Purine and pyrimidine transport and permeation in human erythrocytes

Time courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 microM in suspensions of human erythrocytes at 25 or 30 degrees C. At concentrations above 25 microM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the influx and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 microM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations much less than 1 microM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae.

Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.     

Mechanism of energy coupling for transport of deoxycytidine, uridine, uracil, adenine and hypoxanthine in Escherichia coli.

The transport processes for uridine, deoxycytidine, uracil, adenine and hypoxanthine require an energy source and are active under anaerobic or aerobic conditions. Inhibitory effects of cyanide, arsenate, carbonylcyanide m-chlorophenylhydrazone, 2,4-dinitrophenol and N,N'-dicyclohexylcarbodiimide on the transport of uridine and deoxycytidine differ from the corresponding effects on the transport of uracil, adenine and hypoxanthine. The nature of these inhibitory effects supports the conclusion that uridine and deoxycytidine transport is energized either by electron transport or by ATP hydrolysis via (Ca2+ + Mg2+)-ATPase. The transport or uracil, adenine and hypoxanthine is dependent upon ATP or some high energy phosphate derivative of ATP, but is independent of (Ca2+ + Mg+)-ATPase and electron transport. Uptake of the ribose moiety of uridine by a mutant of Escherichia coli B, which lacks the transport system for uracil and intact uridine, is neither stimulated by energy sources nor inhibited by various inhibitors of energy metabolism under either aerobic or anaerobic conditions.     

Development of competence of Haemophilus influenzae

Spencer, Hugh T. (The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Md.), and Roger M. Herriott. Development of competence of Haemophilus influenzae. J. Bacteriol. 90:911-920. 1965.-A chemically defined nongrowth medium was developed for the induction of competence of Haemophilus influenzae by a stepdown procedure. Cells grown logarithmically in Heart Infusion Broth became competent after being transferred to a medium which consisted of amino acids, sodium fumarate, and inorganic salts. Chloramphenicol (2 mug/ml) or l-valine (1 mug/ml) in the nongrowth medium inhibited development of competence. The inhibitory action of l-valine was reversed by comparable concentrations of l-isoleucine. Kinetic studies of the development of competence showed a variable capacity of competent cells to take up deoxyribonucleic acid and reaffirmed earlier findings that competence was not transmissible in H. influenzae. Addition of nicotinamide adenine dinucleotide, thiamine, calcium pantothenate, uracil, and hypoxanthine to the medium for competence resulted in a minimal growth medium in which reduced levels of competence were developed.     

Control of transformation competence in Azotobacter vinelandii by nitrogen catabolite derepression.

Azotobacter vinelandii (ATCC 12837) became competent to be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Competence in wild-type and nitrogenase auxotrophic (nif-) strains was repressed by the addition of ammonium salts or urea to the transformation medium. Transformation of wild-type cells and nif- strains was optimal on nitrogen-free or nitrogen-limiting medium, respectively. Transformation of wild-type cells also was enhanced when the transformation medium had low molydbate content. During the development of competence, nitrogen was growth limiting, whereas carbon (glucose) was in excess. Carbon source shift-down was not effective in inducing competence. Shifting glucose-grown wild-type cells to medium containing 0.2% beta-hydroxybutyrate initiated encystment and also induced competence. The addition of glucose to this medium blocked encystment and early competence induction and reduced the transformation frequency to the basal level. Cyclic adenosine 3',5'-monophosphate induced competence in wild-type nitrogen-fixing cells and increased the transformation frequency 1,000-fold over the basal level. Exogenous cyclic adenosine 3',5'-monophosphate however, did not reverse nitrogen repression of competence in ammonia-grown wild-type or nif- strains.     

Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation.

The zero-trans uptake of purines and pyrimidines was measured in suspensions of Novikoff rat hepatoma, mouse L, P388 mouse leukemia, and Chinese hamster ovary cells by a rapid kinetic technique which allows the determination of uptake time points in intervals as short as 1.5 s. Kinetic parameters for purine/pyrimidine transport were determined by measuring substrate influx into cells in which substrate conversion to nucleotides was negligible either due to lack of the appropriate enzymes or to depletion of the cells of ATP (5'-phosphoribosylpyrophosphate), and by computer fitting exact, integrated rate equations derived for various carrier-mediated transport models directly to zero-trans influx data. The results indicate that different carriers function in the transport of hypoxanthine/guanine, adenine, and uracil with substrate:carrier association constants (K) at 24 degrees C of 300 to 400 muM, 2 to 3 mM, and about 14 mM, respectively, for Novikoff cells. K and Vmax for hypoxanthine transport by L and P388 cells are similar to those for Novikoff cells, but the transport capacity of Chinese hamster ovary cells is much lower and K = 1500 muM. All transport systems are completely symmetrical. Hypoxanthine transport is so rapid that an intracellular concentration of free hypoxanthine (90%) close to that in the medium is attained within 20 to 50 s of incubation at 24 degrees C, at least at extracellular concentrations below K. In cells in which conversion to nucleotides is not blocked free hypoxanthine accumulates intracellularly to steady state levels with equal rapidity and thereafter the rate of hypoxanthine uptake into total cell material is strictly a function of the rate of phosphoribosylation. The low Km systems for hypoxanthine (1 to 9 muM) and adenine (0.2 to 40 muM) uptake detected previously in many types of cells reflect the substrate saturation of the respective phosphoribosyltransferases rather than of the transport system.     

Entry of Escherichia coli into stationary phase is indicated by endogenous and exogenous accumulation of nucleobases.

Endogenous and exogenous accumulation of nucleobases was observed when Escherichia coli entered the stationary phase. The onset of the stationary phase was accompanied by excretion of uracil and xanthine. Except for uracil and xanthine, other nucleobases (except for minor amounts of hypoxanthine), nucleosides, and nucleotides (except for cyclic AMP) were not detected in significant amounts in the culture medium. In addition to exogenous accumulation of nucleobases, stationary-phase cells increased the endogenous concentrations of free nucleobases. In contrast to extracellular nucleobases, hypoxanthine was the dominating intracellular nucleobase and xanthine was present only in minor concentrations inside the cells. Excretion of nucleobases was always connected to declining growth rates. It was observed in response to entry into the stationary phase independent of the initial cause of the cessation of cell growth (e.g., starvation for essential nutrients). In addition, transient accumulation of exogenous nucleobases was observed during perturbations of balanced growth conditions such as energy source downshifts. The nucleobases uracil and xanthine are the final breakdown products of pyrimidine (uracil and cytosine) and purine (adenine and guanine) bases, respectively. Hypoxanthine is the primary degradation product of adenine, which is further oxidized to xanthine. The endogenous and exogenous accumulation of these nucleobases in response to entry into the stationary phase is attributed to degradation of rRNA.     

Physiological factors involved in the transformation of Mycobacterium smegmatis.

Transfer of streptomycin resistance and changes from methionine and leucine auxotrophy to prototrophy were achieved in Mycobacterium smegmatis by transformation. Recipient cells were more resistant to mitomycin C and methyl methlanesulfonate treatments than were wild-type cells. A high level of calcium ions was essential for transformation, especially during DNA adsorption, whereas the presence of magnesium ions and the exposure of recipient cells to mild doses of UV light enhanced recombination frequencies. Transformants were not isolated when recipient cell-DNA mixtures were first treated with deoxyribonuclease. Recipient cells at various stages of growth showed similar transformabilities. Transformation was successful only when recipient cells were incubated on rich agar medium after mixture with DNA. Exposure of recipient cells to Pronase before treatment with donor DNA did not affect transformation, suggesting the absence of a protein competence factor. Throughout the present experiments, cotransformation frequencies were very low and unselected-marker segregation patterns were independent, indicating that the methionine, leucine, and streptomycin markers are not closely linked in M. smegmatis.     

Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation.

Competent Haemophilus influenzae Rd recipients, either as phage HP1 restricting (r+) or nonrestricting (r-) nonlysogens or defective lysogens, were exposed to deoxyribonucleic acids from various wild-type phage HP1 lysogenic H. influenzae serotype strains (non-encapsulated derivatives of serotypes a,b, c, d, and e), to DNA from lysogenic Haemophilus parahaemolyticus, and to DNA from modified and nonmodified phage HP1. Transformation of antibiotic resistance markers and of prophage markers in homospecific crosses was observed to be unaffected by the recipient restriction phenotype, whereas the transfection response was much reduced in r+ recipients. Heterospecific transformation of prophage markers was reduced by only 80 to 90%, whereas antibiotic resistance marker transformation was 1,000 to 10,000 times lower. Heterspecific transfection was at least 100 times lower than homospecific transfection in both r+ and r- recipients. The general conclusion is that neither class I nor class II restriction enzymes affect significantly the transformation efficiency in homospecific and heterospecific crosses. The efficiency of heterospecific transformation may depend mainly on the deoxyribonucleic acid homology in the genetic marker region.     

CHANGES IN RESPONSE TO PURINE AND PYRIMIDINE DERIVATIVES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURING

1. The growth of the carrot root callus which had been subculturedfor a long period (CCL) was promoted by the addition of 5l0^–8and 5l0^–7 M kinetin, whereas in the callus subculturedfor a short period (CCS) no growth promotion was observed atany concentrations of kinetin tested.
2. CCL showed an increasedgrowth in response to the applicationof kinetin, guanine, adenine,hypoxanthine, uracil, thymine,and cytosine in the presenceof fractions A and C of carrotroot extract, whereas no suchresponse was observed in CCS.CCL required fraction C to respondto uracil and probably purineand pyrimidine derivatives ingeneral.
3. The growth of CCL was promoted by kinetin, guanine,adenine,or hypoxanthine in the medium containing inositol andaminoacids mixture. In this case the growth-promoting actionof guanine,adenine, or hypoxanthine was nullified by kinetin.

(Received December 24, 1964; )     

Interplay between DNA polymerase and proliferating cell nuclear antigen switches off base excision repair of uracil and hypoxanthine during replication in archaea

Archaeal family-B DNA polymerases bind tightly to uracil and hypoxanthine (the deamination products of cytosine and adenine), resulting in profound inhibition of DNA replication. Investigation of the mechanism of inhibition, using single-turnover kinetics with polymerase in excess of DNA, indicated that deoxy-NTPs were efficiently bound to the polymerase-DNA complex but very poorly incorporated into the extending chain. Addition of the processivity factor proliferating cell nuclear antigen (PCNA) resulted in increased affinity of the polymerase for all primer-templates, producing extremely tight complexes when uracil (K_d = 16 pM) or hypoxanthine (K_d = 65 pM) was present. Analytical ultracentrifugation confirmed the stability of these complexes and revealed a polymerase/PCNA/DNA stoichiometry of 1:1:1. However, PCNA had no influence on the ability of the polymerase to read through uracil and hypoxanthine, the same kinetic parameters being observed with or without the processivity factor. The specificity constants determined using single-turnover kinetics showed that uracil and hypoxanthine slowed the polymerase by factors of ∼ 5000 and 3000, respectively. Uracil and hypoxanthine are removed from DNA by base excision repair, initiated by uracil-DNA glycosylase and endonuclease V, respectively. Both enzymes are profoundly inhibited by the simultaneous binding of both PCNA and polymerase to primer-templates, with polymerase alone being much less effective. Thus, when the PCNA-polymerase complex encounters uracil/hypoxanthine in DNA templates, base excision repair is switched off, protecting the complex from a repair pathway that is dangerous in the context of single-stranded DNA formed during replication.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.     

Genetic modifiers of the phenotypic level of deoxyribonucleic acid-conferred novobiocin resistance in Haemophilus

Leidy, Grace (Columbia University, New York, N.Y.), Iris Jaffee, and Hattie E. Alexander. Genetic modifiers of the phenotypic level of deoxyribonucleic acid-conferred novobiocin resistance in Haemophilus. J. Bacteriol. 92:1464-1468. 1966.-An apparent increase in novobiocin resistance in Haemophilus aegyptius after a second exposure to a particular H. influenzae transforming deoxyribonucleic acid was shown to be the result not of multi-step transformation but of the action of a gene functioning as an enhancement modifier. The modifier is very closely linked to a streptomycin resistance gene (which is linked to a novobiocin resistance marker); it affects the natural degree of resistance to both novobiocin and kanamycin to a measurable degree. Evidence of a repressor of the enhancement modifier is reported.     

Optimal conditions for transformation of Azotobacter vinelandii.

Optimal transformation of Azotobacter vinelandii OP required a 20-min incubation of the competent cells with deoxyribonucleic acid at 30 degrees C in buffer (pH 6.0 to 8.0) containing 8 mM magnesium sulfate. Nitrogen-fixing transformants of nitrogen fixation-deficient recipients could be plated immediately on selective medium, but transformants acquiring rifampin and streptomycin resistance required preincubation in nonselective medium. The three phenotypes achieved an approximately equal and stable frequency after 17 h (six generations) of growth in nonselective medium.     

Factors Affecting Competence for Transformation in Staphylococcus aureus

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage phi11 can be transformed. Superinfection of competent cells with phi11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower.     

Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects.Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control.The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine.The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.     

Nucleoside and nucleobase transport and metabolism in wild type and nucleoside transport-deficient Aedes albopictus cells

Nucleoside and nucleobase transport and metabolism were measured in ATP-depleted and normal Aedes albopictus mosquito cells (line C-7-10) by rapid kinetic techniques. The cells possess a facilitated diffusion system for nucleosides, which in its broad substrate specificity and kinetic properties resembles that present in many types of mammalian cells. The Michaelis-Menten constant for uridine transport at 28 degrees C is about 180 microM. However, the nucleoside transporter of the mosquito cells is resistant to inhibition by nmolar concentrations of nitrobenzylthioinosine and the cells lack high affinity nitrobenzylthioinosine binding sites. The cells also possess an adenine transporter, which is distinct from the nucleoside transporter. They lack, however, a hypoxanthine transport system and are deficient in hypoxanthine phosphoribosyltransferase activity, which explains their failure to efficiently salvage hypoxanthine from the medium. The cells possess uridine and thymidine phosphorylase activities and, in contrast to cultured mammalian cells, efficiently convert uracil to nucleotides. An adenosine-resistant variant (CAE-3-6) of the C-7-10 cell line is devoid of significant nucleoside transport activity but transports adenine normally. Residual entry of various nucleosides into these cells and of hypoxanthine and cytosine into wild type and mutant cells is strictly non-mediated. The rate of permeation of various nucleosides and of hypoxanthine into the CAE-3-6 cells is related to their hydrophobicity. Uridine permeation into CAE-3-6 cells exhibits an activation energy of about 20 kcal/mol. At high uridine concentrations permeation is sufficiently rapid to partly overcome the limitation in nucleoside salvage imposed by the nucleoside transport defect in these cells.     

Effects of Acetaldehyde on Growth and Biosynthesis in an Algal Flagellate Polytomella caeca*

SYNOPSIS. Eight mM acetaldehyde prevented growth of Polytomella caeca in acetate medium and differentially changed the labeling by acetate-2-¹⁴C of chromatographically separated RNA hydrolysate products. Four mM acetaldehyde also prevented growth in acetate medium unless uridine, thymidine, guanosine, uracil, thymine or quanine were present; then growth was delayed by 2 or 4 days. Orotidine, orotic acid, dihydroortic acid, cytosine, cytidine, adenosine and adenine had no effect on growth in acetate medium containing 4 mM acetaldehyde. One mM acetaldehyde promoted growth in acetate medium and also could serve as a sole carbon source. One mM propionaldehyde, but not butyraldehyde, was also an adequate carbon source. Four mM acetaldehyde, as a sole carbon source, supported growth only when uridine was present.