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Interconversion Uptakeof Nucleotides, Nucleosides, and ... · PDF file Interconversions of nucleotides, nucleosides, and bases. Weincubated the cells with labeled purine nucleotides,

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  • JOURNAL OF BACTERIOLOGY, May 1982, p. 471 482 Vol. 150, No. 2 0021-9193/82/050471-12$02.00/0

    Interconversion and Uptake of Nucleotides, Nucleosides, and Purine Bases by the Marine Bacterium MB22

    MARTINE FORET AND JAN AHLERS* Institut far Biochemie und Molekularbiologie, Fachbereich Biologie, Freie Universitat Berlin, I Berlin 33,

    Germany Received 17 July 1981/Accepted 12 December 1981

    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 noncompeti- tively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 ,uM and another one for inosine and adenosine with a Km of 1.4 ,uM. 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.

    We recently investigated the kinetic proper- ties of a membrane-bound nucleotidase from different strains of marine bacteria (1, 17). To obtain information about the ability of marine bacteria to use the metabolites formed by the action of this enzyme, we investigated the me- tabolism and transport of nucleosides and purine bases.

    Until now such studies have been performed mainly with Escherichia coli (2, 5, 7, 9, 11, 12, 18) using either the wild types (2, 11, 18), special mutants (3, 4, 7-9), or membrane vesicles (5). The results reported have not yet presented unequivocal proof concerning the mechanism(s) of nucleoside or base transport. Some authors favor a group translocation mechanism (5, 18), whereas others implicate an active transport mechanism (3, 4, 8, 9, 11). Munch-Petersen and Pihl (8) propose a proton motive force as the main energizer of nucleoside transport. Burton (3) presents evidence for porter systems for adenine, hypoxanthine, and uracil dependent on a proton motive force and facilitated by intracel- lular metabolism of the free bases. Most authors propose the existence of more than one trans- port system (4, 7, 8, 11). The interpretation of such transport data is often complicated by several interconversions of substrates and re- sulting products by enzymes located in the cyto- plasm, at the inner or outer surface of the plasma membrane, or in the periplasmic space (2, 3, 5, 18). The data obtained with E. coli cannot be

    readily transfered to other microorganisms. For instance, Pickard et al. (13, 14) described two nucleoside deaminases in Micrococcus sodonen- sis. With E. coli, nucleoside metabolism occurs mainly by the action of phosphorylases (5). Pickard also found that the entry of nucleosides into M. sodonensis was neither saturable, nor was there any competitive inhibition between the nucleosides studied, suggesting the entry by free diffusion. These results suggest the necessity for a gen-

    eral approach to examine the uptake and inter- conversions of nucleotides, nucleosides, and purine bases by marine bacteria, a group of organisms exposed to a completely different environment than E. coli. We have already proven that at least one enzyme involved in the metabolism of these substrates, the nucleotid- ase, has quite different properties than the re- spective enzyme of E. coli (1, 17). This enzyme is not located in the periplasmic space, but rather is firmly attached to the membrane.

    MATERIALS AND METHODS Organism. The marine bacterium MB22 was a kind

    gift from A. J. De Siervo, University of Maine at Orono. Some of the microbiological characteristics of MB22

    were published recently (17). Chemicals. 8-14C-labeled adenine, guanine, hypo-

    xanthine, adenosine, guanosine, AMP, and GMP were obtained from the "Commissariat a l'energie atomi- que" (Gif-sur-Yvette, France), and 'IC-labeled ino-


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    sine was from the Radiochemical Centre, Amersham, England. The unlabeled purine and pyrimidine bases were

    purchased from Pharma-Waldhof, Dusseldorf, Germa- ny; the purine nucleosides were from Boehringer, Mannheim, Germany. Cytidine and uridine were from Serva, Heidelberg, Germany. Yeast extract was from Difco, Detroit, Mich. All other reagents were obtained from Merck, Darmstadt, Germany. Membrane filters (pore size 0.45 ,um) were supplied

    by Schleicher & Schull, Dassel, Germany, and Poly- gram Cel 400 UV254 thin-layer chromatography sheets were by Macherey, Nagel and Co., Duren, Germany.

    Materials used for autoradiography were Kodirex X-ray film (9 by 12 cm), Kodak D 19 developer, and Kodak AL 4 X-ray fixer.

    Culture conditions. The cells were cultivated at 30°C in artificial seawater (ASW), pH 7.4 (0.4 M sodium chloride-0.05 M magnesium sulfate-0.01 M potassium chloride). To the ASW, 2% peptone from casein, 0.005% yeast extract, 0.05% ammonium sulfate, and 0.05% Tris were added, and the mixture was shaken on a rotary shaker. Cells were usually harvested in the stationary phase.

    Preparation of membranes. The cells were treated with lysozyme. After lysis of the spheroplasts by osmotic shock, the plasma membranes were isolated by centrifugation.

    Metabolic conversion studies. Stationary cells (56 mg [wet weight]) were washed twice with ASW and then suspended in 6.4 ml of ASW. The cell suspension was incubated for varying periods of time with 1.6 ml of 150 FM (25 Ci/mol) purine, nucleoside, or nucleotide solution, and 1-ml volumes were centrifuged to sepa- rate the medium from the cells. A 10-,ll portion of the supernatant was applied to polygram Cel 400 UV254 thin-layer chromatography sheets against a reference mixture and developed in 1 M ammonium acetate. The labeled spots were assayed for radioactivity in a Beckman liquid scintillation counter. A pool extraction of the cells was performed by the

    extraction procedure of Nazar et al. (10) and analyzed autoradiographically as described by Seipel and Rei- chert (16).

    Kinetic studies. A 0.5-mg amount of cells (wet weight) generally obtained from the stationary phase was incubated at 25°C for 30 s with 0.03 to 0.2 ,M [8- "C]hypoxanthine, -guanine, -adenine or with 0.5 to 2 FM [8-14C]inosine, -guanosine, or -adenosine in 1 ml of ASW and then collected on membrane filter. The filters were assayed for radioactivity in a Beckman scintillation counter. The scintillation cocktail was 100 g of naphthalene plus 5 g 2,5-diphenyloxazole per liter dioxan. For the study of the different inhibitors, 1 to 10 FM

    unlabeled purine and pyrimidine nucleosides or 0.1 to 0.5 ILM purine and pyrimidine bases were added to the assay. Measurement of accumulation. A 0.5-mg amount of

    cells (wet weight) was incubated at 25°C with 15 ,uM 8-14C-labeled nucleoside or with 2 ,M 8-14C-labeled purine base as described above. Energy poisons (1 mM dinitrophenol or 50 mM sodium azide) were added to the cell suspension 10 min before the labeled compound or at the time indicated in the figure. At different times portions were taken and assayed for radioactivity.

    Analysis of kinetic data and estimation of error. The kinetic constants were obtained from double recipro- cal plots and by least-squares fit to the hyperbolic Michaelis-Menten equation (J. Ahlers, A. Arnold, F. R. V. Dohren, and H. W. Peter, Enzymkinetik, 2nd ed., in press) assuming a constant relative error. All values used for kinetic characterization were averages of four or more experiments performed in duplicate.

    RESULTS Interconversions of nucleotides, nucleosides,

    and bases. We incubated the cells with labeled purine nucleotides, nucleosides, or bases. At different incubation times, medium and cells were separated and analyzed by chromatogra- phy and autoradiography.

    Analysis of the external medium. Figure 1 shows the result of our analysis of the external medium, demonstrating the interconversion of GMP into different metabolites. It can be seen that GMP immediately disappeared from the medium due to its conversion into guanosine. Guanosine, however, was then converted to guanine and finally to xanthine. The results with other substrates are compiled in Table 1. It can be seen that all nucleotides were immediately converted to nucleosides. Interestingly, when AMP was the substrate, an additional deami- nation took place which resulted in the rapid appearance of inosine rather than adenosine. In accordance with Fig. 1, all nucleosides were


    10 x



    5 10 15 20 25 Time (min)

    FIG. 1. Metabolites present in the external medium after incubation of marine bacteria with labeled GMP at 25°C. Symbols: 0, GMP; 0, guanosine; O, guanine; U, xanthine. The different metabolites were separated chromatographically as indicated in the text.


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    TABLE 1. Composition of the external medium'

    Substrate Incubation Presence of metabolitesbtime (min) Guanosine Inosine Adenine Guanine Hypoxanthine Xanthine AMP 1 +++ +++ 0

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