scholarly journals Purine Salvage in Two Halophilic Archaea: Characterization of Salvage Pathways and Isolation of Mutants Resistant to Purine Analogs

1998 ◽  
Vol 180 (3) ◽  
pp. 457-463 ◽  
Author(s):  
Birgitte Stuer-Lauridsen ◽  
Per Nygaard
Keyword(s):  
Reductase Activity ◽  
Nucleoside Analogs ◽  
Purine Salvage ◽  
Nucleotide Synthesis ◽  
Moderate Halophile ◽  
Purine Base ◽  
Purine Bases ◽  
Purine Analogs ◽  
Significant Difference ◽  
Pool Sizes

ABSTRACT In exponentially growing cultures of the extreme halophileHalobacterium halobium and the moderate halophileHaloferax volcanii, growth characteristics including intracellular protein levels, RNA content, and nucleotide pool sizes were analyzed. This is the first report on pool sizes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-α-1-pyrophosphate) in archaea. The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays. The levels varied significantly between the two organisms. The most significant difference was the absence of GMP reductase activity in H. halobium. The metabolism of exogenous purines was investigated in growing cultures. Both purine bases and nucleosides were readily taken up and were incorporated into nucleic acids. Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis.H. volcanii was more sensitive than H. halobium, and purine base analogs were more toxic than nucleoside analogs. Growth of H. volcanii was inhibited by trimethoprim and sulfathiazole, while these compounds had no effect on the growth of H. halobium. Spontaneous mutants resistant to purine analogs were isolated. The most frequent cause of resistance was a defect in purine phosphoribosyltransferase activity coupled with reduced purine uptake. A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosphoribosyltransferase had specificity towards adenine. The differences in the metabolism of purine bases and nucleosides and the sensitivity to purine analogs between the two halobacteria were reflected in differences in purine enzyme levels. Based on our results, we conclude that purine salvage and interconversion pathways differ just as much between the two archaeal species as among archaea, bacteria, and eukarya.

scholarly journals The Purine Efflux Pump PbuE in Bacillus subtilis Modulates Expression of the PurR and G-Box (XptR) Regulons by Adjusting the Purine Base Pool Size

2005 ◽  
Vol 187 (2) ◽  
pp. 791-794 ◽  
Author(s):  
Per Nygaard ◽  
Hans H. Saxild
Keyword(s):  
Bacillus Subtilis ◽  
De Novo ◽  
Efflux Pump ◽  
Purine Base ◽  
Purine Bases ◽  
Purine Analogs ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Transcriptional Fusions

ABSTRACT In Bacillus subtilis, the expression of genes encoding enzymes and other proteins involved in purine de novo synthesis and salvage is affected by purine bases and phosphoribosylpyrophosphate (PRPP). The transcription of the genes belonging to the PurR regulon is negatively regulated by the PurR protein and PRPP. The expression of the genes belonging to the G-box (XptR) regulon, including the pbuE gene, is negatively regulated by a riboswitch-controlled transcription termination mechanism. The G-box regulon effector molecules are hypoxanthine and guanine. pbuE encodes a purine base efflux pump and is now recognized as belonging to a third purine regulon. The expression of the pbuE gene is positively regulated by a riboswitch that recognizes adenine. Here we show that the expression of pbuE′-lacZ transcriptional fusions are induced by adenine to the highest extent in mutants which do not express a functional PbuE pump. In a mutant defective in the metabolism of adenine, the ade apt mutant, we found a high intracellular level of adenine and constitutive high levels of PbuE. A growth test using a purine auxotroph provided further evidence for the role of PbuE in lowering the intracellular concentration of purine bases, including adenine. Purine analogs also affect the expression of pbuE, which might be of importance for the protection against toxic analogs. In a mutant that overexpresses PbuE, the expression of genes belonging to the PurR regulon was increased. Our findings provide further evidence for important functions of the PbuE protein, such as acting as a pump that lowers the purine base pool and affects the expression of the G-box and PurR regulons, including pbuE itself, and as a pump involved in protection against toxic purine base analogs.

The specificity of purine base and nucleoside uptake in promastigotes ofLeishmania braziliensis panamensis

Parasitology ◽  
1982 ◽  
Vol 85 (2) ◽  
pp. 271-282 ◽  
Author(s):  
B. D. Hansen ◽  
J. Perez-Arbelo ◽  
J. F. Walkony ◽  
L. D. Hendricks
Keyword(s):  
Competitive Interactions ◽  
Leishmania Braziliensis ◽  
Purine Base ◽  
Purine Bases ◽  
Uptake Rates ◽  
Nucleoside Uptake ◽  
And Diffusion

SUMMARYPromastigotes ofLeishmania braziliensis panamensisabsorbed the purines adenine, hypoxanthine, adenosine and inosine by a combination of diffusion and mediated components. When the uptake rates for these substrates were corrected for diffusion and compared, the purine bases adenine and hypoxanthine were transported at a significantly slower rate than the purine nucleosides adenosine and inosine. Competitive interactions among those purines tested confirmed the presence of mediated and diffusion components and suggested that three transport loci may be operating (Fig. 6). The first transport locus, designated Locus 1, transported inosine, Locus 2, the purine bases hypoxanthine and adenine and Locus 3, adenosine. In addition, adenine and hypoxanthine inhibited the uptake of one another competitively. A comparison of Kivalues derived from double reciprocal plots of labelled hypoxanthine and adenine uptake in the presence of the unlabelled substrates as inhibitors suggested that adenine has a greater affinity for the transport locus.

scholarly journals Bioreduction of Cr(VI) by Viable Cells and Cell-Free Extract of a Moderate Halophile, Halomonas Smyrnensis KS802 and Evaluation of Their in Vitro Efficacy in Tannery Effluent

2020 ◽  
Author(s):  
Jhuma Biswas ◽  
AK Paul
Keyword(s):  
Reductase Activity ◽  
Basal Medium ◽  
Tannery Effluent ◽  
Chromate Reduction ◽  
Cell Free Extract ◽  
Tannery Effluents ◽  
Moderate Halophile ◽  
Viable Cells ◽  
Reducing Ability ◽  
Halomonas Smyrnensis

Abstract The present study was aimed to characterize the chromate reducing ability of cells and cell-free extract (CFE) of Halomonas smyrnensis KS802 (GenBank Accession No. KU982965) and evaluate their effectiveness in tannery effluents. Viable cells of the strain reduced 200 µM Cr(VI) in basal medium for halophiles (MH) in 10 h and was inversely proportional to Cr(VI) concentrations. The rapid reduction by cells (10⁹ cells/mL) was achieved with 7.5% NaCl, at pH 7 and 37°C which increased with increasing cell density (10¹° cells/mL). While acetate, Cu³⁺, Fe³⁺, SO₄²⁻, and CO₃²⁻ were stimulating the reduction, the inhibitors retarded the process significantly. The NADH-dependent chromate reduction of the CFE was found to be constitutive with Km and Vmax values of 56.58 µM and 3.37 µM/min/mg protein respectively. The optimal reductase activity of the CFE was evident at 200 µM Cr(VI), 10% NaCl, pH 8.0 and at 45°C. A higher concentration of CFE and electron donors increased the enzyme activity but was impacted negatively by toxic metals and anions. Both the cells and CFE were capable of reducing Cr(VI) remarkably from tannery effluent. FTIR and XRD spectra of chromate reducing cells confirmed possible complexation of reduced Cr-species with functional groups on cell surface.

Nitrogen Excretion in Ascidiacea II. Storage Excretion and the Uricolytic Enzyme System

1965 ◽  
Vol 42 (2) ◽  
pp. 299-305
Author(s):  
IVAN GOODBODY
Keyword(s):  
Uric Acid ◽  
Calcium Carbonate ◽  
Protein Metabolism ◽  
Enzyme System ◽  
Purine Metabolism ◽  
Nitrogen Excretion ◽  
Purine Base ◽  
Purine Bases ◽  
Ascidiella Aspersa

1. The evidence for the occurrence of storage excretion in ascidians is reviewed. Most species probably store uric acid or purine bases in some form. 2. The renal concretions of Ascidia nigra and Phallusia mammillata contain 50-60% uric acid, the remainder of the concretion is unidentified but is non-nitrogenous and is not calcium carbonate. In Ascidiella aspersa the concretion is predominantly composed of calcium carbonate and there is no significant quantity of uric acid or purine base. 3. Uric acid is also identified in Molgula manhattensis, Polycarpa obtecta, Pyura vittata and Herdmania momus. 4. Storage excretion probably results from a deficiency in the uricolytic enzyme system. It is concluded that while protein metabolism is ammonotelic, purine metabolism is uricotelic or xanthotelic.

scholarly journals The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

1968 ◽  
Vol 110 (3) ◽  
pp. 407-412 ◽  
Author(s):  
J. L. Holtzman ◽  
T. E. Gram ◽  
P. L. Gigon ◽  
J. R. Gillette
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Reductase Activity ◽  
Mixed Function Oxidase ◽  
Protein Ratio ◽  
Nadph Cytochrome ◽  
Rate Limiting Step ◽  
Significant Difference ◽  
The Difference ◽  
Cytochrome P 450

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3–5), per μg. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1·7) or per unit of NADPH–cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH–cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.

scholarly journals Possible role for mRNA stability in the ammonium-controlled regulation of nitrate reductase expression

1994 ◽  
Vol 297 (3) ◽  
pp. 561-565 ◽  
Author(s):  
A C Cannons ◽  
L C Pendleton
Keyword(s):  
Nitrate Reductase ◽  
Mrna Stability ◽  
Metabolic Regulation ◽  
Reductase Activity ◽  
State Level ◽  
Apparent Difference ◽  
Short Term ◽  
Cell Extracts ◽  
Significant Difference

Ammonium, or a metabolite of ammonium, represses the expression of nitrate reductase (NR) in Chlorella vulgaris. The removal of ammonium and addition of nitrate (induction) resulted in a rapid (20 min) peaked synthesis of NR mRNA. Nitrate reductase protein and activity increased at a much lower rate, reaching their maxima by 8 h. Ammonium added to nitrate-grown cells resulted in a dramatic decrease in NR mRNA from a steady-state level to undetectable levels within 15 min of ammonium addition. Nitrate reductase activity and protein levels decreased to 20% and 40% of initial levels respectively over 8 h. The half-life for NR mRNA under these conditions was estimated to be less than 5 min, compared with 120 min for NR protein. Such rapid decreases in NR mRNA suggested a degradation and/or cessation in NR mRNA transcription. No apparent difference in NR mRNA-specific RNAase activity of crude cell extracts (NR-induced or repressed) was observed. However, a significant difference in the susceptibility to degradation of NR mRNA from long-term nitrate-grown cells compared with the NR mRNA isolated from short-term induced cells (20 min in nitrate) was observed. NR mRNA isolated from long-term-nitrate-grown cells was completely degraded by RNAases in cell extracts under conditions in which the NR mRNA isolated from short-term induced cells was resistant to degradation. These results suggest that mRNA stability may be an important factor in the metabolic regulation of assimilatory nitrate reductase in Chlorella.

Measurement of endogenous allantoin excretion in sheep urine

1982 ◽  
Vol 98 (1) ◽  
pp. 221-223 ◽  
Author(s):  
Anna M. Antoniewicz ◽  
P. M. Pisulewski
Keyword(s):  
Nucleic Acid ◽  
Rumen Microorganisms ◽  
Purine Base ◽  
Purine Bases ◽  
Tissue Turnover ◽  
Micro Organisms ◽  
Sheep Urine

Urinary allantoin, an end product of purine base metabolism, originates in sheep from three possible sources: exogenous, purine bases of rumen microorganisms and feed purines and ureides, and endogenous, purines catabolized in tissue turnover. Earlier studies suggested that nucleic acid purines of rumen micro-organisms may be a predominant source of urinary allantoin (Antoniewicz, Heinemann & Hanks, 1979, 1981).

Expression of the high-affinity purine nucleobase transporter in mutant mouse S49 cells does not require a functional wild-type nucleoside-nucleobase transporter

1987 ◽  
Vol 7 (1) ◽  
pp. 97-103
Author(s):  
B Ullman ◽  
J Patrick ◽  
K McCartan
Keyword(s):  
Genetic Locus ◽  
Nucleoside Transport ◽  
Recessive Trait ◽  
Wild Type ◽  
Purine Base ◽  
Purine Bases ◽  
High Affinity ◽  
Low Concentrations ◽  
Guanine And Adenine ◽  
Mutant Cells

A novel type of somatic mutation that causes the expression of a high-affinity purine base permease (B. Aronow, D. Toll, J. Patrick, P. Hollingsworth, K. McCartan, and B. Ullmann, Mol. Cell Biol. 6:2957-2962, 1986) has been inserted into nucleoside transport-deficient S49 cells. Two classes of mutants expressing this nucleobase permease were generated. The first, as exemplified by the AE1HADPAB2 cell line, possessed an augmented capacity to transport low concentrations of the three purine bases, hypoxanthine, guanine, and adenine. The second class of mutants, as typified by the AE1HADPAB5 clone, possessed an augmented capability to translocate low levels of hypoxanthine and guanine, but not adenine. Neither the AE1HADPAB2 nor the AE1HADPAB5 cells could transport nucleosides, suggesting that the expression of the high-affinity base transporter did not reverse the mutation in the nucleoside transport system. The transport of purine bases by both AE1HADPAB2 and AE1HADPAB5 cells was much less sensitive than that by wild-type cells to inhibition by dipyridamole, 4-nitrobenzylthionosine, and N-ethylmaleimide, potent inhibitors of nucleoside and nucleobase transport in wild-type S49 cells. Fusion of the AE1HADPAB2 and AE1HADPAB5 cell lines with wild-type cells indicated that the expression of the high-affinity base transporter behaved in a dominant fashion, while the nucleoside transport deficiency was a recessive trait. These data suggest that the high-affinity purine base transporter of mutant cells and the nucleoside transport function of wild-type cells are products of different genes and that expression of the former probably requires the unmasking or alteration of a specific genetic locus that is silent or different in wild-type cells.

Stimulated transport of hypoxanthine in Crithidia luciliae: relationship to purine stress

Parasitology ◽  
1997 ◽  
Vol 114 (1) ◽  
pp. 19-27 ◽  
Author(s):  
R. E. DAY ◽  
A. M. GERO
Keyword(s):  
Substrate Specificity ◽  
Transport System ◽  
Diffusion System ◽  
Purine Nucleoside ◽  
Transport Mechanisms ◽  
Nucleotidase Activity ◽  
Purine Salvage ◽  
Purine Bases ◽  
Crithidia Luciliae ◽  
Adenosine Transport

Hypoxanthine transport in the insect trypanosome, Crithidia luciliae, was activated in purine depleted conditions. The existence of 2 hypoxanthine transport mechanisms was established. The first, a non-saturable diffusion system, present in purine replete conditions, exhibited properties that were different from the second transport system which was evident only during purine depleted conditions (purine stress). The rate of transport under purine stress was elevated approximately 8-fold over that in replete conditions. This transporter was saturable with a Km of 3·9 μM for hypoxanthine. The transporter substrate specificity included other purine bases, e.g. adenine and guanine, and the purine nucleoside, adenosine. These inhibited hypoxanthine transport competitively with Ki values of 2 μM, 3 μM and 42 μM respectively. Coincident with the increase of hypoxanthine transport under purine stress, the transport of adenosine increased 4-fold and the activity of the 3′-nucleotidase ectoenzyme also increased significantly. Under purine stress the concurrent increase of hypoxanthine and adenosine transport and the increase in 3′-nucleotidase activity could be repressed with either the supplementation of excess purines or by cycloheximide. This study of purine salvage mechanisms in Crithidia luciliae illustrates the successful adaptation of the parasite to nutritional insufficiency.

scholarly journals Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.)

1975 ◽  
Vol 146 (1) ◽  
pp. 79-85 ◽  
Author(s):  
T Suzuki ◽  
E Takahashi
Keyword(s):  
De Novo ◽  
Tea Plant ◽  
Shoot Tips ◽  
Purine Nucleotides ◽  
Purine Salvage ◽  
Nucleotide Synthesis ◽  
Caffeine Biosynthesis ◽  
Tea Shoot ◽  
Tea Plants ◽  
Almost All

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.

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