Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides I. Role of non-photosynthetic CO2-fixation in chloroplast regeneration

Carbon dioxide enhanced chloroplast regeneration in glucose-bleachedcells of Chlorella protothecoides in the presence of CMU inthe light. Both the formation of chlorophyll and the synthesesof RNA and protein were considerably enhanced. The CO₂ metabolism of algal cells during greening was investigatedusing ¹⁴C-bicarbonate as the tracer. Radiocarbon was largelyincorporated into purine and pyrimidine bases in nucleic acidand the arginine in protein, specifically at the crabon atomsderived from carbamylphosphate. ¹Part of this investigation was reported at the conference onthe "Autonomy and biogenesis of mitochondria and chloroplasts"held at Canberra in 1969 (4). (Received August 19, 1975; )     

Temperature inducible protochlorophyllide reduction in darkness in a pigment mutant of Scenedesmus obliquus

Accumulation of chlorophyll and protochlorophyllide (PChlide) was followed during beterotrophic growth of the pigment mutant C-2A' of Scenedesmus obliquus L. in the darkness at 30 and 20°C. At 30°C the cells remained yellow with accumulation of protochlorophyllide, whereas they became green at 20°C with only traces of protochlorophyllide. The capacity of mutant cells to reduce PChlide to chlorophyllide (Chlide) in the dark with or without addition of 5-aminolevulinic acid as measured in isolated membranes, was high in cells grown at 20°C but negligible at 30°C. The high capacity to reduce PChlide created in cells growing at 20°C was only slightly diminished by exposure of cells to 38°C for 3 h. Mechanisms of temperature-sensitive chlorosis in algae and higher plants are discussed in relation to the results with pigment mutant C-2A' of Scenedesmus obliquus . It is assumed that either an activator of NADPH protochlorophyllide oxidoreductase (EC 1.6.99.1) or a different enzyme system can be activated by lower temperature as by light.     

Effect of Ammonia on Cellular pH of Anabaena cylindrica Determined with 31P NMR Spectroscopy

The pH changes in the blue-green alga (cyanobacterium) Anabaenacylindrica caused by addition of ammonia were investigated using³¹P NMR spectroscopy. A pH shift of 0.9 or more was observedwhen 30 nM NH₄OH was added to the cell suspension, but no significantcellular pH change was observed with 50 mM NH₄CI, a concentrationhigh enough to stimulate dark CO₂ fixation of this alga. Thechange in cellular pH does not seem to cause ammonia-inducedstimulation of dark CO₂ fixation. (Received June 22, 1985; Accepted January 10, 1986)     

13C-NMR evidence of bacteriochlorophyll a formation by the C5 pathway in Chromatium

The 13C-NMR spectra of bacteriochlorophyll a formed in the presence of L-[1-13C]glutamate and [2-13C]glycine in Chromatium vinosum strain D were analyzed. The isotope in the glutamate was specifically incorporated into eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid (ALA), and the 13C in glycine was incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of bacteriochlorophyll a. These labeling patterns provide evidence for the exclusive operation of the C5 pathway in ALA biosynthesis in the bacterium. The 13C chemical shifts of two quaternary carbons (C-9 and C-16) of bacteriochlorophyll a were reassigned in the present study.     

13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of L-[1-13C]glutamate and [2-13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2-13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5-aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium. The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5-[1,4,5-13C]aminolevulinic acid formed by the C5 pathway from [1,2,5-13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2-[1,2,5-13C]oxoglutaric acid, arising by carboxylation of [1,4-13C]succinyl-CoA with 13CO2 catalysed by 2-oxoglutaric acid synthase activity, and that the labelled succinyl-CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle. The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C-2 and C-4) were reassigned.     

Role of Light in 5-Aminolevulinic Acid Formation in Wild Strain and Mutant C-2A' Cells of Scenedesmus obliquus

In dark-grown wild strain cells of Scenedesmus obliquus, 5-aminolevulinicacid (ALA) formation was induced by irradiation with a weakblue light, as in its mutant C-2A' cells. The induction wasinhibited by distamycin A, 6-methylpurine, cycloheximide andchloramphenicol. After the light induction, the ALA formationcould proceed in the dark as well as in the light, in such heterotrophicallygrown wild type cells, but not in the greening mutant C-2A'cells. In the latter, ALA formation was dependent on red light,as well as on blue light, in the presence of CMU. The amountsof protochlorophyll in the mutant cells increased upon cessationof illumination and decreased with subsequent irradiation withblue and red light. The possible role of protochlorophyll asa photoreceptor in regulation of ALA formation in the mutantcells is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otuka, Hachioji, Tokyo 192-03, Japan. (Received January 17, 1981; Accepted April 30, 1981)     

Characterization of the process of 5-aminolevulinic acid formation from glutamate via the C5 pathway in Clostridium thermoaceticum.

1. In vitro formation of 5-aminolevulinic acid (ALA) from glutamate required two enzyme fractions, separable on Blue Sepharose affinity chromatography, and a tRNA fraction, which can be replaced by Escherichia coli tRNA(Glu) in the reconstituted assay. 2. Gabaculine was shown to inhibit ALA formation in the complete assay as well as in a defined system consisting of only glutamate-1-semialdehyde and the enzyme fraction not retained on Blue Sepharose. 3. The results indicate that the enzyme system supporting ALA formation in Clostridium thermoaceticum is very similar to the tRNA(Glu)-dependent C5 pathway in plant plastids.     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides II. Role of carbamylphosphate in chloroplast regeneration

Levels of the activities of glutamine-dependent carbamylphosphatesynthetase, ornithine-and aspartate-transcabamylase and phosphoenolpyruvatecarboxylase were followed in greening cells of Chlorella prolothecoides.Among the enzymes examined the activity of carbamylphosphatesynthetase was extremely low, especially at the early phaseof greening. Arginine (but not ornithine or aspartate), when administeredto algal cells at the 24th hour of greening, stimulated thesyntheses of RNA, protein and chlorophyll in the subsequentperiod. It also affected the metabolic pathway of the ¹⁴CO₂supplied simultaneously with arginine in the presence of CMU.Arginine produced a decreased incorporation of ¹⁴C into proteinand an increased incorporation into nucleic acid. The mechanismof the action of CO₂ on chloroplast regeneration is discussed.We concluded that chloroplast regeneration in glucose-bleachedcells is limited by the synthesis of carbamylphosphate, especiallyin the early phase of greening. (Received August 19, 1975; )