Mechanism of Action of Showdomycin

¹⁴C-Labelled showdomycin was rapidly taken up by Escherichia coli K-12 cells. The showdomycin uptake was highly temperature dependent, sensitive to azide and N-ethyl-maleimide, but was only partially inhibited by treatment with high concentration of iodoacetic acid.

The uptake of showdomycin was inhibited by a wide variety of nucleosides but not by purine and pyrimidine bases, nucleotides, ribose or ribose-5-phosphate. The inhibition of showdomycin uptake by adenosine was of a competitive type.

Since nucleosides inhibited the uptake of showdomycin but did not facilitate its efflux, they must play a role of inhibitors to the entry of the antibiotic into cells.

Removal of extracellular showdomycin by washing, or inhibition of its subsequent entry into cells by the addition of nucleosides or sulfhydryl compounds resulted in a rapid decrease in the intracellular level of the antibiotic during subsequent incubation.     

Nucleoside transport systems in Escherichia coli K12: Specificty and regulation

Two nucleoside transport systems have been verified and separated by mating and recombination experiments. The recipient strain was a mutant which is negative for transport of all nucleosides. The two systems differ in specificity and in regulation. One system transports pyrimidine and adenine in specificity and in regulation. One system transports pyrimidine and adenine nucleosides, but not guanine nucleosides. It is regulated by the cytR gene. The other system transports all nucleosides and is regulated by the cytR as well as by the deoR genes. Enzyme assays performed on whole cells of strains, able or unable to transport nucleosides, indicate that the nucleoside catabolizing enzymes are located inside the permeability barrier of the cell.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.     

Mechanism of Action of Showdomycin

Showdomycin, 2-β-d-ribofuranosylmaleimide, inhibited the incorporation of amino acids and purine and pyrimidine bases into macromolecules in E. coli K-12 cells at low concentrations. The inhibitory action of showdomycin could be reversed by the addition of a nucleoside or a sulfhydryl compound. In marked contrast to common nucleosides, the pseudouridine showed no such effect. This may indicate that the N-glycosyl linkage in the nucleoside is a structural requirement for its reversing activity on the inhibitory action of showdomycin.

N-Ethylmaleimide, which has structural similarity to showdomycin, inhibited the incorporation of amino acids and purine and pyrimidine bases as well as showdomycin. The inhibitory action of N-ethylmaleimide, however, was not reversed by the addition of a nucleoside. This may indicate that there may be difference in the mechanism of the inhibitory action between N-ethylmaleimide and showdomycin.     

Purine and pyrimidine metabolism in cultured white spruce (Picea glauca) cells: Metabolic fate of 14C-labeled precursors and activity of key enzymes

In order to examine the biosynthesis, interconversion, and degradation of purine and pyrimidine nucleotides in white spruce cells, radiolabeled adenine, adenosine, inosine, uracil, uridine, and orotic acid were supplied exogenously to the cells and the overall metabolism of these compounds was monitored. [8‐¹⁴C]adenine and [8‐¹⁴C]adenosine were metabolized to adenylates and part of the adenylates were converted to guanylates and incorporated into both adenine and guanine bases of nucleic acids. A small amount of [8‐¹⁴C]inosine was converted into nucleotides and incorporated into both adenine and guanine bases of nucleic acids. High adenosine kinase and adenine phosphoribosyltransferase activities in the extract suggested that adenosine and adenine were converted to AMP by these enzymes. No adenosine nucleosidase activity was detected. Inosine was apparently converted to AMP by inosine kinase and/or a non‐specific nucleoside phosphotransferase. The radioactivity of [8‐¹⁴C]adenosine, [8‐¹⁴C]adenine, and [8‐¹⁴C]inosine was also detected in ureide, especially allantoic acid, and CO₂. Among these 3 precursors, the radioactivity from [8‐¹⁴C]inosine was predominantly incorporated into CO₂. These results suggest the operation of a conventional degradation pathway. Both [2‐¹⁴C]uracil and [2‐¹⁴C]uridine were converted to uridine nucleotides and incorporated into uracil and cytosine bases of nucleic acids. The salvage enzymes, uridine kinase and uracil phosphoribosyltransferase, were detected in white spruce extracts. [6‐¹⁴C]orotic acid, an intermediate of the de novo pyrimidine biosynthesis, was efficiently converted into uridine nucleotides and also incorporated into uracil and cytosine bases of nucleic acids. High activity of orotate phosphoribosyltransferase was observed in the extracts. A large proportion of radioactivity from [2‐¹⁴C]uracil was recovered as CO₂ and β‐ureidopropionate. Thus, a reductive pathway of uracil degradation is functional in these cells. Therefore, white spruce cells in culture demonstrate both the de novo and salvage pathways of purine and pyrimidine metabolism, as well as some degradation of the substrates into CO₂.     

Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

Synthesis of Some Purine Carbocyclic Isosteres of 2′,3′-Dideoxy-3′-C-Hydroxymethyl Nucleosides as Potential Inhibitors of HIV

Abstract

The synthesis of some enantiomerically pure carbocyclic 2′,3′-dideoxy-3′-C-hydroxymethyl derivatives of adenine, inosine and guanine is described. The Mitsunobu reaction was used in the coupling procedure giving exclusively N⁹-coupling. The nucleosides were tested for inhibition of HIV multiplication in vitro and were found to be inactive in the assay.     

Promotion or retardation of the growth of adenine auxotrophs ofCandida albicans by purines,pyrimidines and nucleosides

A study has been made of the growth responses to purine and pyrimidine metabolites shown by sixteen ultraviolet-induced adenine requiring mutants ofCandida albicans blocked at early stages in purine biosynthesis. The salient findings establish that, inC. albicans, (1) the pathway for the conversion of adenine to guanine is not reversible, (2) exogenous nucleotides are not utilized, and the purine and pyrimidine components of exogenous nucleosides must be converted to the free base form before utilization and (3) cytosine and guanine competitively inhibit different steps in the utilization of exogenous adenine.     

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.     

Purine metabolism in Toxoplasma gondii

We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.     

Utilization of adenine and guanine as nitrogen sources by Chlamydomonas reinhardtii cells

Chlamydomonas reinhardtü Dangeard, adenine or guanine can be used as the sole nitrogen source for growth by means of an inducible system which is repressed by ammonia. Cells grown on either adenine or guanine were able to take up both purines, although the adenine uptake rate was always about 40% of the guanine uptake rate. Both adenine and guanine were taken up by an inducible system(s) exhibiting hyperbolic kinetics with identical apparent A, values of 3-2 mmol m^?3 for adenine and 3-2mmol m^?3 for guanine. Adenine and guanine utilization depended on pH, with similar optimal pH values of 7·3 and 7·4, respectively. Adenine and guanine each acted as a competitive inhibitor of the other's uptake, and their utilization was also inhibited by hypoxanthine, xanthine and urate. Inhibition of adenine uptake by guanine and hypoxanthine was competitive, with A′, values of 5·5 and 1. 6 mmol m^?3 respectively. Guanine uptake was also inhibited competitively by adenine (K₁= 1·3mmol m^?3) and hypoxanthine (K₁= 3. 3 mmol m^?3). Utilization of both adenine and guanine was inhibited by cyanide, azide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,4-dinitrophenol and carbonylcyanide m-chlorophenylhydrazone, and was also sensitive to p-hydroxymercuribenzoate and N-ethyl-maleimide. On the basis of these results, taken together, the possibility that adenine and guanine are translocated into Chlamydomonas by a common system is discussed.     

Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

Entamoeba histolytica: uptake of purine bases and nucleosides during axenic growth.

A comparison was made of the uptake mechanisms of selected purine bases and nucleosides by axenically grown Entamoeba histolytica. Adenine, adenosine, and guanosine were taken up, in part, by a “carrier”-mediated system. Guanine, hypoxanthine, and inosine entered amoebas via diffusion. Inhibitor studies support the presence of individual transport sites for adenine-adenosine and adenosine-guanosine. Additional sites for transport of adenine, adenosine, and guanosine are implied by “non-productive binding” involving guanine, hypoxanthine, and inosine. Uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N-ethylmaleimide. Ribose failed to inhibit uptake of purine nucleosides.     

Mechanism of Action of Showdomycin

The effect of showdomycin on the syntheses of deoxyribonucleotides from various pyrimidine and purine derivatives was studied in cell-free systems from E. coli.

The formations of deoxycytidine phosphates, deoxyuridine phosphates, deoxyguanosine phosphates and deoxyadenosine phosphates from the corresponding ribonucleoside diphosphates were all inhibited by low concentrations of showdomycin. The formation of deoxythymidine phosphates from dUMP was also very susceptible to the antibiotic. These inhibitory actions of showdomycin could be reversed by a sulfhydryl compound (mercaptoethanol) but not by nucleosides, in contrast to a previous finding that the inhibitory action of this antibiotic on the cell growth was reversed by compounds belonging to both of these groups.

N-Ethylmaleimide (NEM), a thiol reagent which has a structure related to the aglycone moiety of showdomycin, was also found to be a potent inhibitor of both the reduction of CDP and the methylation of dUMP as showdomycin. A mercurial thiol reagent, p-chloromercuribenzoic acid (PCMB), however, was found to be inactive against the methylation of dUMP although the salvage synthesis of dUMP was inhibited by low concentrations of this reagent.

The formations of deoxythymidine phosphates and of deoxyuridine phosphates from their respective pyrimidine bases and a deoxyribosyl donor were quite resistant to showdomycin.     

A comparison of purine metabolism and nucleotide pools in normal and hypoxanthine-guanine phosphoribosyltransferase-deficient neuroblastoma cells

Purine nucleotide synthesis and interconversion were examined over a range of purine base and nucleoside concentrations in intact N4 and N4TG (hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient) neuroblastoma cells. Adenosine was a better nucleotide precursor than adenine, hypoxanthine or guanine at concentrations greater than 100 μM. With hypoxanthine or guanine, N4TG cells had less than 2% the rate of nucleotide synthesis of N4 cells. At substrate concentrations greater than 100 μM the rates for deamination of adenosine and phosphorolysis of guanosine exceeded those for any reaction of nucleotide synthesis. Labelled inosine and guanosine accumulated from hypoxanthine and guanine, respectively, in HGPRT-deficient cells and the nucleosides accumulated to a greater extent in N4 cells indicating dephosphorylation of newly synthesized IMP and GMP to be quantitatively significant. A deficiency of xanthine oxidase, guanine deaminase and guanosine kinase activities was found in neuroblastoma cells. Hypoxanthine was a source for both adenine and guanine nucleotides, whereas adenine or guanine were principally sources for adenine (>85%) or guanine (>90%) nucleotides, respectively. The rate of [¹⁴C]formate incorporation into ATP, GTP and nucleic acid purines was essentially equivalent for both N4 and N4TG cells. Purine nucleotide pools were also comparable in both cell lines, but the concentration of UDP-sugars was 1.5 times greater in N4TG than N4 cells.     

Genes conferring resistance to 8-aza adenine in Neurospora crassa and the variability of resistant alleles in the aza-1 locus with respect to excretion of purines

Summary Strains inhibited by analogues of aromatic amino acids due to mutations in the 5mt locus were also sensitive to the purine analogues, 8-aza adenine, 8-aza guanine and 2,6-diaminopurine. Adenine or hypoxanthine and the respective ribosides relieved inhibition due to purine analogues but guanine or guanosine were not effective. Using resistance to 8-aza adenine and sensitivity to DL-4-methyl tryptophan as criteria aza-1 ^r and aza-2 ^r mutants were isolated and both loci were located in linkage group I the former was 10–20 units distal to the mating type and the latter approximately 2 units in the vicinity of mating type. The four allelic aza-1 ^r strains were resistant to 8-aza adenine and 2,6-diaminopurine but one of them showed increased resistance to 8-aza guanine also. The aza-2 ^r strain was resistant to both 8-aza analogues but was comparable to the parental aza-2 ^s strain in its sensitivity to 2,6-diaminopurine. One aza-1 ^r was distinguished by its ability to excrete hypoxanthine and/or inosine and this ability always segregated with the aza-1 ^r allele and appears to be a consequence of a single mutation in the aza-1 locus. In heterokaryon one aza-1 ^r and one aza-2 ^r alleles were found to be recessive to the respective wild type alleles. Uptake of exogenous adenine was reduced in germinating conidia of one aza-1 ^r and one aza-2 ^r strain when compared to a parental aza-1 ^s, aza-2^s strain but the low affinity of 8-aza adenine to compete with adenine even in the sensitive strain indicated a need for direct study of the transport and metabolic reactions of the analogue. If an altered purine phosphoribosyl transferase is involved in the aza-1 ^r phenotype, an analogy with the Lesch-Nyhan syndrome of man, with respect to analogue-resistance and altered regulation of purine metabolism, seems to be appropriate.     

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.