATP had little stimulatory effect on the activity of this enzyme. 4. AMP competed with 5-phosphoribosyl pyrophosphate in the reaction catalysed by adenine phosphoribosyltransferase. Liver extracts containing enzyme with a low value of K_m for 5-phosphoribosyl pyrophosphate (3μm) had a K_m/K_i ratio approximately half that of extracts with a high value of K_m (10μm). 5. The results indicate that two different forms of adenine phosphoribosyltransferase can exist in mouse liver at different stages of development. The physiological significance of these findings is discussed.     

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaminase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.     

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.     

Isolation of a Chinese hamster cell mutant with low intracellular phosphoribosylpyrophosphate concentration

A tritium-adenine suicide procedure was used to select for mutants with reduced uptake of adenine from a population of Chinese hamster V79 cells mutagenized with ethyl methane sulfonate. In one of the mutant lines isolated, designated KC62, the uptake of adenine, hypoxanthine, and guanine was reduced by approximately 70%. The specific activities, Km values, and Vmax values of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase were the same in extracts from KC62 and from the parental cell line. Metabolic fate studies of incorporated [3H]adenine and 3[H]hypoxanthine revealed a metabolic block at the level of phosphoribosylation. Determination of phosphoribosylpyrophosphate pool size showed that the mutant contained only 25% of the phosphoribosylpyrophosphate found in the parent. Its reduced availability in KC62 appears to result in a decreased ability to salvage adenine, hypoxanthine, and guaninine via phosphoribosylation. Phosphoribosylpyrophosphate synthetase from KC62 was shown to have an increased sensitivity to inhibition by a variety of nucleotides.     

Lesch-Nyhan mutation: the influence of population density on purine phosphoribosyltransferase activities and exogenous purine utilization in monolayer cultures of skin fibroblasts

In extracts of normal and Lesch-Nyhan (LN) heterozygous skin fibroblast monolayer cultures, hypoxanthine-guanine (H-G) and adenine (A) phosphoribosyltransferase (PRT) activities are correlated. These activities vary concomitantly during the life cycle of a culture: Peak activities occur during exponential growth. The ratio of H-GPRT to APRT activity can manifest heterozygosity at the LN locus when H-GPRT activity, per se, is close to and unreliable different from the normal range. If 2 or 8 × 10⁴ cells are planted per 35 mm petri dish, a strain with clearly heterozygous levels of H-GPRT activity in its cell extract may not reveal its genotype by simultaneous measurement of adenine and hypoxanthine incorporation into its acid-insoluble fraction one day after subculture. In contrast, a 1:1 coculture of normal and mutant cells yields the ratio of adenine to hypoxanthine incorporation expected from the behavior of each cell type separately. In heterozygous cultures at the higher population density, the incorporation of hypoxanthine relative to adenine is 28% greater than in those at the lower population density. A 1:1 mixture of normal and mutant cells exhibited a 20% increase in the relative incorporation of hypoxanthine over the same four fold increase in population density. These observations appear to provide a quantitative expression of the phenomenon of metabolic cooperation between contacting H-GPRT-negative and H-GPRT-positive cells.     

Metabolism of hypoxanthine in isolated rat hepatocytes.

The hepatic metabolism of hypoxanthine was investigated by studying both the fate of labelled hypoxanthine, added at micromolar concentrations to isolated rat hepatocyte suspensions, and the kinetic properties of purified hypoxanthine/guanine phosphoribosyltransferase from rat liver. More than 80% of hypoxanthine was oxidized towards allantoin; less than 5% of the label was incorporated into the purine mononucleotides, and a similar proportion appeared transiently in inosine. The maximal velocity of oxidation (approx. 750nmol/min per g of cells) was in close agreement with the known activity of xanthine oxidase in liver extracts. In contrast, the maximal velocity of the incorporation of labelled hypoxanthine into mononucleotides reached only 30nmol/min per g of cells, compared with an activity of hypoxanthine/guanine phosphoribosyltransferase, measured at substrate concentrations analogous to those prevailing intracellularly, of 500nmol/min per g of cells. Hypoxanthine incorporation into the mononucleotides was decreased by allopurinol, anoxia and ethanol, despite inhibition of its oxidation under these conditions; it was increased by incubation of the cells in supraphysiological concentrations of Pi. Allopurinol and anoxia decreased the concentration of phosphoribosyl pyrophosphate inside the cells by respectively 40 and 60%, ethanol had no effect on the concentration of this metabolite and Pi increased its concentration up to 10-fold. The kinetic study of purified hypoxanthine/guanine phosphoribosyltransferase showed that a mixture of ATP, IMP, GMP and GTP, at the concentrations prevailing in the liver cell, decreased the V max. of the enzyme 6-fold, increased its Km for hypoxanthine from 1 to 4 microM and its Km for phosphoribosyl pyrophosphate from 2.5 to 25 microM. In the presence of 5 microM-hypoxanthine and 2.5 microM-phosphoribosyl pyrophosphate, the mixture of nucleotides inhibited the activity of purified hypoxanthine/guanine phosphoribosyltransferase by 95%. It is concluded that this inhibition results in a limited participation of hypoxanthine/guanine phosphoribosyltransferase in the control of the production of allantoin by the liver.     

The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine-xanthine-guanine phosphoribosyltransferase produced by this organism.     

Adenine phosphoribosyltransferase mutants in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae deficient in adenine phosphoribosyltransferase (A-PRT, EC 2,4,2,7) have been isolated following selection for resistance to 8-azaadenine in a prototrophic strain carrying the ade4-su allele of the gene coding for amidophosphoribosyltransferase (EC 2,4,2,14). The mutants were recessive and defined a single gene, apt1. They did not excrete purine when combined with ade4+. The mutants appeared to retain some A-PRT activity in crude extracts, and strains of the genotype ade2 apt1 responded to both adenine and hypoxanthine. Mutants deficient in adenine aminohydrolase (EC 3,5,4,2) activity, aah1, and hypoxanthine:guanine phosphoribosyltransferase (EC 2,4,2,8) activity, hpt1, were used to synthesize the genotypes apt1 hpt1 aah+ and apt1 hpt+ aah1. The absence of A-PRT activity in strains with these genotypes confirmed the hypothesis that the residual A-PRT activity of apt1 mutants was due to adenine aminohydrolase and hypoxanthine:guanine phosphoribosyltransferase acting in concert.     

Enzymes for purine synthesis and scavenging in pathogenic mycobacteria and their distribution in Mycobacterium leprae

Three enzymes catalysing the synthesis of four intermediates (phosphoribosylglycinamide, phosphoribosylaminoimidazole-succinocarboxamide, phosphoribosylaminoimidazole-carboxamide and AMP) in the purine biosynthetic pathway were detected in extracts of Mycobacterium microti and M. avium, even when the organisms had been grown in mice. However only one of the three enzymes, adenylosuccinate AMP-lyase (catalysing the synthesis of the last two of the four intermediates listed above) was detected in M. leprae. Phosphoribosyltransferases, which convert adenine, guanine and hypoxanthine to the corresponding nucleoside monophosphates, and adenosine kinase were the major enzymes for purine scavenging in all mycobacteria studied. In contrast to enzymes in the synthetic pathway, evidence for metabolic regulation of the purine-scavenging enzymes was obtained. In particular, 20-80-fold differences in the activities of guanine phosphoribosyltransferase and adenosine kinase were observed when M. microti was grown in media with or without purines, or in mice. In M. leprae, activities of all phosphoribosyltransferases were low in comparison with activities in M. microti and M. avium (specific activity less than 2% when comparisons were made between extracts of host-grown mycobacteria). However, activity of adenosine kinase was higher in host-grown M. leprae than in host-grown M. microti or M. avium.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.     

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.