Inhibitory effect of hypergravity on photosynthetic carbon dioxide fixation in Euglena gracilis.

Photosynthesis, the conversion of light energy into chemical energy, is a critical biological process, whereby plants synthesize carbohydrates from light, carbon dioxide (CO2) and water. The influence of gravity on this biological process, however, is not well understood. Thus, centrifugation was used to alter the gravity environment of Euglena gracilis grown on nutritive agar plates illuminated with red and blue light emitting diodes. The results showed that hypergravity (up to 10xg) had an inhibitory effect on photosynthetic CO2 fixation. Chlorophyll accumulation per cell was essentially unaffected by treatment; however, Chl a/Chl b ratios decreased in hypergravity when compared to 1xg controls. Photosynthesis in Euglena appears to have limited tolerance for even moderate changes in gravitational acceleration.     

A possible ribosomal-directed regulatory system in Euglena gracilis. Carbon dioxide fixation

It was shown that cycloheximide inhibits CO(2) fixation in Euglena cells in the dark, but no effect of chloramphenicol was found. The light-dependent CO(2) fixation was inhibited by chloramphenicol and by large amounts of cycloheximide, but was stimulated by small amounts of cycloheximide. The presence of the stimulatory concentration of cycloheximide abolished the inhibition effect of high concentrations of chloramphenicol. The results indicate that the light-dependent CO(2) fixation is controlled by a repression-derepression regulatory system, which seems to be independent of chlorophyll synthesis.     

The photoreceptor protein of Euglena gracilis

We isolated the photoactive protein Erh, isolated from the photoreceptor of the unicellular photosynthetic flagellate Euglena gracilis. It is a 27 kDa protein with a photocycle resembling that of sensory rhodopsin, but with at least one stable intermediate. We recorded the absorption spectrum of the parent form of this protein both under native form and in the presence of hydroxylamine and sodium borohydride, and the fluorescence spectra of both the parent and intermediate forms. We suggest that Erh is a rhodopsin-like protein and propose a simple photocycle. This protein shows optical bistability, without thermal deactivation.     

Growth and cell volume of Euglena gracilis in different media.

It is necessary to propagate Euglena gracilis cells for several days after transfer from one medium to another to establish the steady state of balanced growth. Steady-state growth was established in minimal and in complex medium. Specific growth rates and cell volume distributions were computed for each culture medium. Mean cell volume of E. gracilis is not uniquely correlated with the specific growth rate.     

Characterization of an aldehyde dehydrogenase from Euglena gracilis

The free-living protist Euglena gracilis showed an enhanced growth when cultured in the dark with high concentrations of ethanol as carbon source. In a medium containing glutamate/malate plus 1% ethanol, E. gracilis reached a density of 3 x 10(7) cells/ml after 100 h of culture, which was 5 times higher than that attained with glutamate/malate or ethanol separately. This observation suggested the involvement of a highly active aldehyde dehydrogenase in the metabolism of ethanol. Purification of the E. gracilis aldehyde dehydrogenase from the mitochondrial fraction by affinity chromatography yielded an enrichment of 34 times and recovery of 33% of the total mitochondrial activity. SDS-PAGE and molecular exclusion chromatography revealed a native tetrameric protein of 160 kDa. Kinetic analysis showed Km values of 5 and 50 microM for propionaldehyde and NAD(+), respectively, and a Vm value of 1,300 nmol (min x mg protein)(-1). NAD(+) and NADH stimulated the esterase activity of the purified aldehyde dehydrogenase. The present data indicated that the E. gracilis aldehyde dehydrogenase has kinetic and structural properties similar to those of human aldehyde dehydrogenases class 1 and 2.     

Microsomal ethanol-oxidizing system in Euglena gracilis. Similarities between Euglena and mammalian cell systems.

1. ADH activity of Euglena grown with 50 mM ethanol decreased, but MEOS activity increased with a corresponding increase in the total amount of cytochrome P-450. 2. Phenobarbital treatment increased the total amount of cytochrome P-450. 3. CO and KCN, cytochrome P-450 ligands, diminished acetaldehyde formed from ethanol oxidation by MEOS. 4. The amounts of NAD(P)H cytochrome c reductases and cytochrome b5 type, components of microsomal monooxygenase reaction, have been spectrophotometrically measured. 5. NAD(P)H cytochrome c reductases activities were induced by phenobarbital. 6. DMSO, an inhibitor of rabbit MEOS, inhibited O2 consumption (11-20%) by Euglena grown with an ethanol, but not a lactate medium. 7. These studies indicate the presence of cytochrome P-450-dependent MEOS in Euglena similar to that in the mammalian hepatic cell.     

Light regulation of the cell cycle in Euglena gracilis bacillaris

We have studied the light regulation of the cell division cycle in the photosynthetic alga Euglena gracilis bacillaris. Euglena grown under phototrophic conditions are easily synchronized to a 12 h light-12 h dark regime. By inoculating stationary phase, nondividing cells into fresh media and exposing the diluted cells to either light or darkness, we have determined that initiation of DNA synthesis for the cell division cycle is light dependent. By varying the length of time in light to which synchronized cells are exposed, we have shown that commitment to the cell cycle requires exposure to more than 6 h of light. We propose that this is to allow the accumulation, through photosynthetic electron transport, of an initiating factor that will enable DNA synthesis to begin. Flow cytometry analysis also shows that once cells are committed to the cell cycle, they complete the cycle in the dark, so mitosis is a light-independent step.     

Growth and cell volume of Euglena gracilis in different media.

It is necessary to propagate Euglena gracilis cells for several days after transfer from one medium to another to establish the steady state of balanced growth. Steady-state growth was established in minimal and in complex medium. Specific growth rates and cell volume distributions were computed for each culture medium. Mean cell volume of E. gracilis is not uniquely correlated with the specific growth rate.     

Purification and some properties of cytosolic cobalamin-binding protein in Euglena gracilis.

Naturally prepared radiolabelled pulmonary surfactant can be rapidly cleared from the alveolar surface to the lung tissue after intratracheal instillation into experimental rats. This clearance is both time- and dose-dependent, a large dose (10 mg/animal) becoming associated with lung tissue more rapidly than a smaller more physiological dose (0.75 mg/animal). The data indicate that extracellular dipalmitoyl-phosphatidylcholine, the major component of pulmonary surfactant, is not catabolized at the alveolar surface. Alveolar free cells (mainly macrophages) appear to play a minor role in surfactant clearance. Quartz-induced phospholipidosis does not lead to an alteration in the rate of bulk surfactant clearance from the alveolar surface, although the initial distribution of the removed phospholipid complex may change in relation to the enlarged heterogenous free cell population.     

Biosynthesis of polyamines in Euglena gracilis

In Euglena gracilis Z the biosynthesis of spermidine and spermine closely resembles the pathways occurring in mammalian tissues and in most microorganisms. l-Ornithine and not l-arginine, as is the case in most plants, is the main precursor of putrescine, and S-adenosylmethionine donates the propylamino moiety for the biosynthesis of spermidine and spermine. Cell-free extracts of Euglena synthesized sym-norspermidine and sym-norspermine from 1,3-diaminopropane and labelled S-adenosylmenthionine. The synthases for the biosynthesis of these two polyamines have a pH optimum of 7.6, like that of spermidine and spermine synthases. Ion exchange chromatography showed two peaks corresponding to the retention times of 2,4-diaminobutyric acid and 1,3-diaminopropane, lower homologues of ornithine and putrescine, respectively. Experiments with dl-2,4-diaminobutyric acid-[4-¹⁴C] did not result in significant incorporation of the label into 1,3-diaminopropane.     

Control of chloroplast formation in Euglena gracilis. Antagonism between carbon and nitrogen sources

1. Cells of Euglena gracilis grown in the dark on high ratios of carbon source to nitrogen source (;high-carbon cells') are unable to form chlorophyll during a subsequent incubation in the light; cells grown in the dark on low ratios of carbon to nitrogen (;low-carbon cells') synthesize chlorophyll at a rapid rate during the subsequent incubation in the light. High-carbon cells will form chlorophyll rapidly if supplied with a nitrogen source during the incubation in the light: of the nitrogen sources tested, ammonium sulphate was the most effective at overcoming the block in chlorophyll synthesis. The nitrogen source does not have to be present during the actual incubation in the light: a 5hr. exposure of high-carbon cells to ammonium sulphate in the dark, followed by removal of the nitrogen source, is sufficient to bring about rapid chlorophyll synthesis during a subsequent incubation in the light. 2. The synthesis of chlorophyll by low-carbon cells exposed to the light is strongly repressed by the addition of ethanol or other utilizable carbon sources during the incubation in the light. Chlorophyll synthesis ceases altogether between 5 and 10hr. after the addition of the carbon source. Carotenoid synthesis is also inhibited, but to a smaller extent. The inhibitory effects of ethanol are prevented if ammonium sulphate is added at the same time. 3. High-carbon cells contain about four times as much carbohydrate per cell and about twice as much lipid per cell as low-carbon cells. The content per cell of total protein, soluble protein and DNA are about the same in both types of cell. The low-carbon cells sometimes, but not always, contain more RNA than the high-carbon cells. Analysis of cold-acid extracts indicates that the two kinds of cells contain about the same concentrations of pool amino acids, but that the low-carbon cells contain somewhat higher concentrations of peptides in the pool. Ion-exchange analysis of pool extracts shows a number of differences between high-carbon and low-carbon cells with respect to the concentrations of individual amino acids: in particular low-carbon cells contain higher concentrations of alanine. High-carbon cells have approximately twice as much protease activity as low-carbon cells. 4. The possible biochemical basis for the differing ability of high-carbon and low-carbon cells to form chloroplasts in the light is discussed.     

The light-harvesting system of Euglena gracilis during the cell cycle

The apoproteins of the light-harvesting chlorophyll-protein complexes LHCI and CP29 (apparent molecular weights of 27 kDa and 29 kDa, respectively) of Euglena gracilis were identified immunologically. Both complexes are present in the thylakoids of autotrophically cultured Euglena cells during the whole cell cycle. The relative amount of each apoprotein tends to increase towards the end of the cell cycle. The light-harvesting chlorophyll-protein complex of photosystem II, LHCII, of E. gracilis contains chlorophyll a, chlorophyll b, neoxanthin, diadinoxanthin and beta-carotene. Its chlorophyll a/b ratio is about 1.7 during the whole cell cycle. About 9 h after cell division the ratio of diadinoxanthin to chlorophyll a is doubled for a time of 3–4 h. The relevance of this increase during one developmental stage is discussed in relation to the insertion and-or assembly of newly synthesized LHCII.Abbreviations LHCP light-harvesting chlorophyll-protein complex - PS photosystem This research was partly supported by the Deutsche Forschungsge meinschaft.