Separate regulation of purA and purB loci of Escherichia coli K-12.

We isolated a strain of Escherichia coli K-12 in which the lac structural genes are fused to the purB control region and used this strain to study the regulation of the purA and purB loci. The purA locus was derepressed in response to either limiting adenine or guanine growth conditions in the presence of excess guanine or adenine, respectively. The presence of hypoxanthine in the culture medium did not have any effect on the expression of the purA locus. The purB locus responded to limiting adenine growth conditions in the presence of either excess hypoxanthine or guanine alone but not when both hypoxanthine and guanine were present.     

Inhibition of purine utilization by adenine in Alcaligenes eutrophus H16

With 0.5% substrate present in mineral medium, cells of Alcaligenes eutrophus H 16 were able to grow heterotrophically at the expense of guanine, hypoxanthine and xanthine, but not of adenine as sole sources of carbon and nitrogen. An increase in cell counts, however, was observed at lower adenine concentrations (0.1%). Similarly, adenine was only respired if present at low concentrations. Higher amounts of adenine were inhibitory to the utilization of adenine, guanine, hypoxanthine, xanthine, allantoin and glyoxylate, but not to that of fructose or glycerate. The adenine-dependent inhibition of adenine utilization was not overcome by the addition of thiamine, uridine or cytidine. The enzyme glyoxylate carboligase, usually formed in presence of metabolisable purines and of allantoin, was synthesized only at low adenine concentrations. Higher amounts were inhibitory even with allantoin present as additional substrate. According to these resutls, the utilization of purine derivatives and of allantoin as sources of carbon and energy is repressed by adenine in cells of A. eutrophus H 16.     

Production of Inosine from Hydrocarbons by Corynebacterinm petrophilum

Using the adenine auxotroph of hydrocarbonoclastic microorganism, Corynebacterium petrophilum, the effects of glucose on the inosine productivity were investigated. The mutant did not produce inosine from glucose as the sole source of carbon. Production of inosine in n-C₁₆ medium was found to be inhibited by the addition of glucose. To obtain information on such effect of glucose, several characters were compared between the cells grown in glucose medium and those grown in n-C₁₆ medium. Intracellular content of UV-absorbing materials of the glucose-cells was higher than that of hydrocarbon-cells. The glucose-celle could not grow in media containing adenosine or 5′-AMP. On the other hand, hydrocarbon-cells were able to achieve growth, with adenine, adenosine and 5′-AMP contained in the hydrocarbon medium, but, in the case of glucose medium, the cells could grow only in the presence of adenine. Furthermore, the growth of this mutant in n-C₁₆ medium was found to be inhibited by a larger amount of adenine than that required for the maximum growth, and this inhibition was overcome by the addition of guanine. The significance of the effect of guanine was discussed.     

Guanine uptake and metabolism in Neurospora crassa

Guanine is transported into germinated conidia of Neurospora crassa by the general purine base transport system. Guanine uptake is inhibited by adenine and hypoxanthine but not xanthine. Guanine phosphoribosyltransferase (GPRTase) activity was demonstrated in cell extracts of wild-type germinated conidia. The Km for guanine ranged from 29 to 69 micro M in GPRTase assays; the Ki for hypoxanthine was between 50 and 75 micro M. The kinetics of guanine transport differ considerably from the kinetics of GPRTase, strongly suggesting that the rate-limiting step in guanine accumulation in conidia is not that catalyzed by GPRTase. Efflux of guanine or its metabolites appears to have little importance in the regulation of pools of guanine or guanine nucleotides since very small amounts of 14C label were excreted from wild-type conidia preloaded with [8-14C]guanine. In contrast, excretion of purine bases, hypoxanthine, xanthine, and uric acid appears to be a mechanism for regulation of adenine nucleotide pools (Sabina et al., Mol. Gen. Genet. 173:31-38, 1979). No label from exogenous [8-14C]guanine was ever found in any adenine nucleotides, nucleosides, or the base, adenine, upon high-performance liquid chromatography analysis of acid extracts from germinated conidia of wild-type of xdh-l strains. The 14C label from exogenous [8-14C]guanine was found in GMP, GDP, GTP, and the GDP sugars as well as in XMP. Xanthine and uric acid were also labeled in wild-type extracts. Similar results were obtained with xdh-l extracts except that uric acid was not present. The labeled xanthine and XMP strongly suggest the presence of guanase and xanthine phosphoribosyltransferase in germinated conidia.     

Nitrogen Metabolism in Paramecium aurelia

Nitrogen in cell fractions of Paramecium aurelia varied according to the growth medium. Trichloroacetic acid-soluble fractions of cells were chromatographer. Adenine, adenosine, guanine, guanosine, hypoxanthine, aspartic acid, glutamic acid, histidine, lysine, proline, and phenylalanine were identified. Fyrimidines and xanthine, or their respective ribosides and ribotides, were not detected. Ammonia was released into the medium by both actively growing and "resting" cells. Culture fluids of "resting"cells also contained hypoxanthine and lesser amounts of adenine and guanine. Urea, uric acid, creatine, cretonne, and ailantoin were absent.
Pyrimidine nitrogen seems excreted as dihydrouracil. The following enzymes were detected in homogenates and cell-free preparations: nucleotidases, nucleoside hydrolases, and cytidine deaminase. Urease, uricase, adenase, guanase, xanthine oxidase, adenosine deaminase, and 5'-adenylic acid deaminase were not present in this organism.
Purine and pyrimidine incorporation into nucleic acids was investigated by the use of radioactive tracers. Guanosine gives rise to nucleic-acid guanine and adenine; adenosine was precursor to nucleic acid adenine only. Formate was incorporated into purines; glycine was not. P. aurelia can interconvert cytidine and uridine; both give rise to nucleic acid thymine. The methyl group of thymine may be derived from formate.     

Purine metabolism by intracellular Chlamydia psittaci.

Purine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined deficiencies in purine metabolism. C. psittaci AA Mp cannot synthesize purines de novo, as assessed by its inability to incorporate exogenous glycine into nucleic acid purines. C. psittaci AA Mp can take ATP and GTP, but not dATP or dGTP, directly from the host cell. Exogenous hypoxanthine and inosine were not utilized by the parasite. In contrast, exogenous adenine, adenosine, and guanine were directly salvaged by C. psittaci AA Mp. Crude extract prepared from highly purified C. psittaci AA Mp reticulate bodies contained adenine and guanine but no hypoxanthine phosphoribosyltransferase activity. Adenosine kinase activity was detected, but guanosine kinase activity was not. There was no competition for incorporation into nucleic acid between adenine and guanine, and high-performance liquid chromatography profiles of radiolabelled nucleic acid nucleobases indicated that adenine, adenosine, and deoxyadenosine were incorporated only into adenine and that guanine, guanosine, and deoxyguanosine were incorporated only into guanine. Thus, there is no interconversion of nucleotides. Deoxyadenosine and deoxyguanosine were cleaved to adenine and guanine before being utilized, and purine (deoxy)nucleoside phosphorylase activity was present in reticulate body extract.     

Purine metabolism in human lymphocytes.

In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with 14C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No perferential uptake for one of the purine bases was observed. In all cases only traces of 14C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predomonance of adenine nucleotide formation in normal human lymphocytes.     

Flavinogenesis and regulation of purine biosynthesis de novo in Pichia guilliermondi mutants resiatant to 8-azaguanine]

93 mutants resistant to 8-azaguanine (AGR-mutants) were derived from the strain of Pichia guilliermondii with blocked guanine deaminase (EC 3.5.4.3.) by UV-irradiation. The mutants retained the ability to uptake 8-azaguanine and guanine but could not deaminate guanine. Some of the AGR-mutants were found to accumulate large amounts of hypoxanthine and small amounts of guanine in the cultural medium. The inhibitory effect of guanine and 8-azaguanine but not adenine on the purine biosynthesis de novo was considerably decreased. It was established observing the rates of 5 amino 4-imidazoleribotide accumulation in purine-requiring AGR-mutants in the presence of different purines. The regulation of the activity and biosynthesis of IMP-dehydrogenase (EC 1. 2. 1. 14) with guanine compounds in AGR-mutants was completely preserved. Under cultivating in iron-rich medium all the AGR-mutants accumulated more riboflavin than the strain H-101 and the wild type strain. That occured as a result of the increase of flavinogenesis velocity in AGR-mutants during late logarithmic and negative growth acceleration phases. Some of mutants also synthesized more riboflavin in iron-deficient medium. Depression of riboflavine synthetase was not observed in the iron-rich cells of AGR-mutants.     

Characterization of a guanine-sensitive mutant defective in adenylo-succinate synthetase activity

A contingent auxotrophic mutant of CHO-Kl cell is described. This mutant grows in minimal medium. Its growth is inhibited by the exogenous addition of guanine at levels which do not affect the wild type parent. Adenine reverses the guanine effect. This mutant does not complement ade-H (defective in adenylosuccinate synthetase) and has been denoted as ade-HG because of its guanine sensitivity. Some partial revertants of ade-H are found to be also sensitive to guanine, suggesting a close relationship between the ade-H locus and the guanine sensitivity. Studies of 14C-hypoxanthine incorporation into nucleotides indicated that ade-HG has some adenylosuccinate synthetase activity whether it is pre-exposed to guanine or not. Early de novo purine synthesis in ade-HG, however, is greatly inhibited when pre-exposed to guanine. This inhibition of purine synthesis by guanine is reversible and its recovery is facilitated by adenine.     

Thymidine uptake in bacteria: the effect of purine nucleosides.

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 microng/ml while the initial concentration of purine nucleosides ranged from 25 to 250 microng/ml. Adenine nucleosides when present at a concentration more than 50 microng/ml strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial concentration of deoxyadenosine (or adenosine) of 250 microng/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase. Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 microng/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides. It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

A novel synergistic effect of alanosine and guanine on adenine nucleotide synthesis in mammalian cells. Alanosine as a useful probe for investigating purine nucleotide metabolism

A novel synergistic effect of the antitumor agent alanosine (2-amino-3-(hydroxynitrosoamino) propionic acid), which specifically inhibits the enzyme adenylosuccinate synthetase (ASS) and guanine on the growth of Chinese hamster ovary (CHO) cells and human diploid fibroblasts (HDF) has been observed. In the presence of subinhibitory concentrations of alanosine, both CHO cells and the HDF show excessive sensitivity to exogenous guanine—a phenotype which closely resembles that seen with some of the mutants containing reduced enzymatic activity of ASS. The growth inhibitory effects of alanosine, or alanosine and guanine, on CHO cells are completely reverted by the addition of adenine to the culture medium, and the synergistic effect of guanine is not observed in mutants which lack the enzyme hypoxanthine-guanine phosphoribosyl transferase. These results suggest that guanine nucleotides exert a regulatory effect on the activity of the enzyme adenylosuccinate synthetase. The ability to confer the guaninesensitive phenotype and its modulation by subinhibitory concentrations of alanosine in different cell types indicates that alanosine provides a useful probe for investigating the regulation of purine nucleotide metabolism in mammalian cells.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Fluorometric determination of adenine and its derivatives by reaction with glyoxal hydrate trimer

A fluorometric determination of adenine has been devised. It was found that adenine gives strong fluorescence when reacting with glyoxal hydrate trimer in an acidic medium. The maximum wavelengths of excitation and emission spectra were 328 and 382 mμ, respectively. This reaction was successfully applied to determination of 0.2–1.0 or 2.0–10.0 mμmoles of adenine and its derivatives, in which the hydrogen atom at position 9 is substituted with ribose or substituted ribose. Other nucleic acid bases, guanine, uracil, thymine, and cytosine, did not interfere with this fluorometric determination of adenine.     

Further characterization of a depurinated DNA-purine base insertion activity from cultured human fibroblasts.

The purification from cultured human fibroblasts of a protein that binds specifically to partially depurinated DNA and inserts purines into those sites is described. The purine insertion, but not the binding, requires K+. The DNA binding can be saturated with increasing apurinic sites and is weakened by the presence of adenine or guanine. Base insertion into depurinated DNA is specific for adenine or guanine; none is observed with dATP or dGTP. When the depurinated DNA substrate is specifically cleaved with apurinic endonuclease, no purine insertion occurs. Guanine insertion does not occur into tRNA or depyrimidinated DNA, and thymine is not inserted into either depyrimidinated DNA or depurinated DNA. Purine insertion activity follows Michaelis-Menten kinetics with respect to purintes; the apparent Km values for both adenine and guanine are 5 microM. The enzyme binds the purine bases very tightly. Adenine binding saturates at less than 1 microM adenine, perhaps reflecting the low intracellular adenine concentration. The binding protein specific for UV-irradiated DNA (Feldberg, R.S., and Grossman, L. (1976) Biochemistry 15, 2402-2408) had no detectable purine or pyrimidine base insertion activity with depurinated or depyrimidinated DNAs.     

Adenine uptake in Neurospora crassa mycelium]

The uptake of 8-C14-adenine in N. crassa strain Lindegren (+) was studied. The ability of N. crassa cells to uptake adenine from the medium reaches maximum at the very beginning of the logarithmic stage of growth. Adenine enters the mycelium against the concentration gradient. The uptake of adenine is maximal at 25-30 degrees C, pH 4,6-4,8, and adenine concentration in the medium about 2-15X10(-6) M. The entry of adenine into the cells follows normal Michaelis-Menten kinetics, the apparent Km=0.83+/-0.02 micron. The uptake is inhibited at higher concentrations (10(-3)-10(-4) M) of adenine. 2,6-Diaminopurine, hypoxanthine, guanine, 8-azaadenine and 8-azaguanine inhibit the transport of adenine into the cell. Xanthine and cytosine do not affect the uptake of adenine. Adenine taken up into the cell is rapidly metabolized to AMP, ADP and ATP.     

Electrochemical determination of antioxidant capacities in flavored waters by guanine and adenine biosensors

The immobilization and electro-oxidation of guanine and adenine as DNA bases on glassy carbon electrode are evaluated by square wave voltammetric analysis. The influence of electrochemical pretreatments, nature of supporting electrolyte, pH, accumulation time and composition of DNA nucleotides on the immobilization effect and the electrochemical mechanism are discussed. Trace levels of either guanine or adenine can be readily detected following short accumulation time with detection limits of 35 and 40 ngmL(-1) for guanine and adenine, respectively. The biosensors of guanine and adenine were employed for the voltammetric detection of antioxidant capacity in flavored water samples. The method relies on monitoring the changes of the intrinsic anodic response of the surface-confined guanine and adenine species, resulting from its interaction with free radicals from Fenton-type reaction in absence and presence of antioxidant. Ascorbic acid was used as standard to evaluate antioxidant capacities of samples. Analytical data was compared with that of FRAP method.     

The effects of added purines on urate and purine synthesis de novo by isolated chick liver, kidney and lymphoid cells.

1. Isolated chick lymphoid cells, together with isolated chick liver and kidney cells, incorporate [1-14C]glycine or [14C]formate into urate. 2. Of the cell types used, bursal cells incorporate 14C into urate at the fastest rate, although the output of total urate by bursal cells is only 10% that of liver cells. 3. When suspended in Eagle's medium the incorporation of 14C into urate is inhibited by adenine and guanine up to 1 mM. In contrast, the addition of 1 mM-AMP or -GMP results in a relatively large stimulation of this incorporation. 4. Added adenine is rapidly taken up by liver cells and then released in an unmetabolized form; AMP is taken up more slowly and is rapidly metabolized. The metabolites (possibly including adenine) are then released. 5. Intracellular liver 5-phosphoribosyl 1-pyrophosphate is approx. 0.7mM and remains constant or falls slightly during a 3 h incubation of the cells. 6. The addition of adenine or guanine, AMP or GMP, does not alter liver intracellular 5-phosphoribosyl 1-pyrophosphate concentrations. Added 5-phosphoribosyl 1-pyrophosphate is not taken up by liver cells. 7. The results are discussed in the context of the control of urate and purine synthesis de novo in the chick.     

Nutritional induction and suppression of fruiting in Myxococcus xanthus FBa

A defined agar medium (A agar) containing 15 amino acids in concentrations between 0.5 and 2 mm was developed for studying the fruiting cycle of Myxococcus xanthus FBa. Cells grew only vegetatively in this medium unless the initial concentration of one of nine required or stimulatory amino acids was lowered about 50-fold. In the latter circumstance, fruiting bodies developed after several days of vegetative growth. The conclusion was that fruiting occurred when any amino acid required for normal growth became limiting in the environment. High concentrations (10 mm) of phenylalanine, tryptophan, or methionine prevented fruiting without affecting growth. Mutants requiring arginine, thymidine, or adenine could not be induced to fruit by limiting their unique requirement although they responded to the same deprivations which brought about fruiting of the wild type. A histidine auxotroph formed fruiting bodies when histidine was lowered to growth-limiting concentrations, provided that the medium was supplemented with purines. A uracil auxotroph was isolated that, perhaps secondarily, had lost some of the mechanisms which control the formation of fruiting bodies; if uracil was present, it formed fruits even when no amino acid was limiting. No concentration of uracil was sufficient to prevent fruiting. Fruiting bodies were formed when mixtures of the uracil auxotroph and wild-type cells were inoculated on A agar plus uracil, even when 75% of the cells were wild type. Microcysts of both strains were present in the fruiting bodies.