STUDIES ON THE METABOLISM OF UREA AND OTHER NITROGENOUS COMPOUNDS IN CHLORELLA ELLIPSOIDEA: III ASSIMILATION OF UREA

Assimilation of urea by Chlorella ellipsoidea was investigatedby using ¹⁴C-urea. It was revealed that urea carbon metabolizedby the N-starved cell was almost quantitatively recovered incarbon dioxide under dark-aerobic conditions. The rate of ureaconsumption was considerably influenced by the N-content ofalgal cells, practically no metabolism of urea being observedwith the cells containing more than 7 per cent N on a dry weightbasis. Similar results were also obtained when urea was replacedwith ammonia as nitrogen source. Based on the results obtained,a tentative scheme for the assimilation process of urea in Chlorellawas proposed. (Received February 7, 1960; )     

Fractionation of Nitrogen Isotopes during the Uptake and Assimilation of Ammonia by Plants

Two varieties (Nihonbare and Koshihikari) of rice plants (Oryzasativa L.) were grown hydro-ponically with two levels (20 and100 mg N liter ^–1) of ammonia. Variations in levels ofnatural abundance of ¹⁵N (¹⁵N) were analyzed in the ammoniaand organic nitrogen of shoots and roots, as well as in theammonia in the culture solution. There was substantial fractionationof nitrogen isotopes during the uptake of ammonia. When plantsabsorbed a large proportion of ammonia from a solution witha low concentration, less negative ¹⁵N values in plants andhigh positive ¹⁵N values in the ammonia remaining in solutionwere observed. The reverse was found when a smaller fractionof ammonia was absorbed from a solution with a higher concentrationof ammonia. The ^l5N values of ammonia in shoots and roots werehigher than in the respective constituent organic nitrogen,suggesting the fractionation of nitrogen isotopes during theassimilation of ammonia. Wild-type and mutant cells of the cyanobacterium(blue-green alga) Synechococcus PCC 7942 were grown in nitrate-or ammonia-containing medium as the source of nitrogen. Fractionationof nitrogen isotopes during the uptake of nitrate was limited,whereas that during the uptake of ammonia was considerable. ¹ In this report, the term ammonia refers indiscriminately toboth NH₃ or NH₄⁺. (Received June 13, 1991; Accepted September 12, 1991)     

Metabolism of Urea by Chlorella vulgaris

Urea metabolism was studied with nitrogen-starved cells of Chlorella vulgaris Beijerinck var. viridis (Chodat), a green alga which apparently lacks urease. Incorporation of radioactivity from urea-(14)C into the alcohol-soluble fraction was virtually eliminated in cell suspensions flushed with 10% CO(2) in air. This same result was obtained when expected acceptors of urea carbon were replenished by adding ornithine and glucose with the urea. Several carbamyl compounds, which might be early products of urea metabolism and a source of the (14)CO(2), were not appreciably labeled. If cells were treated with cyanide at a concentration which inhibited ammonia uptake completely and urea uptake only slightly, more than half of the urea nitrogen taken up was found in the medium as ammonia. Cells under nitrogen gas in the dark were unable to take up urea or ammonia, but the normal rate of uptake was resumed in light. Since 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not selectively inhibit this uptake, an active respiration supported by light-dependent oxygen evolution in these cells was ruled out. A tentative scheme for urea metabolism is proposed to consist of an initial energy-dependent splitting of urea into carbon dioxide and ammonia. This reaction in Chlorella is thought to differ from a typical urease-catalyzed reaction by the apparent requirement of a high energy compound, possibly adenosine triphosphate.     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

Differential Cellular Isotope Effects of Deuterium on Photosynthetic Metabolism of Carbon in Chlorella ellipsoidea

Isotope effects of deuterium on photosynthetic metabolism ofcarbon in Chlorella ellipsoidea were investigated. Photosyntheticfixation of ¹⁴C in D₂O was about a half of that in H₂O. Eachstep in the photosynthetic metabolism of carbon was affecteddifferently by D₂O in the medium and constitutive D. (Received June 15, 1989; Accepted October 23, 1989)     

Growth of Phaseolus vulgaris on Various Nitrogen Sources: The Importance of Urease

Rhizobium-inoculatcd plants of Phaseolus vulgaris L. were grownwith different N-sources (nitrate, ammonium, urea) and differentconcentrations of urea. The distribution of growth between plantparts varied with N-sources. Nitrate and ammonium were moreinhibitory to nodulation than urea, which at 40 mol m^–3N had no effect. Urease activity varied in amount and locationover a range of urea concentrations. At higher concentrations,more urea was transported to and increased urease activity wasfound in the shoot Lower levels of activity in plants relianton N₂-fixation were consistent with a ureide-degradation pathwaynot involving urea. Moderate doses of urea could be assimilatedconcomitantly with N₂-fixation. At higher levels of appliedurea, nodulation and ureide transport to the shoots were reduced,although increased growth could not be maintained at concentrationsof applied urea greater than 6.0 mol m^–3 urea N. Key words: Phaseolus vulgaris, growth, nitrogen source, urease     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach     

Phytoplankton uptake of ammonium and urea during growth on oxidized forms of nitrogen

Uptake capabilities for ammonium (NH₄⁺) and urea by diatoms(Thalassiosira pseudonana and Skeletonema costatum) growingon oxidized forms of nitrogen were studied in short-term uptakeexperiments. Even when nutrient-saturated, an enhanced uptakecapability not coupled with the growth rate was present forNH₄⁺ and urea. No such enhanced uptake ability was seen forNO₂^– or NO₃^– under either nutrient-saturated ornutrient-depleted conditions. The presence of NH₄⁺ decreasedthe enhanced ability to take up urea, but the urea uptake ratein 5 min incubations remained greater than the growth rate evenwhen NH₄⁺ was present.     

Taurine Transport in N-deprived Chlorella fusca

The development of taurine uptake into the unicellular greenalga Chlorella fusca 211-8b was characterized as a specificresponse to either nitrate or sulphate limitation. Taurine transportunder nitrogen starvation was stimulated by low pH and showeda biphasic kinetics with K_m-values of 1.1 x 10^–3 mol dm^–3and 1.0 x 10^–2 mol dm^–3. Uptake was substantiallyinhibited by all - and ß-amino acids tested, whereassulphonate analogues failed to diminish taurine accumulation.Thus, uptake seemed to be mediated by a ‘general aminoacid permease’, unable to discriminate between carboxyland sulphonyl groups. However, Chlorella fusca could not catabolizethis unusual ß-amino acid and mobilize the amino-boundnitrogen for growth. Only a small group of -amino acids supportedthe growth of Chlorella fusca as an efficient nitrogen source. Key words: Taurine uptake, nitrogen starvation, amino acid uptake, Chlorella fusca.     

Uptake of 15N by Kiwifruit Vines from Applications of Nitrogen Fertilizer Prior to Budbreak

Mature field-grown kiwifruit vines (Actinidia deliciosa var.deliciosa cv. Hayward) were fertilized with ¹⁵N-labelled fertilizer(ammonium sulphate, 10 atom % ¹⁵N, 50 kgN ha^-1) to investigatethe timing of uptake of fertilizer nitrogen (N) and its availabilityfor new season's growth. Treatments were applied on four occasions,representing 2, 6, 10 and 14 weeks prior to budbreak. Samplesof root, stem, cordon, fruiting cane, vacuum-extracted xylemsap, and new season's growth were collected at fortnightly intervalfrom early winter until 2 months after budbreak. Two weeks after application of each treatment, ¹⁵N equivalentto an average of 7% of the applied label was recovered in rootmaterial. Although label was taken up by roots, there was nomovement of ¹⁵N within the plant until about 1 month prior tobudbreak when it was measured in the stem and cordon. Fertilizernitrogen was not detected at the distal end of fruiting canes,and in new season's growth until 3-4 weeks after budbreak. Beforebudbreak, all nitrogen in the xylem sap was in amino forms.Nitrate appeared 4 weeks after budbreak, and although more enrichedwith ¹⁵N than the amino nitrogen, accounted for only 19% ofthe label. Eight weeks after budbreak, nitrate nitrogen accountedfor 57% of the label. There were no major treatment effects of ¹⁵N on vines in eitherspring or at harvest, although enrichments in fruit and leavesfrom the earliest treatment tended to be less at the end ofthe season than those from the later applications.Copyright1993, 1999 Academic Press Actinidia deliciosa, kiwifruit, nitrogen, ¹⁵N, nutrient uptake     

Carbon and Nitrogen Assimilation by Vicia faba L. at Low Temperature: the Importance of Concentration and Form of Applied-N

Low temperature (6 C) growth was examined in two cultivarsof Vicia faba L. supplied with 4 and 20 mol m^–3 N as nitrateor urea. Both cultivars showed similar growth responses to increasedapplied-N concentration regardless of N-form. Total leaf areaincreased, as did root, stem and leaf dry weight, total carboncontent and total nitrogen content. In contrast to findingsat higher growth temperatures, 20 mol m^–3 urea-N gavesubstantially greater growth (all parameters measured) than20 mol m^–3 nitrate-N. The increased carbon content per plant associated with increasedapplied nitrate or urea concentration, or with urea in comparisonto nitrate, was due to a greater leaf area per plant for CO₂uptake and not an increased CO₂, uptake per unit area, carbon,chlorophyll or dry weight, all of which either remained constantor decreased. Nitrate reductase activity was substantial inplants given nitrate but negligible in plants given urea. Neitherfree nitrate nor free urea contributed greatly to nitrogen levelsin plant tissues. It is concluded that there is no evidence for a restrictionin nitrate reduction at 6 C, and it is likely that urea givesgreater growth than nitrate because of greater rates of uptake. Vicia faba, broad bean, low temperature growth, carbon assimilation, nitrogen assimilation     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Synthetic rates of chloroplast and cytoplasmic ribosomal ribonucleic acids during the cell cycle of Chlorella

By feeding radioisotopic precursors of RNA ([5-³H]uracil and[5-³H]uridine) to cells of Chlorella ellipsoidea at variousstages in the cell cycle effected by autotrophic synchronousculture, we examined synthetic rates of the chloroplast andthe cytoplasmic ribosomal ribonucleic acids (chl-rRNA and cyt-rRNA,respectively). The net incorporation of the precursors intochl-rRNA was higher than that into cyt-rRNA in the early stagesof the cell cycle, and vice versa in the late stages. The specificactivity of chl-rRNA was extremely high, and this phenomenonwas likely to be intrinsic to small cells at the start of thecell cycle under autotrophic conditions, namely, cell-cyclestagespecific. We conclude that algal cells grown autotrophicallysynthesize chl-rRNA at a distinctly higher rate than cyt-rRNAin the early stages of the cell cycle. (Received July 21, 1978; )     

The Transport and Metabolism of Urea in Chara australis: I. PASSIVE DIFFUSION, SPECIFIC TRANSPORT AND METABOLISM OF UREA, THIOUREA AND METHYLUREA

Most of the urea entering Chara australis cells is rapidly metabolizedto produce CO₂, which diffuses out of the cells into the surroundingmedium. A simple and convenient apparatus to measure both the¹⁴C-urea retained by cells and the ¹⁴CO₂ released into the mediumwas developed and used in a study of urea transport in Chara.The permeability coefficient for urea in the Chara plasmalemmawas estimated from the slope of an uptake versus concentrationfunction as 85 nm s^-1. Computer modelling of urea uptake andmetabolism suggests that this could be a 20% underestimate ofthe true value.The corresponding permeability coefficients forthiourea and N-methyl-urea were estimated in the same way as34 and 35 nm s^-1, respectively. These permeabilities are muchgreater than expected on the basis of either/water partitioncoefficients for the solutes and are consistent with the diffusionof urea and its similarly-sized analogues through aqueous poresin the plasmalemma.At external concentrations of urea less than20 mmol m^-3, the bulk of the uptake is effected by a specifictransport mechanism with an apparent K_m for urea of less than1.0 mmol m^-3. This transport system operates most rapidly withexternal pH in the range 6.5–7.5 and is influenced bythe nitrogen status of the cell.Evidence is produced here suggestingthat the specific transport of urea may be an active process. Key words: Chara, urea uptake, metabolism, diffusion, specific transport     

Accumulation of cadmium by Dunaliella tertiolecta Batcher

Cultures of Dunaliella tertiolecta were exposed to five concentrationsof cadmium in solution (0.1, 0.5, 1, 5 and 10ppm(mg 1^-1)). Theaccumulation of cadmium by the algae was found to have two phases,an initial rapid uptake followed by a stabilisation of the cellularcadmium levels. D. tertiolecta concentrated cadmium from solution(cone, factor approx. 1350) at exposures up to 1 ppm Cd butexposure to the higher concentrations caused no further increasein the accumulated cadmium concentration of the algae whichreached a nmriimim at about 1.5 µg Cd mg¹ Dunaliella.     

Effcts of Blue Light and Ammonia on Nitrogen Metabolism in a Colorless Mutant of Chlorella

Illumination of a colorless mutant of Chlorella vulgaris 1lh(M125) with blue light enhanced both the uptake of nitrate andthe release of ammonia. These effects were not observed underillumination with red light. The release of ammonia was alsoenhanced by the addition of methionine sulphoximine (MSX), aninhibitor of glutamine synthetase (GS). Addition of MSX to culturesin the dark increased the rate of breakdown of starch. Algal cells grown in nitrate-containing medium did not showthe aminating activity of glutamate dehydrogenase (GDH). Additionof large (millimolar) amounts of ammonia in the dark resultedin the induction of NADPH-GDH activity and, in addition, a decreasein GS activity. From these results it appears that GS catalyzesthe primary step in the assimilation of ammonia in algal cellsgrown in nitrate-containing medium. Two isoforms (GS1 and GS2)of GS have been separated by ion exchange chromatography. Theactivities of both isoforms were decreased upon the additionof ammonia. Illumination of the alga with blue light at intensities up to10,000 mW m^–2 enhanced the measurable activity of GS invitro, while higher intensities were ineffective. In red lightno such effect was observed. The effects of blue light and ammonia on nitrogen metabolismin algal cells are discussed. (Received November 25, 1988; Accepted March 6, 1989)     

Dinitrocresol, Cyanide, and the Pasteur Effect in Yeast

At pH 5·0 the respiration of yeast is stimulated by lowconcentrations of 3:5-dinitro-o-cresol, reaching a peak levelof 170 per cent, at 10⁵ M. Concentrations above this inhibitoxygen uptake and cause aerobic fermentation to appear, whichin turn reaches a peak value and is then inhibited. The rateof carbohydrate breakdown, or glycolysis, calculated from therates of respiration and aerobic fermentation, increases steadilyup to 3 x 10^–5 M., at which concentration it is 5 timesfaster than the control: higher concentrations depress the rateof glycolysis. The rate of fermentation under nitrogen is abouttwice that of respiration, and it is inhibited over the sameconcentration range as aerobic fermentation. It was found earlier that oxidative assimilation of glucoseby yeast is progressively inhibited by increasing concentrationsof dinitrocresol, and it is now shown that this parallels theincrease in the rate of aerobic glycolysis. It is argued thatdinitrocresol is here acting as an uncoupling agent and thatboth oxidative assimilation and the rate of glycolysis are controlledby the level of energy-rich phosphate. With cyanide there is no stimulation of oxygen uptake, aerobicfermentation only appears when respiration becomes inhibited,and after an initial slight decrease the rate of glycolysisrises to 575 per cent. of the control value at 5 x 10^–4M. It is suggested that the rate of glycolysis only increaseswhen respiration has been inhibited sufficiently to reduce therate of formation of energy-rich phosphate.     

STEROLS OF CHLORELLA. I. THE NATURALLY OCCURRING STEROLS OF CHLORELLA VULGARIS, C. ELLIPSOIDEA, AND C. SACCHAROPHILA

The characteristics of the sterols naturally occurring in threespecies of Chlorella were examined. The algae were grown heterotrophicallyon glucose. Sterols were extracted and isolated from the lipidfraction and were characterized by means of chemical and physicaltests. Chlorella vulgaris contained three sterols. Only the principalone, chondrillasterol, was identified. Chondrillasterol hasbeen isolated previously from the genus Scenedesmus. Chlorella ellipsoidea and Chlorella saccharophila were foundto contain sterols with ß-oriented alkyl groups atC-24 in contrast to the -oriented groups commonly found in higherplants. Poriferasterol was identified as the principal sterolof both algae. Clionasterol and 22-dihydrobrassicasterol wereidentified as the two secondary sterols present. None of thesesterols have previously been reported to occur in plants. Theisolation of 22-dihydrobrassicasterol has not been previouslyreported from any natural source. ¹Scientific Article A1153, Contribution No. 3623 of the Universityof Maryland Agricultural Experiment Station. ²This work has been supported in part by a grant from the NationalAeronautics and Space Administration.     

Properties of Chlorella cells grown under various photo-heterotrophic conditions

1. Chlorella ellipsoidea was cultured under photo-heterotrophicconditions. Cells showed most favorable growth when 0.2% glucosewas added at the start to the inorganic nutrient medium. 2. Treatment with a mixed solvent, methanol/hexane: 4/3, gavehighly decolorized cells. Molasses alone and pyruvate in combinationwith glucose or with molasses and glucose were effective inobtaining a high yield of chlorophyll and carotenoid pigments. 3. The nitrogen content was lower in bleached cells than innormal cells. Protein yield obtained by the urea soaking methodwas higher with bleached cells than with normal cells. 4. Electron microscopic studies revealed that the outer partof normal cells consists of two layers, an electron-dense innerlayer and an electron-lucent outer layer, whereas, that of bleachedcells consists of only an electron-dense layer. (Received August 28, 1969; )     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)