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.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

Utilization of organic compounds as the sole source of nitrogen by Thiobacillus thiooxidans

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.Non-standard abbreviation U-¹⁴C uniformly labeled by ¹⁴C     

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

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

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

Absorption and metabolism of purines by the lower intestine of the chicken.

1. Absorption of purines and their metabolism by the lower intestine were estimated by using the everted gut sacs from the colo-rectum and caecum of the chicken. 2. Adenine, hypoxanthine and uric acid were appreciably absorbed from the colo-rectum and caecum, and an especially high rate was observed in the absorption of uric acid from the colo-rectum. 3. Guanine was not absorbed unchanged from either the colo-rectum or the caecum and a small amount of xanthine was absorbed only from the caecum. 4. Hypoxanthine was also absorbed in uric acid form, to a much lesser extent, in xanthine form from the colo-rectum and caecum, adenine and xanthine in uric acid form from the colo-rectum and adenine in hypoxanthine form from the colo-rectum and caecum. 5. Adenine was metabolized to hypoxanthine and xanthine, guanine and hypoxanthine to uric acid and xanthine, and xanthine to adenine, in both mucosal fluids of the colo-rectum and caecum. The conversion of guanine to uric acid in the caecum was most active, being almost twice as much as that in the colo-rectum.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Absorption and metabolism of purines by the small intestine of the chicken.

1. Absorption of purines and their metabolism by the small intestine were estimated by using the everted gut sacs from the duodenum, jejunum and ileum of the chicken. 2. When no purine was added to the mucosal fluid, large amounts of uric acid, much less but appreciable adenine, hypoxanthine and xanthine and no detectable guanine were released from both sides of all segments of the small intestine, and these released amounts were largest in the duodenum. 3. Similar absorption rates of adenine from the jejunum and ileum were about 1.7-3.0 times as high as those of hypoxanthine and uric acid from these intestines and those of adenine and uric acid from the duodenum (P less than 0.05). 4. Guanine was not absorbed unchanged from any segments of the intestine and a little xanthine was absorbed only from the jejunum and ileum. 5. Guanine and xanthine seem to be absorbed in uric acid form, hypoxanthine in xanthine and uric acid forms and adenine in hypoxanthine form, from the small intestine especially from the jejunum. 6. Adenine, guanine, xanthine and hypoxanthine were greatly metabolized in the mucosa of the duodenum, and the conversions of hypoxanthine to xanthine and uric acid were most active.     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Purification and Properties of Uricase from Enterobacter cloacae

Uricase activity was found in Enterobacter cloacae KY3074 grown on guanine, hypoxanthine, uric acid, and xanthine media. The enzyme was purified from cells grown on uric acid as a source of nitrogen. The purification procedure included ammonium sulfate fractionation, gel filtration on Sephadex G-150, and column chromatography on DEAE-cellulose and DEAE-Sephadex. The enzyme had a molecular weight of about 105,000 and was specific for uric acid. The optimum pH was around 9.5, and the activity was inhibited by the presence of potassium cyanide, Ag⁺ or Cu²⁺. This uricase can be used for estimation of uric acid.     

Riboflavin-binding sites associated with flagella of Euglena: A candidate for blue-light photoreceptor?

The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena gracilis. Published evidence shows that the phototactic stimulus — with a flavin-type action spectrum — is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euglena gracilis by a combined cold and Ca²⁺ shock. Saturable binding of [¹⁴C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for RF were high: K _D (oxidized)=0.08 M, and K _D (reduced)=0.7 M. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 10⁶. This number is in line with published estimates of chromophores bound in or at the PFB.Abbreviations FAD flavin adenine dinucleotide - FMN flavin mononucleotide - PFB paraflagellar body - RF riboflavin This work was supported by the Deutsche Forscungsgemeinschaft.     

Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine,xanthine, uric acid,putrescine and primary n-alkylamines as the sole source of carbon,nitrogen and energy

A new yeast species, Trichosporon adeninovorans, was isolated from soil by the enrichment culture method. Apart from adenine, the strain utilized uric acid, guanine, xanthine, hypoxanthine, 6,8-dihydroxypurine, putrescine, propylamine, butylamine, pentylamine, hexylamine and octylamine as sole source of carbon, nitrogen and energy.The structure of the cell wall of Tr. adeninovorans was ascomycetous. On the subcellular level growth on adenine or uric acid was accompanied with the development of microbodies in the cell. These cell organelles probably were the site of urate oxidase, an enzyme that, after growth on purine substrates, together with allantoinase was present at high activities. Low activities of adenine amidohydrolase and xanthine dehydrogenase were also demonstrated.     

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.     

Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN ⁶-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective K_i values were 3.5×10^–6 M and 9.5×10^–6 M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10^–4 M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers.     

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

Uric acid synthesis by rat liver supernatants from purine bases,nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0·5 mM and guanosine and guanine, 0·1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g^?1 of wet liver min^?1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylo-succinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min^?1 per mg^?1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0·1 and 0·05 mM concentrations was also determined. ATP (1 nM) strongly inhibited uric acid synthesis from 0·05 mM AMP (91 per cent) and from 0·05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM ) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0·05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0·012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.     

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes

The ³¹P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pK_a, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pK_a < 7 allowed an easy and simultaneous ³¹P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum.     

Identification of xanthine and hypoxanthine as components of assembly pheromone in excreta of argasid ticks

In addition to guanine, xanthine and hypoxanthine were identified in white spherules in excreta of five species ofArgas andOrnithodoros ticks by a reverse-phase high-performance liquid chromatography (HPLC) and a gas chromatographic method with mass spectrometric detection, (GC/MS). The mutual relationships of these purines in excreta ofArgas (Persicargas) persicus were found to be less than 1.5% for hypoxanthine, less than 9.0% for xanthine and 89.8–98.6%, for guanine. In excreta of other species, the relationships of purines were similar, with the exception ofArgas (A.) reflexus andA. (A.) polonicus, where the amount of hypoxanthine was rather elevated. Uric acid was also identified in some cases. The assembly efficacy of xanthine and hypoxanthine is similar to that of guanine, but xanthine significantly enhances the assembly efficacy of commercial guanine when mixed in ratio of about 125. Thus, xanthine seems to be the second important component of assembly pheromone of argasid ticks.     

Distinction between Hypoxanthine and Xanthine Transport in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii cells consumed hypoxanthine and xanthine by means of active systems which promoted purine intracellular accumulation against a high concentration gradient. Both uptake and accumulation were also observed in mutant strains lacking xanthine dehydrogenase activity. Xanthine and hypoxanthine uptake systems exhibited very similar Michaelis constants for transport and pH values, and both systems were induced by either hypoxanthine or xanthine. However, they differed greatly in the length of the lag phase before uptake induction, which was longer for hypoxanthine than for xanthine. Cells grown on ammonium and transferred to hypoxanthine media consumed xanthine before hypoxanthine, whereas cells transferred to xanthine media did not take up hypoxanthine until 2 hours after commencing xanthine consumption. Metabolic and photosynthetic inhibitors such as 2,4-dinitrophenol, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, and carbonylcyanide m-chlorophenylhydrazone inhibited to a different extent the hypoxanthine and xanthine uptake. Similarly, N-ethylmaleimide abolished xanthine uptake but slightly affected that of hypoxanthine. Hypoxanthine consumption was inhibited by adenine and guanine whereas that of xanthine was inhibited only by urate. We conclude that hypoxanthine and xanthine in C. reinhardtii are taken up by different active transport systems which work independently of the intracellular enzymatic oxidation of these purines.