Inactivation and repression by ammonium of the nitrate reducing system in chlorella

of crude, cell-free extract in a total volume of 3.0 ml of 0.05 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. The reaction was allowed to proceed for 5 and 10 min at 24 C and then stopped by adding 0.5 ml of a 1 % solution of sulfanilamide in 3 M HCl. Then, 0.5 ml of 0.02% aqueous solution of N(1-naphthyl)ethylenediamine HCl solution was added. After 10 min, the color was read on a Zeiss PMQ-II spectrophotometer at 540 nm against a blank prepared by adding the sulfanilamide-HCl solution before the enzyme. The absorbancy was compared to a standard curve prepared with known concentrations of nitrite in the standard reaction mixture.Nitrate Uptake Assay. The uptake assay is based on the observation that nitrate in acid solution absorbs strongly at 210 nm (6). The mycelium was washed with 0.1 M potassium phosphate buffer, pH 6.0, and resuspended in the same buffer at 24 C at a density of 2 g wet weight/ 100 ml. The suspension was divided into two parts. To one part (30 ml), 0.3 ml of 10 mM KNO, was added. Periodically, 4.5-ml samples were filtered rapidly with suction. To the filtrates, 0.5 ml of concentrated HCl was added, and the absorbance was read at 210 nm on a Zeiss PMQ-II spectrophotometer against a blank prepared in the same way with no nitrate added. Samples were also taken periodically from the minus nitrate suspension to correct for leakage of UV-absorbing material from the mycelia during the 7-min uptake period. Under the above conditions, 0.1 mM NO3-has an absorbance of 0.74 in a 1-cm cuvette. Uptake rates were calculated from the rate at which the absorbance at 210 nm decreased and are reported in terms of ,umoles X g dry weight mycelium-' x min-1. One gram wet weight of mycelium is equivalent to 0.14 g dry weight.Methylamine-"C Transport Assay. The specific activity of the NH,' transport system was measured using methylamine-

Section: Resultsmentioning

confidence: 75%

Regulation of Nitrate Uptake in Penicillium chrysogenum by Ammonium Ion

Goldsmith¹,

Livoni²,

Norberg³

et al. 1973

“…This result was the opposite of what has been observed in Chlorella cultures where NH,+ added after the development of NR activity had begun prevented any further increase in the enzyme (19). Thus the synthesis of NR in rose cells was stimulated by NH4', and the mechanism responsible for this appears to be characteristic of higher plants (1,12,13), but not algae and fungi (14,17,19).…”

Section: Discussionmentioning

confidence: 70%

Ammonium Influence on the Growth and Nitrate Reductase Activity of Paul's Scarlet Rose Suspension Cultures

Mohanty¹,

Fletcher²

1976

Suspension cultures of Paul's Scarlet rose were grown in two defined media which differed only in their inorganic nitrogen content. Both possessed equal amounts of NO3' (24 mM), but differed in that NH4' (0.91 mM) was present in control medium; whereas, no NH4( was present in the test medium. A comparison of fresh weight increases over a 14-day growth period showed that NH4+ caused a 2-fold stimulation in growth and governed the pattern of development.Ammonium also caused a 2-fold increase in nitrate reductase activity but had little influence on the activity of representative enzymes from the Embden-Meyerhof pathway or citric acid cycle. Thus NH,4 enhanced the nitrate reductase activity which was correlated with increased growth.Ammonium had no influence on the in vitro activity of nitrate reductase which suggested that the stimulatory influence was due to an increased synthesis of the enzyme. The enhanced synthesis did not appear to be due to an increased availability of NO3+ since the uptake of NO3' by intact cells was not influenced by the presence of NH4+ during the period of most rapid increase in nitrate reductase activity.In previous work with suspension cultures of Paul's Scarlet rose, cells were shown to grow with only NO3+ serving as a nitrogen source, but maximum growth required a supplemental amount of either NH4+ or glutamine (22). Similar requirements were reported for suspension cultures of soybean (1, 23). Thus, in these tissue culture systems a reduced form of nitrogen such as NH4+ is required for the maximum utilization of NO3+ provided in the medium.It is well accepted that NO3+ induces the synthesis of NR1, the first enzyme in the pathway of NO3+ reduction. However, the regulatory role played by NH4+, end-product of NO3+ reduction, is less clearly understood. Ammonium has been shown to repress the induction of NR in Neurospora, Chlorella, and Lemnaceae (2). In most higher plants NH4+ either has no influence or it enhances the induction of NR by NO3-(2, 11).Based on extensive studies with genetic hybrids of corn and wheat, Hageman and colleagues (7,8)

“…It is possible that the plasmalemma and cytoplasmic forms of NR are regulated separately and that the NAD(P)H:diaphorase subunit of the plasmalemma NR (that which is assayed in the experiments described above) is synthesized and maintained in the plasmalemma even in the presence of ammonium. Possible support for this proposition is provided by the observation of Funkhouser and Ramadoss (8), who noted that ammonium-grown Chlorella cells contain proteins that cross-react with anti-NR; we believe these might be constitutively produced NR evident that the NAD(P)H:diaphorase partial activity is not repressed as rapidly or to the same extent as the complete NAD(P)H:NR activity (15).…”

mentioning

confidence: 87%

Plasmalemma Redox Activity in the Diatom Thalassiosira

Jones¹,

Morel²

1988

Plasmalemma redox activity in the diatom Thalassiosira is competitively inhibited by antiserum prepared against algal nitrate reductase (NR), and fluorescent labeling experiments reveal the binding of NR antiserum to the cell surface. Furthermore, the external electron acceptor Cu bathophenanthroline disulfonate causes immediate inhibition of intracellular primary amine production. A model is proposed in which plasmalemmabound nitrate reductase reduces extracellular electron acceptors and intracellular nitrate and also acts as a trans-plasmalemma proton pump.A considerable body of literature has accumulated in the past 10 to 15 years concerning the redox systems of higher plants and animals (6, 7). Plasmalemma redox enzymes can transfer electrons from an internal or, in some cases, an external reductant to artificial electron acceptors such as DCPIP,3 ferricyanide, and Cyt c. Often, transplasmalemma electron transport appears to be accompanied by proton extrusion from the cell (16,22,24). Based on the latter observation, it has been proposed that the plasmalemma redox enzyme(s) may act as an alternate proton pump and form the energetic basis for active transport of nutrients across the plasmalemma (7).Enzymically mediated redox activity at the plasmalemma of eukaryotic phytoplankton has recently been demonstrated in our laboratory (12,13). Redox activity in phytoplankton exhibits saturation kinetics (half-saturation constants in the range 2 to 20 ,UM for Cu [II].phen derivatives) is inhibited by mild heat treatment and membrane-impermeable thiol binding reagents, and the enzyme active site has a standard reduction potential within the range -0.1 to + 0.1 V. We noted (12) (Pharmacia). The purified antiserum was passed through a Sephadex G-25 column, and the collected preparation was used without further purification. In redox inhibition experiments with anti-NR, 25 ,uL crude antiserum or 75 ,uL purified antiserum was added to 1.5 mL culture and incubated for 15 min before commencing the assay measurements. Previous studies using this antiserum (8) showed that purified anti-NR gave only one precipitin band in Ouchterlony assays with Chlorella whole cell extracts and that the band showed identity with that obtained with purified NR. Further tests of cross-reactivity of the crude and purified anti-NR with the diaphorase moiety of cellular redox enzymes were made using a range of redox enzymes purchased from Sigma; these were lipoamide dehydrogenase (type V from Torula yeast), glutathione reductase (type III from baker's yeast), Cyt c reductase (type I from porcine heart), and malic dehydrogenase (from porcine heart mitochondria). Only the diaphorase activity of lipoamide dehydrogenase was inhibited by anti-NR, and this inhibition was strictly non-competitive (data not shown) and thus was not due to anti-NR binding at the metal reduction site of the enzyme.Cells were prepared for fluorescence microscopy by treatment with purified anti-NR as described above. After incubation, the cells were collected by cent...