Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions

Cobalamin is essential for growth of Euglena gracilis and photosynthesis. Methylcobalamin in Euglena chloroplasts (Y Isegawa, Y Nakano, S Kitaoka, 1984 Plant Physiol 76: 814-818) functions as a coenzyme of methionine synthetase. The requirement of cobalamin for photosynthesis appeared remarkably high in Euglena grown under the dark-precultured condition. The required amount of cobalamin for normal photosynthetic activity was 7.4 × 10⁻¹¹ molar, while 7.4 × 10⁻¹⁰ molar cobalamin was required for normal growth. The lowered photosynthetic activity in cobalamin-limited cells was restored 20 hours after feeding cyanocobalamin or methionine to cobalamin-limited cells. Lowering of photosynthetic activity was due to loss of photosystem I activity. This photosynthetic activity was recovered after supplementation by methionine or cobalamin. The results suggest that methionine serves for the stabilization of photosystem I. This paper is the first report of the physiological function of cobalamin in chloroplasts of photosynthetic eukaryotes.     

Isolation and some properties of soluble and membrane-bound cobalamin binding proteins of Euglena mitochondria

Cobalamin binding activity occurred in the soluble fraction (69%) and the membrane fraction (31%) of Euglena mitochondria. The mitochondrial soluble cobalamin binding protein was purified about 580-fold in a yield of 34%; the membrane-bound cobalamin binding protein was solubilized with 2 M urea and partially purified. Both purified mitochondrial cobalamin binding proteins showed low pH dependency for activity. The pH optima of the soluble and membrane-bound cobalamin binding proteins were in the vicinity of 7.0 and 6.0–8.0, respectively. The K _s values of the soluble and membrane-bound cobalamin binding proteins for cyanocobalamin were 0.3 and 0.9 nM, respectively. Neither mitochondrial cobalamin binding proteins required metal ions for activity, but the activity of the soluble and membrane-bound cobalamin binding proteins was inhibited by 1 mM Mn²⁺, 48% and 89%, respectively. Molecular weight of the soluble cobalamin binding protein was calculated to be 93,000. The physiological roles of both mitochondrial cobalamin binding proteins were discussed on the basis of their properties and location in Euglena mitochondria.Abbreviations Cbl cobalamin - Ado-Cbl 5-deoxyadenosylcobalamin - CN-Cbl cyanocobalamin - Me-Cbl methylcobalamin - OH-Cbl hydroxocobalamin - 2-AMP-Cbl 2-amino-2-methylpropanolylcobalamin     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

A Human Vitamin B12 Trafficking Protein Uses Glutathione Transferase Activity for Processing Alkylcobalamins

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5′-deoxyadenosylcobalamin by MMACHC are 11.7 ± 0.2 and 0.174 ± 0.006 h⁻¹ at 20 °C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B₁₂-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.     

Separation and Ultrastructure of Proplastids from Dark-grown Euglena Cells

A procedure for the separation of proplastids free of mitochondria from dark-grown Euglena cells has been developed. A fraction enriched in proplastids was used for freeze-etching study of proplastid structure. The prolamellar body in freeze-etched replicas appeared sponge-like, with thylakoids, often vesicular, emerging from it. The prolamellar body and the thylakoids were covered by particles of about 100Å in diameter. No larger particles, typical of light-grown chloroplasts, were observed.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Further studies on the bicarbonate stimulation of photophosphorylation in isolated chloroplasts

The bicarbonate effect in stimulating the rate of photophosphorylation by isolated spinach (Spinacia oleracea var. Virginia blight-resistant savoy) chloroplasts at a pH below the optimum has been re-examined. Its seasonal nature may be related to the hormonal status of the plants. Bicarbonate anions stimulate adenosine 5′-triphosphate synthesis if added in the final, adenosine 5′-triphosphate-forming stage of either a postillumination or an acid-base experiment. They also stimulate the membrane-bound, Mg²⁺-dependent adenosine 5′-triphosphatase of chloroplasts, and the Ca²⁺-dependent adenosine 5′-triphosphatase of detached coupling factor. These and other data point to the interaction between energized thylakoid membranes and the coupling factor as the probable site of action of bicarbonate anions when they stimulate photophosphorylation.     

Studies of sulfate utilization of algae: 15. Enzymes of assimilatory sulfate reduction in euglena and their cellular localization

Crude extracts of wild-type Euglena grown in the light (WTL) or in the dark (WTD) and a mutant lacking detectable plastid DNA (W₃BUL) contain adenosine 5′-phosphosulfate (APS) sulfotransferase. Isotope dilution experiments indicate that adenosine 3′-phosphate 5′-phosphosulfate (PAPS) sulfotransferase is absent.     

Evidence for Abasic Site Sugar Phosphate-Mediated Cytotoxicity in Alkylating Agent Treated Saccharomyces cerevisiae

To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in Saccharomyces cerevisiae, we replaced the RAD27^FEN1 open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In S. cerevisiae, Rad27^Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in S. cerevisiae, and this partially rescued the MMS hypersensitivity observed with rad27^fen1-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in rad27^fen1-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27^Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.     

Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C(4) Plant, Panicum miliaceum L. : Identification and Kinetic Characterization

The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-³H]1,2-(2,2′ -disulfo-4,4′ -diisothiocyano)diphenylethane ([³H]H₂DIDS) and by using SDS-PAGE. Assay of ³²Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C₃ counterpart and can be regarded as C₄ types.     

Diversity in Guanosine 3′,5′-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants

The guanosine 3′,5′-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a K_i of 2.8 μm relative to the substrate GMP, whereas the K_m of this enzyme for GMP was 73 μm. The IC₅₀ of ppGpp for GKpm was ∼10 μm. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers'' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.     

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.     

Preparation of chloroplasts from euglena highly active in protein synthesis

Chloroplasts can be obtained by gentle lysis or mild shear of spheroplasts of vitamin B₁₂-deficient Euglena gracilis and then purified by isopycnic sedimentation on gradients of Ludox AM or Percoll. The chloroplasts appear compact and highly refractile by phase contrast or Hoffmann contrast microscopy. Upon incubation with [³H]leucine or [³⁵S]methionine, the chloroplasts incorporate the amino acids into protein at rates that are 100-fold faster than we had previously observed with Euglena and up to 8-fold faster than with chloroplasts of spinach. Euglena chloroplasts prepared by the current procedure are thus qualitatively superior to those previously available from Euglena and at least as active in protein synthesis as chloroplasts from higher plants.     

The epsilon Subunit of the Chloroplast Coupling Factor 1 from Euglena gracilis: A Possible Role in Controlling ATPase Activity

The coupling factor from chloroplasts (CF₁) of Euglena gracilis Z strain is an active ATPase in situ, and its activity cannot be increased by treatment with trypsin or heating as is the case with the CF₁ from other sources. The smallest subunit of CF₁, the ε subunit, is supposed to be involved in controlling the ATPase activity. We have devised a simple technique for rapid and large-scale isolation of this subunit. The ε subunit from Euglena CF₁, although having only a limited inhibitory effect on Euglena CF₁, drastically inhibited the ATPase activity of heat-activated spinach CF₁. The inhibition of spinach CF₁ could be reversed by passage through Sephadex G-50 or by a second heat activation. An antibody to the ε subunit of Euglena CF₁ cross-reacted only weakly with CF₁ from spinach, Sorghum, Kalanchoë, or Anacystis nidulans, but reacted well with whole Euglena CF₁ in addition to its ε subunit. The antibody increased the ATPase activity of Euglena and Anacystis CF₁ and of unactivated or partially activated spinach CF₁. The results suggest that the function of the ε subunit in Euglena CF₁ is similar to its function in CF₁ from other sources. The data also suggest that changes induced in spinach CF₁ by activation involves modifications in subunits other than the ε one.     

Co-localization of mitochondria with chloroplasts is a light-dependent reversible response

Co-localization of mitochondria with chloroplasts in plant cells has long been noticed as beneficial interactions of the organelles to active photosynthesis. Recently, we have found that mitochondria in mesophyll cells of Arabidopsis thaliana expressing mitochondrion-targeted green fluorescent protein (GFP) change their distribution in a light-dependent manner. Mitochondria occupy the periclinal and anticlinal regions of palisade cells under weak and strong blue light, respectively. Redistributed mitochondria seem to be rendered static through co-localization with chloroplasts. Here we further demonstrated that distribution patterns of mitochondria, together with chloroplasts, returned back to those of dark-adapted state during dark incubation after blue-light illumination. Reversible association of the two organelles may underlie flexible adaptation of plants to environmental fluctuations.Key words: Arabidopsis thaliana, blue light, chloroplast, green fluorescent protein, mesophyll cell, mitochondrion, organelle positioningHighly dynamic cell organelles, mitochondria, are responsible not only for energy production, but also for cellular metabolism, cell growth and survival as well as gene regulations.^1,2 Appropriate intracellular positioning and distribution of mitochondria contribute to proper organelle functions and are essential for cell signaling.^3,4 In plant cells operating photosynthesis, the co-localization of mitochondria with chloroplasts has been a well known phenomenon for a long period of time.^5,6,7 Physical contact of mitochondria with chloroplasts may provide a means to transfer genetic information from the organelle genome,⁸ as well as to exchange metabolite components; a process required for the maintenance of efficient photosynthesis.^9,10,11Using Arabidopsis thaliana stably expressing mitochondrion-targeted GFP,¹² we have recently examined a different aspect of mitochondria positioning. Although mitochondria in leaf mesophyll cells are highly motile under dark condition, mitochondria change their intracellular positions in response to light illumination.¹³ The pattern of light-dependent positioning of mitochondria seems to be essentially identical to that of chloroplasts.¹⁴ Mitochondria occupy the periclinal regions under weak blue light (wBL; 470 nm, 4 µmol m⁻²s⁻¹) and the anticlinal regions under strong blue light (sBL; 100 µmol m⁻²s⁻¹), respectively. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts in the periclinal regions with time period of wBL illumination clearly demonstrates that the co-localization of these two organelles is a light-induced phenomenon.¹³In the present study, to ask whether the light-dependent positioning of mitochondria is reversible or not, a time course of mitochondria redistribution was examined transferring the sample leaves from light to dark conditions. The representative results (Fig. 1) clearly show that mitochondria re-changed their positions within several hours of dark treatment. Immediately after dark adaptation, mitochondria in the palisade mesophyll cells were distributed randomly throughout the cytoplasm (Fig. 1A and ^{ref. 13}). Chloroplasts were distributed along the inner periclinal walls and the lower half of the anticlinal walls. On the contrary, mitochondria accumulated along the outer (Fig. 1B) and inner periclinal walls when illuminated with wBL. Chloroplast position was also along the outer and inner periclinal walls. Many of the mitochondria located near the chloroplasts lost their motility. When wBL-illuminated leaves were transferred back to dark condition, the numbers of mitochondria and chloroplasts present on the periclinal regions began to decrease within several hours (Fig. 1C). After 10 h dark treatment, distribution patterns of mitochondria as well as chloroplasts almost recovered to those of dark-adapted cells (Fig. 1D).Open in a separate windowFigure 1Distribution of mitochondria and chloroplasts on the outer periclinal regions of palisade mesophyll cells of A. thaliana under different light conditions. Mitochondria (green; GFP) and chloroplasts (red; chlorophyll autofluorescence) were visualized with confocal microscopy after dark adaptation (A), immediately after wBL (470 nm, 4 µmol m⁻²s⁻¹) illumination for 4 h (B), after dark treatment for 6 h (C) and 10 h (D) following the 4-h wBL illumination, respectively. Bar = 50 µm.To our knowledge, this may be the first report that directly demonstrates that wBL regulates mitochondria and chloroplast positioning in a reversible manner, though the nuclei in A. thaliana leaf cells were also found to reverse their positions when transferred from sBL to dark conditions.¹⁵ Reversible regulation of organelle positioning in leaf cells should play critical roles in adaptation of plants to highly fluctuating light conditions in the nature. Since distribution patterns of mitochondria under wBL and sBL are identical to those of chloroplasts, we can assume that phototropins, the BL receptors for chloroplast photo-relocation movement,¹⁶ may have some role in the redistribution of mitochondria. On the other hand, we also found that red light exhibited a significant effect on mitochondria positioning (Islam et al. 2009), suggesting an involvement of photosynthesis. These possibilities are now under investigation.     

The adaptive behaviour of isoenzyme forms of rat liver alanine amino transferases during development

1. The activities of the mitochondrial and cytosol isoenzyme forms of l-alanine–glyoxylate and l-alanine–2-oxoglutarate aminotransferases were determined in rat liver during foetal and neonatal development. 2. The mitochondrial glyoxylate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. The cytosol glyoxylate aminotransferase and the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities first appear prenatally, increase further after birth and then rise to the adult values during weaning. 4. In foetal liver the mitochondrial glyoxylate aminotransferase and the cytosol 2-oxoglutarate aminotransferase activities are increased after injection in utero of glucagon, dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) or thyroxine. The cytosol glyoxylate aminotransferase and the mitochondrial 2-oxoglutarate aminotransferase activities are increased after injection in utero of cortisol or thyroxine. 5. After birth the further normal increases in the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities can be hastened by cortisol injection, whereas the increase in cytosol glyoxylate aminotransferase activity requires cortisol treatment together with the intragastric administration of casein. 6. The results are discussed with reference to the metabolic patterns and the changes in regulatory stimuli (hormonal and dietary) that occur during the period of development.     

Tryptophan aminotransferase and tryptophan dehydrogenase activities in some cell compartments of spinach leaves: The effect of calcium ions on tryptophan dehydrogenase

The content of spinach-leaf cells was compartmented by differential centrifugation. Three fractions were obtained,i.e. chloroplasts, pellet of remaining organelles sedimenting at 97 000g and cytosol. Enzyme activities of L-tryptophan aminotransferase (TAT) as well as L-tryptophan dehydrogenase (TDH) were demonstrated in all cell fractions. The highest activities of both enzymes were found in the pellet of organelles followed by the enzyme activities in the chloroplasts. The cytosol had the lowest enzyme activities. Chloroplasts are characterized by a relatively higher TDH activity, organelles sedimenting at 97 000g were marked by a relatively higher TAT activity. In all fractions both pyridine nucleotide coenzymes catalyzed the TDH activity. Ca²⁺ in a concentration of 0.8 minol l⁻¹ increased markedly the TDH activity in both directions of its activity. An erratum to this article is available at .     

In Vivo 23Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

Sodium gradients (ΔpNa) were measured in resting cells of Fibrobacter succinogenes by in vivo ²³Na nuclear magnetic resonance using Tm(DOTP)⁵⁻ [thulium(III) 1,4,7,10-tetraazacyclododecane-N′,N′′,N′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ΔpNa of −55 to −40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na⁺ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).