Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH₃ assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH₃ is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Regulation of the assimilation of nitrate in Chlamydomonas reinhardii

In Chlamydomonas, The assimilation of ammonia proceeds through the glutamine synthetaseglutamate synthase pathway. The primary target in the regula     

Effect of NaCl on the activities of glutamate synthase from a halophyte Suaeda maritima and from a glycophyte Phaseolus vulgaris

The action of NaCl on the activity of root and leaf glutamate synthase is compared in a halophyte, Suaeda maritima var. macrocarpa and in a glycophyte Phaseolus vulgaris. The addition of salt in the nutrient medium lowers the activity of glutamate synthase from Phaseolus without affecting that of Suaeda. This result, attributed to the fact that glutamate synthase is stimulated while glutamate dehydrogenase is partly inhibited in the halophyte grown in presence of high NaCl concentrations, suggests that the GS-GOGAT pathway is the primary route for ammonia assimilation. This pathway is especially active in the leaves. In vitro, NaCl (25–300mM) reduces the activity of glutamate synthase in Phaseolus as well as in Suaeda. Comparison with results obtained in situ suggests that there are differences in intracellular compartmentalization between the two types of plant.     

A kinetic investigation of the aps-kinase from Chlamydomonas reinhardii CW 15

The reaction kinetics of APS-kinase from Chlamydomonas reinhardii showed that the enzyme formed PAPS from APS upon the addition of ATP. Evidence for a ³⁵S-labelled protein intermediate between APS and PAPS has been obtained. The APS-kinase activity could only be measured in the presence of low concentrations of APS (20 ± 10 μM) and of ATP (0.2 ± 0.05 mM) due to substrate inhibition. The inhibition was partially overcome by low concentrations of 3′,5′-PAP (10,μM). The rates of PAPS formation obtained with cell extracts from the alga varied from 2 to 6 nM PAPS/mg protein/min (33–100 × 10^?12 kat/mg).     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Glutamine synthetase and glutamate synthase from Sclerotinia sclerotiorum

Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg²⁺-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of ¹⁵N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.     

Isolation and characterisation of metribuzin-resistant Chlamydomonas reinhardii cells

Chlamydomonas reinhardii cells were treated with 5-fluorodeoxyuridine and ethylmethanesulfonate to induce mutagenesis. The mutant cells were analyzed for resistance against metribuzin (4-amino-6-(t-butyl)-3-methylthio-1,2,4-triazine-5-one). Clones with normal growth were isolated and the mutant cells further characterized. The photosynthetic rates of the mutant cells were about 20% lower than those of wild-type cells. The mutant cells were not only resistant against metribuzin (pI₅₀ lowered from 6.65 to 3.41) but also against bromacil, atrazine, phenisopham and tolerant against 3-(3,4-dichlorophenyl)-1,1-dimethylurea. However, the mutant was more susceptible to phenolic electron-transport inhibitors like bromonitrothymol, ioxynil and i-dinoseb. 2,4-Dinitrophenyl-2′-iodo-3′-methyl-4′-nitro-6′-isopropyl phenyl ether inhibited the wild-type thylakoids more than the mutant. The analysis of the electron transport with artificial electron donors and acceptors showed that only Photosystem II was affected by the mutation and not Photosystem I. Binding experiments with isolated thylakoids of resistant and susceptible cells using [¹⁴C]metribuzin and [³H]-i-dinoseb revealed that metribuzin did not bind specifically to the thylakoids of the mutant cells, but that i-dinoseb did bind to the thylakoids of the mutant, and even better than to the thylakoids of the wild-type cells. Fluorescence studies confirmed these results.     

Organization of chlorophyll-protein complexes of Photosystem I in Chlamydomonas reinhardii

The polypeptide pattern, chlorophyll-protein complexes, fluorescence emission spectra and light intensity required for saturation of electron flow via Photosystem (PS) II and PS I in a pale-green photoautotrophic mutant, y-lp, were compared to those of the parent strain, Chlamydomonas reinhardii y-1 cells. The mutant exhibits a 686 nm fluorescence yield at 25°C and 77 K 2–6-fold higher than that of the parent strain cells, and is deficient in thylakoid polypeptides 14, 17.2, 18 and 22 according to the nomenclature of Chua (Chua, N.-H. (1980) Methods Enzymol. 60C, 434–446). All chlorophyll-protein complexes ascribed to PS II and the CP I complex were present in both type of cells. However, a chlorophyll-protein complex CP Ia containing — in the parent strain — the 66–68 kDa polypeptides of CP I and the four above-mentioned polypeptides, was absent in the mutant. It was previously reported that a chlorophyll-protein complex, CP O, obtained from C. reinhardii contains five polypeptides, namely, 14, 15, 17.2, 18 and 22 (Wollman, F.A. and Bennoun, P. (1982) Biochim. Biophys. Acta 680, 352–360). A CP O-like complex was present also in the mutant y-lp cells but it contains only one polypeptide, 15. Energy transfer from PS II to PS I was not impaired in the mutant, although a 4-fold higher light intensity was required for the saturation of PS I electron flow in the y-lp cells as compared with the parent strain. No difference was found in the light saturation curves for PS II activity between the mutant and parent strain cells. Based on these and additional data (Gershoni, J.M., Shochat, S., Malkin, S. and Ohad, I. (1982) Plant Physiol. 70, 637–644), it is concluded that the chlorophyll-protein complexes of PS I in Chlamydomonas comprise a reaction center-core antenna complex containing the 66–68 kDa polypeptides (CP I), a connecting antenna consisting of four polypeptides (14, 17.2, 18 and 22), and a light-harvesting antenna containing one polypeptide, 15. These appear to be organized as a complex, CP Ia. The interconnecting antenna is deficient in the y-lp mutant and thus the CP Ia complex is unstable and energy is not transferred from CP O to CP I. The effective cross-section of PS I antenna is thus reduced and a high fluorescence is emitted at 686 nm.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardii

After transition to anaerobiosis an oscillatory fermentation of plastidic starch is found in the green alga Chlamydomonas reinhardii. Oscillations are investigated by analysis of fluctuations in starch, fermentation products and metabolites. Period length, amplitudes and phase relationships are determined by a biometrical processing of the biochemical signals, which allows statistical treatment even under limitation of data points. The mean period length is 59 min and oscillations are detectable within 3 h after the onset of anaerobiosis. Metabolized starch carbon is found by 90% in the accumulating fermentation products as formate, acetate and ethanol. From the intermediates only pyruvate increases during fermentative oscillations. A phase shift of 180° between ATP and both, AMP and ADP, indicates activity of adenylate kinase and values for energy charge fluctuates between 0.70 and 0.89. Phase relations of glucose-6-P to both AMP and pyruvate are 190° and 56°, respectively, whereas that of pyruvate to ATP is 134°. Glucose-6-P concentration does not follow the rate of starch degradation. Phase relations of fermentation products to pyruvate indicates enzyme activities of formate pathway. High period length as observed during algal fermentation is thought to be generated by a complex rate control in a different compartmentalized glycolytic pathway.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Dimeric H-ATP synthase in the chloroplast of Chlamydomonas reinhardtii

H⁺-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane.By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF₁ part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.     

Purification and properties of glutamate synthase from Streptomyces lincolnensis

Ｉｎｍｏｓｔｐｒｏｃａｒｙｏｔｅｓ,ｔｈｅｅｎｚｙｍｅｇｌｕｔａｍａｔｅｓｙｎｔｈａｓｅ[Ｌｇｌｕｔａｍａｔｅ:ＮＡＤ(Ｐ)+ｏｘｉｄｏｒｅｄｕｃｔａｓｅ(ｔｒａｎｓａｍｉｎａｔｉｎｇ)(ＥＣ1.4.1.13ｏｒ14)](ＧＯＧＡＴ),ｔｏｇｅｔｈｅｒｗｉｔｈｇｌｕｔａｍｉｎｅｓｙｎｔｈｅｔａｓｅ[Ｌｇｌｕｔａｍａｔｅ:ａｍｍｏｎｉａｌｉｇａｓｅ(ＡＤＰｆｏｒｍｉｎｇ)(ＥＣ6.3.1.2)](ＧＳ),ｈａｓｂｅｅｎｃｏｎｓｉｄｅｒｅｄｔｏｂｅａｎａｌｔｅｒｎａｔｉｖｅｔｏｇｌｕｔａｍａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅｉｎａｍｍｏｎｉａ…     

Purification and properties of the allophanate hydrolase from Chlamydomonas reinhardii

Allophanate hydrolase was purified to homogeneity from extracts of Chlamydomonas reinhardii grown phototrophically using urea as sole source of nitrogen. The following sequence of steps comprised the purification procedure: (1) protamine sulfate precipitation; (2) ammonium sulfate fractionation; (3) poly(ethylene glycol) fractionation; (4) batch-wise DEAE-cellulose adsorption; (5) Sepharose 6-B gel filtration; (6) hydroxyapatite chromatography. This procedure yielded an allophanate hydrolase preparation which was homogenous as judged by polyacrylamide gel electrophoresis. The molecular weight, as determined by gradient polyacrylamide electrophoresis and gel filtration, was 110 000 and 100 000, respectively. The pH optimum of this enzyme was approximately 9.0, while the K_m for allophanate was 0.55 mM. Allophanate hydrolase was sensitive to N-ethylmaleimide but was protected from this inhibition by allophanate. Malonic acid, oxaloacetic acid, and acetoacetic acid were inhibitory to allophanate hydrolysis.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.