Presence Of Adenine Research Articles

Biosynthesis of Cytokinins in Soil

AbstractCytokinin biosynthesis in soil by rhizosphere microorganisms may increase upon the addition of physiological precursors and affect plant growth. This study was conducted to establish the effects of the purine, adenine (ADE), the isoprenoid, isopentyl alcohol (IA), and a cytokinin‐producing bacterium, on cytokinin biosynthesis in an Arlington soil (coarse‐loamy, mixed thermic Haplic Durixeralf). Various concentrations of ADE were applied, separately and in combination with 8.81 × 10−2 g IA kg−1 soil, and an inoculum of Azotobacter chroococcum (ATCC 9043), as soil treatments. Cytokinin bioactivity, isolation, and quantification of soil extracts were monitored over a period of 7 d using the radish cotyledon bioassay and HPLC‐UV spectrometry. Cytokinin production was evident as early as 2 d and reached a maximum at 5 d post‐treatment. An application of 1.35 × 10−4 g ADE kg−1 soil, 8.81 × 10−2 g IA kg−1 soil, and A. chroococcum enhanced cytokinin production of zeatin riboside and t‐zeatin in this soil up to 1.50‐ and 1.39‐fold, respectively, in comparison to controls containing no precursors. The production of zeatin riboside (78.0 µg kg−1) and t‐zeatin (22.1 µg kg−1) in the presence of ADE and IA was 1.35‐ and 2.44‐fold greater with the inoculation of A. chroococcum, when compared to the indigenous microflora alone. This study provides information on extraction and analytical detection of cytokinins in soil and reveals that microbial biosynthesis of cytokinins can be enhanced by applying physiological precursors to soil.

Comparison of the effects of adenine-ribose with adenosine for maintenance of ATP concentrations in 5-day hypothermically perfused dog kidneys

The quality of preservation of kidneys is dependent upon a number of factors, one of which may be the concentration of adenine nucleotides in the tissue during long-term perfusion preservation. In this study we have investigated how adenine (5 m M) and ribose (5 m M) in combination affect the concentration of adenine nucleotides in dog kidney cortical tissue after 5 days of continuous hypothermic perfusion preservation. These results were compared to kidneys perfused with adenosine and without any added purine precursors of adenine nucleotide synthesis. Additionally, we investigated how these conditions affected renal tissue slice function after 5 days of preservation and how adenine plus ribose affected renal function after autotransplantation in the dog. Adenosine is nearly completely degraded during 5 days of perfusion but there was little loss of adenine (10%). The adenosine triphosphate concentration in kidney cortical tissue was higher in adenine/ribose-perfused kidneys (1.41 ± 0.19 μmol/g) than in adenosine-perfused kidneys (0.71 ± 0.1 μmol/g) after 5 days of preservation. Tissue slices prepared from kidneys preserved in the presence of adenine plus ribose were metabolically more functional (slice volume control and electrolyte pump activity) than slices from adenosine-perfused kidneys. Adenine plus ribose had no detrimental effects on kidneys preserved for 3 days as tested in the autotransplant model but did not yield successful 5-day preservation. Because of some potentially detrimental factors in using adenosine as an adenine nucleotide synthesis precursor, we have now switched to the combination of adenine and ribose for perfusion preservation of kidneys both in the laboratory and in the clinic.

Electron transfer mechanisms of some biochemically active purines

Free purines are found in some of the biological Systems. Some types of purines as consti- tuents of large molecules however are found in every living celi. Uric acid is a purine derivative which was discovered över one hundred years ago. It is found in blood, cerebral spinal fluid and milk. Bird excrement, guano contains a significant amount of uric acid. Uric acid as well as other oxypurines are the majör products of purine metabolism in man. Presence of adenine and guanine in nucleic acids is important in transfer of genetic infor- mation. Oxidation and reduction of purine derivatives in human and animal metabolisms have been studied extensively. Electron transfer mechanisms of purines, are therefore important in stud- ying their role in biological Systems. In the present study the electron transfer mechanisms of some biologically active purines are discussed.

Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli.

Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA+ uvr+ bacteria, plasmid pIC80, mucAB+ mediated UV mutagenesis more efficiently than did plasmid pSE117, umuDC+. A similar result was obtained in lexA41 (Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recA142 mutant, pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA+ uvrB5 bacteria, plasmid pSE117, umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These negative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA+ uvrB5 cells. In contrast, overproduction of MucAB protein in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis.

Adenylate Metabolism in Phosphate-depleted Isolated Soybean Leaf Cells and Wheat Leaf Fragments

Summary The influence of phosphate deficiency on adenylate degradation and on adenine incorporation into adenylates was examined in isolated soybean leaf cells and in wheat leaf fragments. Phosphate deficiency was obtained either by sequestration with non-metabolizable sugars (glucosamine or mannose) or by cultivating the plants on phosphate-deprived media. Adenylate catabolism was studied on adenine- 14 C prelabelled plant material submitted to phosphate sequestrating treatments. Adenine incorporation into adenylates was studied by measuring adenylate- 14 C labelling rates in the presence of adenine- 14 C on already phosphate-deficient plant material. Phosphate sequestration resulted in enhanced adenylate degradation rates with an accumulation of purine oxidation products. Phosphate-deficient plant material showed lower adenine- 14 C incorporation rates into adenylates. Then, the lower adenylate levels observed in phosphate-deficient plants are very likely due to the combined effect of lower adenylate synthesis rates and higher degradation rates.

INHIBITION OF DE NOVO PURINE SYNTHESIS BY METHYLTHIOADENOSINE: 72

De novo purine synthesis in human B lymphoblast cell lines was inhibited by methylthioadenosine (MTA) or adenine in the medium. Two human leukemic T-cell lines lacking methylthio-adenosine phosphorylase (MTAP) activity showed increased rates of de novo purine synthesis which were resistant to inhibition by MTA but were inhibited by adenine in a similar fashion to MTAP+ cells. The presence of MTA in culture media inhibited growth in a dose-dependent manner in MTAP+ and MTAP− cell lines. Similar inhibition of growth was effected by the presence of adenine in the medium. These results suggest that the inhibition of de novo purine synthesis by MTA is due to cleavage of the MTA to adenine by the MTAP enzyme. The adenine so formed can be converted to 5'-AMP in a PRPP-dependent reaction catalysed by the purine salvage enzyme APRT. Inhibition of growth of the MTAP− cell lines by MTA suggests that MTA may also exert effects on cell metabolism by a mechanism unrelated to its degradation.

Mononuclear cell-surface antigens during storage of banked blood.

The surface antigens present on lymphocyte subpopulations and monocytes in whole blood stored under standard blood bank conditions were analyzed with a series of monoclonal antibodies and a fluorescence-activated cell sorter. During the first week of storage, the percentage of viable cells bearing T lymphocyte markers declined from 66% to 29% (P less than 0.001). Within the T cell subset, there was a disproportionate decrease in the percentage of cells reacting with anti-Leu 3a (a helper T cell marker) resulting in a reduction in the measured ratio of helper-to-suppressor T cells (P less than 0.01). The relative percentage of cells bearing B lymphocyte markers increased from 11% to 31% (P less than 0.001). The proportion of HLA-DR-positive cells that were B cells increased during the first week of storage from 38% to 65% (P less than 0.01). The degree of residual antigen expression as measured by the remaining median intensity of fluorescence was significantly greater for B cell and HLA-DR antigens as compared with T cell antigens. The measured changes during storage in the relative proportions of mononuclear cells expressing cell-surface antigens probably result from a combination of differential residual antigen expression and differential survival of mononuclear subpopulations. The presence of adenine in the anticoagulant-preservative solution had no measurable effect. Storage at 4 C, however, was shown to result in a 70% decrease in the proportion of antigen-bearing T cells, even as early as 24 hr of storage. The findings may have bearing on the beneficial effect of blood transfusion in renal transplant recipients, as well as on experimental models of the immune response to transfusion.

Restoration of RecA protein activity by genetic complementation.

Bacteria carrying either recA430 or recA453-441 mutations are sensitive to UV-irradiation since they amplify the synthesis of RecA protein either poorly or not at all. We show here that, in a recA453-441 (recA430) heterodiploid, UV-resistance and amplification of RecA430 protein were restored, indicating that the cellular level of RecA-associated protease activity was high enough to inactivate LexA repressor. Prophage 434 repressor was also extensively inactivated, whereas RecA430 protein alone cannot cleave this substrate. On the other hand, during growth of the recA453-441(recA430) heterodiploid at 42 degrees C in the presence of adenine, a treatment activating only RecA441 protein, RecA441 protease activity was as high as in a recA441 haploid. In contrast, following this inducing treatment, there was no complementation between RecA441 and RecA+ proteins in a recA453-441(recA+) heterodiploid. These results indicate that multimerization of RecA protein molecules results in a functional interaction that, in some combination between RecA protein subunits, may enhance RecA-associated protease activity.

Search for heterocyclic radiolytic products in aqueous solutions of cyanides

This work is a part of a systematic search for biologically significant compounds which are formed during radiolysis of aqueous solutions of cyano compounds. Aqueous solutions of HCN (0.2M, O 2-free, natural pH) were irradiated at 18 Mrad dose of 60Co gammas, and the non-volatile radiolytic products concentrated in a dry residue. An aliquot of the dry residue was hydrolyzed with formic acid and the hydrolyzate selectively absorbed on active charcoal. The bulk of products from the charcoal was passed through a SP Sephadex C-25 column and the fractions analyzed by means of high performance liquid chromatography on a reverse phase column. Tentative assignments are based on retention times in the HPLC procedure and their pH dependence, and the coinjection of standards. The presence of adenine is certain. There is supporting evidence for uracil, thymine and cytosine. Experimental results also strongly support the presence of triazines.

Differential capacitance of mercury in the absence and the presence of adenosine in aqueous electrolytes

The differential capacitance of mercury in the absence and the presence of adenosine was traced in 1.0 N aqueous solutions of Na 2SO 4, NaClO 4, KNO 3, KCl, KBr and H 2SO 4 over appropriate potential ranges at 1 kHz and 25 °C. The results are informative with respect to the adsorption, reorientation and association of adenosine in Na 2SO 4 and NaClO 4, which are assumed not to exhibit specific anion adsorption. The area occupied by the molecule in the vertical orientation was calculated from the relative surface coverage by a monolayer of adenosine and the associated minimum capacitance. The measurements provide evidence of the effect of both ClO - 4 and Br - anions on the stacking interactions between the adsorbed molecules, which prevent the formation of multilayers in these electrolytes. Measurements of the differential capacitance of mercury in the presence of adenine, ribose sugar and a mixture of both lead to the conclusion that the purine part plays the major role in the reorientation of adenosine.

Regulation of purE transcription in a purE::lac fusion strain of Escherichia coli

A purE::lac fusion strain was isolated by using a special Mu phage developed by M. Casadaban. In the presence of adenine (100 micrograms/ml), beta-galactosidase synthesis was repressed by greater than 90%. beta-Galactosidase activity could be detected 6 to 8 min after the removal of adenine and increased linearly for at least 20 min. purR- mutants were isolated and synthesized 1.7- to 1.8-fold-higher levels of beta-galactosidase compared with purR+ cells. Azaserine derepressed purE transcription approximately 1.7-fold by lowering purine nucleotide pools. Glutamine and pyrimidine supplementation or starvation had no effect on purE transcription. A comparison of the rate of de novo purine biosynthesis and purE transcription indicated that the in vivo rate of de novo purine biosynthesis was more sensitive to the inhibitory effects of adenine than was transcription at the purE locus.

S-adenosylhomocysteinase from mouse liver. Inactivation of the enzyme in the presence of metabolites

1. 1. S-Adenosylhomocysteinase ( S-adenosylhomocysteine hydrolase, EC 3.3.1.1) was slowly inactivated in the presence of adenine and adenine nucleotides (Ueland & Saebø, 19790). 2. 2. The enzyme was stabilized by 2-mercaptoethanol and dithiothreitol, and was slowly inactivated at 37°C in the absence of reducing agents and rapidly inactivated in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The inactivation (both in the absence and presence of DTNB) was partly prevented by adenine, AMP and ADP. 3. 3. A slow decrease in enzyme activity was observed in the presence of AMP, ADP and ATP, and this process was enhanced by sulfhydryl compounds like l-homocysteine, l-cysteine, 2-mercapthoethanol and dithiothreitol. 4. 4. Inactivation of the enzyme by adenine was independent of sulfhydryl compounds, and was characterized by an initial phase showing first-order kinetics and saturability with respect to adenine. 5. 5. Inorganic phosphate nearly abolished the inactivation of l-adenosylhomocysteinase induced by both adenine nucleotides and adenine. 6. 6. The enzyme activity was recovered when adenine was removed by dilution or gel filtration. Attempts to reverse the effect of adenine nucleotides on the enzyme were not successful. 7. 7. The effect of adenine nucleotides was a V max-effect, and the inactivation was not associated with dissociation or polymerization of the enzyme or dissociation of enzyme-bound NAD.

Mechanism of adenine toxicity in Escherichia coli.

The mechanism of adenine toxicity in an hpt gpt strain of Escherichia coli that is extremely sensitive to adenine inhibition was investigated. Adenine-resistant derivatives had secondary mutations in adeninephosphoribosyltransferase or the purR repressor. Growth studies with various purine salvage pathway mutants and the ability of guanosine to prevent adenine toxicity indicated that adenine exerts its toxic effects by depleting guanine nucleotide pools. In the presence of adenine, ATP pools increased twofold in wild-type cells and stabilized after 5 min. In contrast, ATP pools continued to rise in hpt gpt cells up to 25 min and increased sevenfold after adenine addition. hpt gpt cells were shown to have higher levels of adeninephosphoribosyltransferase than did the wild-type cells. In response to adenine addition, GTP pools dropped three- to fourfold in all strains tested. Although GTP levels returned to near normal values in wild-type cells after 35 min, no restoration of GTP pools was observed in the hpt gpt strain during this period. Measurements of guanine pools before and after the addition of adenine indicated that guaninephosphoribosyltransferase plays an important role in maintaining GTP pools by converting the free guanine to GMP during guanine nucleotide depletion.

Changes in nucleotide pools during conidial germination in Neurospora crassa.

The acid-soluble nucleotide pools of wild type and several adenine auxotrophs of Neurospora crassa were studied immediately prior to and during conidial germination in the presence of adenine. A two- to four-fold increase in most nucleotide pools was observed after 6 h of germination at 33 °C indicating a general increase in nucleotide pools during this developmental period. The largest components of the acid-soluble nucleotide pools were uracil-containing nucleotide–sugars, which are precursors of chitin and glucan, the major constituents of cell walls. On removal of adenine, the UDP–sugar pools decreased significantly, in adenine auxotrophs, while the pools of UTP increased significantly. ATP levels increased approximately twofold in the first 6 h of germination. After 1 h without exogenous adenine, ATP dropped twofold or more in adenine auxotrophs but not in wild type. There was a net decrease in all adenine nucleotide pools during adenine starvation and a much greater decrease was seen in adenine auxotrophs than in wild type. The adenylate energy charge remained stable despite major changes in the adenylate pools.Accumulation of intermediates was observed in germinated conidia from purine auxotrophs blocked at various steps in the purine pathway. IMP accumulated in ungerminated and in starved conidia of the adenine-8 (ad-8) strain. Ungerminated conidia of the ad-5 strain contained a large pool of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) while the ad-1 strain had smaller amounts of AICAR, but significantly more than any other strain tested. The AICAR pools disappeared from ad-5 and ad-1 in the presence of 50 mg histidine/100 mL. Similarly the IMP pools in ad-8 decreased markedly in the presence of histidine, indicating that the contribution from the histidine biosynthetic pathway to purine nucleotide formation is significant.

Morphogenetic effects of light and guanine derivatives on the fruiting myxobacterium Stigmatella aurantiaca.

When low cell densities of the myxobacterium Stigmatella aurantiaca were starved on an inorganic salts and agar medium, cell aggregation and fruiting body formation showed a striking dependency upon the presence of light. This dependency was not manifested when sufficient amounts of guanosine or guanine nucleotides were added to the medium. Light interacted cooperatively with suboptimal concentrations of guanine compounds to promote development. None of the other purine or pyrimidine derivatives, with the exception of adenine, stimulated development. However, aggregates that formed in the presence of adenine did not mature into fruiting bodies and instead disaggregated.

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

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

Metabolism of Okazaki fragments during simian virus 40 DNA replication.

Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.

Adenine inhibition of the synthesis of photosynthetic membranes in the chloroplast of Scenedesmus obliquus grown in the dark

In the presence of adenine, heterotrophically grown Scenedesmus obliquus lost most of its photosynthetic membranes. The rudimentary membranes appeared to be intact both functionally and morphologically.

Cow red blood cells. I. Effect of purines, pyrimidines, and nucleosides in bovine red cell glycolysis

Cow red cells, under in vitro incubation conditions, exhibit a comparatively low glycolytic rate of 0.56 +/- 0.05 micromol/(ml cells.h), with a ratio of lactate formed to glucose consumed of 1.58. It has been found that this low glycolytic rate can be stimulated 50--60% above the basal level in the presence of a variety of purine and pyrimidine compounds including adenosine, inosine, adenine, hypoxanthine, xanthine, and uracil. In contrast, calf red cells, which have a much higher glycolytic rate, display no discernible response to these agents. In attempts to elucidate the mechanism by which this stimulation takes place, both glucose transport and glycolytic enzyme activities were determined in the presence of these stimulators. Glucose influx in cow red cells, measured using the glucose analog 3-O-methyl-glucose, exhibits both a low Km of 117 microM and a Vmax of 0.38 micromol/(ml cells.min), and is unaltered in the presence of adenosine. On the other hand, hexokinase, which in normal hemolysates of cow red cells has an activity of 0.49 +/- 0.03 micromol/(g Hb.min). was found to be stimulated to 0.73 micromol/(g Hb.min) in the presence of adenine. Both pyruvate kinase and phosphofructokinase were unaffected by this compound. These data suggest that certain purines and pyrimidine compounds may exert their stimulatory effect on hexokinase activity, resulting in an augmentation of cow red cell glycolysis.

Role of adenine in pathogenesis of Mycoplasma pneumoniae infections of tracheal epithelium.

Adenine sulfate and several related compounds were evaluated for their ability to retard the cytotoxic effect which normally accompanies M. pneumoniae infections of hamster tracheal explants. Adenine sulfate, at the 0.01 mM level, was found to exert a significant protective effect. Little or no ciliostasis or loss of cell viability was detectable when organ cultures were infected with 10(7) CFU of virulent M. pneumoniae in the presence of the adenine supplement. Mycoplasmas grew in broth and on plastic surfaces in the presence of adenine, and no significant diminution of growth rate or cell yield was detectable. Organisms adhered to the tracheal epithelial surface regardless of the presence or absence of adenine. When explants were incubated in the presence of 14C-(8)-adenine, rinsed, and then infected with M. pneumoniae, the adenine label could be recovered from the mycoplasmas 20 h after the infection. These data are compatible with the known nucleic acid requirement of mycoplasmas and with a model which ascribes a role for purine/pyrimidine competition and/or depletion in the infective process.