Enzymatic conversion of glutamate to delta-aminolevulinic acid in soluble extracts of Euglena gracilis

Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis. delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA. The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant. Dithiothreitol was also required for activity after carbon treatment. delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp. PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum. E. coli glutamate-specific tRNA was inhibitory. Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract. RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly. delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells. Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration. Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration.     

Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L-glycerophosphatidyl residue attached via a phosphodiester linkage to C-1 of GlcNAc as the reducing end of the ECA sugar chain. ECA of P. shigelloides showed the best-resolved 13C-NMR spectra, especially after the removal of non-stoichiometric O-acetyl groups at C-6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100 degrees C, 10 min). Subsequent 13C-NMR studies were therefore carried out with the mild-acid-treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C-NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA. The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95-104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co-exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non-amphiphilic (cyclic) polysaccharide. The finding that the sugar chain of ECA is attached to an L-glycerophosphatidyl residue is in full corroboration with serological, enzymatic and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Enterobacteriaceae and P. shigelloides.     

Chemical analysis of Azospirillum lipopolysaccharides

Lipopolysaccharides (LPS) were extracted by hot phenol-water from five strains each of Azospirillum lipoferum and Azospirillum brasilense. Rhamnose, glucose, glucosamine and 3-deoxy-d-mannooctulosonic acid were comon sugar constituents of all LPS preparations. 2-O-Mefucose, 3-O-Me-fucose, 3-O-Me-rhamnose and 2-O-Megalactose were found in LPSs of some A. brasilense strains. Fatty acid spectra from all LPSs studied were almost identical with predominance of 3-hydroxymyristic and 3-hydroxypalmitic acids. 3-Hydroxypalmitic acid was the only amide-linked fatty acid. Lipopolysaccharides isolated from A. brasilense showed higher heterogeneity in sugar composition than those from A. lipoferum.Abbreviations glc gas liquid chromatography - ms mass spectrometry - LPS lipopolysaccharide - dOclA 3-deoxy-d-mannooctulosonic acid - 3-OH-16:0 3-hydroxypalmitic acid - nir^- nitrite reductase negative - nir⁺ nitrite reductase positive     

The cytoplasmic pH in photosynthesizing cells of the green alga Chlorella fusca,measured by P-31 NMR spectroscopy

P-31 NMR investigations were performed with the green alga Chlorella fusca under anaerobic conditions in the dark and in the light.In spectra of cells in the dark the signal of intracellular, nonvacuolar P_i indicates a pH in its chemical environment of 7.0–7.2. Upon illumination this signal looses intensity and shifts to lower field, corresponding to a pH of 7.7. Further downfield no other signal that could be attributed to a P_i-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this P_i-signal was assigned to the cytoplasm. The pH increase in the cytoplasm upon transfer of cells from the dark to the light is the same as that previously observed upon transfer of cells from anaerobic to aerobic conditions.In cells performing only cyclic photophosphorylation the cytoplasmic pH is lower than in photosynthesizing cells but still 0.2 pH units higher than in the cells in the dark. The reasons for the missing of a signal of stromal P_i and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.Non-standard abbreviations 2dG 2-Deoxyglucose - dG-6-P 2-deoxyglucose-6-phosphate - DCMU 3,4-dichlorophenyl-dimethylurea - MOPSO 3-(N-morpholino)-2-hydroxypropane sulfonic acid - P-31 NMR P-31 nuclear magnetic resonance     

Ammonium Triggers Uptake of NO-3 by Chenopodium rubrum Suspension Culture Cells and Remobilization of their Vacuolar Nitrate Pool

Photoautotrophic cell suspension cultures of Chenopodium rubrumrequire high concentrations of nitrate and ammonium. Duringthe growth phase total NH₄⁺ and the greater portion of NH₃^–were consumed. During the stationary phase nitrate uptake continuedbut at a substantially smaller rate than during the growth phase.During growth the bulk of the absorbed N was incorporated intoprotein, the amount of which was then maintained constant untilsenescence. NH₃^– was accumulated upon transition betweenthe growth and the stationary phase. NH₃^–, like the freeamino acids, was deposited in the vacuole but, unlike thesecompounds, could not be remobilized upon transfer of the cellsinto N-free medium. Readdition of NH₄⁺ to the medium, however,resulted in a mobilization of the vacuolar NH₃^–-pool.Reutilization of both vacuolar N-storage pools must have beenaccomplished by recycling from the vacuole to the cytoplasmbecause N-metabolizing enzymes could not be detected in isolatedvacuoles. Transfer of the cells of the stationary phase intomedium containing NH₃^– and NH₄⁺ resulted in an inductionof nitrate uptake by the cells, but only after a lag phase of4–5 days. It is conceivable that NH₄⁺ induces NH₃^–-translocatingsystems in the plasmalemma and in the tonoplast. (Received December 19, 1988; Accepted March 2, 1989)     

Isolation of a natural cytotoxic IgM "antibody" in human serum sensitizing L cells to complement

An IgM fraction of human serum was isolated and purified. A portion of this fraction firmly attaches to L cells' surfaces, which sensitizes these cells to the lytic action of low concentrations of serum C. It contains the natural cytotoxic "antibody" to L cells.     

Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors.

Mouse fibroblasts (3T3-L1 cells) accumulate detergent- and salt-insoluble aggregates of proteins conjugated to ubiquitin when incubated in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. These ubiquitin-protein conjugates co-fractionate with lysosomes on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. Both E-64 and ammonium chloride increase the intracellular concentration of free ubiquitin, but only E-64 leads to the formation of insoluble lysosomal ubiquitin-protein conjugates. The results are discussed in relation to the possible intracellular roles of ubiquitin conjugation.     

N-15 in vivo NMR spectroscopic investigation of nitrogen deprived cell suspensions of the green alga Chlorella fusca

The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K¹⁵NO₃ after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected. Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids. The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo NMR spectroscopy can be applied to the investigation of N metabolism of the cells.     

Chemical studies on the lipopolysaccharide of Rhizobium meliloti 10406 and its lipid A region

The structure of the lipopolysaccharide from Rhizobium meliloti 10406, a derivative of the wild-type strain MVII-1, was examined. The compositional analysis of its polysaccharide moiety demonstrated lack of heptose(s), but high contents in glucose, galacturonic acid and 2-keto-3-deoxy-octonate (dOclA) as characteristic features. The lipid A moiety consisted of a -1,6 linked glucosamine disaccharide carrying ester (at C-4) and glycosidically (at C-1) linked phosphate residues, both present exclusively as monoester phosphates but not as phosphodiesters. Ester- and amidelinked 3-hydroxy fatty acids were mostly present as non-3-O-acylated residues. Laser desorption mass spectrometry (LD-MS) revealed heterogeneity in the fatty acid substitution, as was also indicated by the non-stoichiometric ratios obtained by quantitative fatty acid analysis. The predominating lipid A structure contained at the reducing glucosamine residue ester-linked 3-hydroxy-tetradecanoic acid (3-OH-14:0) and amide-linked 3-OH-18:0, or 3-OH-18:1, respectively. The distal (non-reducing) glucosamine carried ester-bound the recently discovered 27-hydroxyoctacosanoic acid and 3-OH-14:0 and, as amide-linked fatty acid, mostly 3-hydroxy-stearic acid (3-OH-18:0).The isolated lipopolysaccharide exhibited a high extent of lethal toxicity in galactosamine-treated mice, comparable to that of enterobacterial lipopolysaccharide. The structural relationship of LPS and lipid A of Rhizobium meliloti to other rhizobial lipopolysaccharides and lipid A's with respect to questions of taxonomy and of phylogenetic relationships will be discussed.Abbreviations LPS lipopolysaccharide - dOclA 3-deoxy-D-mannooctulosonic acid (KDO) - GalA galacturonic acid - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - LD-MS laser desorption-mass spectrometry