Localization of vitamin B12 binding in Euglena gracilis

Different fractionation procedures were used to determine the location of vitamin B12 binding sites in Euglena gracilis. Using uptake measurements, cell fractionation, and light and electron microscopy, the cuticle of the cell was found to be the fraction containing the majority of B12 binding sites. The apparent distribution of vitamin binding sites differed according to the cell lysis method used. The cuticle fraction was responsible for the binding of 80% of the vitamin taken up by the cell during both the rapid and the slow phase of uptake. These results suggest that vitamin B12 binding is regulated, in part, at the cuticle level, and support our previous conclusion that the secondary phase of uptake represents the synthesis of new receptor sites and not the unloading of vitamin inside the cell.     

Ribonucleotide reductase activity in vitamin B12-deficient Euglena gracilis.

The ribonucleotide reductase activities in vitamin B12-sufficient and -deficient cells of Euglena gracilis were measured. We found that the cells progress into vitamin B12 deficiency the enzyme activity increases, reaching a maximum value of 20-fold in advanced deficiency. No signigicant differences in the activities were found to result as a consequence of different growth conditions. We propose that the increased activity in vitamin B12-deficient cells is due to an increase in enzyme protein.     

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.     

The role of vitamin B12 in the metabolism of Euglena gracilis var. bacillaris

1. The concentrations of RNA, DNA and protein are decreased in cells of Euglena gracilis var. bacillaris grown on suboptimum concentrations of vitamin B(12). 2. The addition of vitamin B(12) to deficient cells stimulates the incorporation of [(14)C]formate into the above cell components as well as into thymine of DNA and serine and methionine of protein. 3. In a cell-free system from vitamin B(12)-deficient cells, the incorporation of labelled formate into thymidylate is decreased to a greater extent with uridine than with deoxyuridine as the substrate. 4. The addition of unlabelled glutamate dilutes the radioactivity incorporated into thymine from labelled formate. 5. These results are interpreted to mean that, in DNA synthesis, vitamin B(12) has a greater role in the reduction of ribotides to deoxyribotides than in the reduction of formate to thymine methyl and that the vitamin B(12)-dependent conversion of glutamate into beta-methylaspartate also contributes to thymine synthesis.     

Sequential changes in cell volume distribution during vitamin B12 starvation of Euglena gracilis.

During vitamin B12 starvation of Euglena, a new peak appears in the cell volume distribution. Some cells are inhibited at a unique point in the cell cycle between the initiation of DNA synthesis and nuclear division. The mechanism of inhibition of other cells differs.     

Factors affecting division rhythmicity in Euglena gracilis

In phototrophic culture of Euglena gracilis, good synchrony was found only under rather restricted programs of light-dark cycles, and rather narrow ranges of temperature and light intensity, when cultures were flushed with air fortified with adequate amounts of CO₂. When flushed with air alone, CO₂ was found to be limiting, and while cell divisions were rhythmic, less than a doubling of cell number occurred in division bursts. With air as gas phase, rhythmic division activity was maintained over wide ranges of temperature, light intensity, and the ratio of light:dark in a given program; all these factors affected the amplitude of the division burst, however.     

Synthesis of Receptor Sites for Vitamin B12 in Euglena gracilis

In Euglena gracilis, vitamin B₁₂ uptake follows a biphasic patternconsisting of an initial rapid phase followed by a slower secondaryphase. Chase experiments showed that vitamin B₁₂ was tightlybound to its receptor-sites during either rapid or slow phaseof uptake. The slow phase was markedly inhibited when Euglenacells were preincubated with cycloheximide for 30 min at a concentrationof 100µg/ml. When the preincubation time was longer than30 min, a gradual inhibition of the rapid phase occurred andreached 84% after 4 h. This inhibitory effect of cycloheximideis reversible. On the other hand, tunicamycin at 1 µ/mlirreversibly inhibited the B₁₂ uptake suggesting that the receptor-sitefor B₁₂ is glycoprotein in nature. These results suggest thatthe rapid phase is also dependent on protein synthesis and representsB₁₂ binding on preexisting free receptors whereas the secondaryslow phase represents B_l2 binding on newly synthesized receptors.Both phases of uptake seem to be controled by the same receptor.The half-life of the free receptor is estimated to be 66 minwhereas the B₁₂ receptor complex is very stable. (Received October 3, 1988; Accepted February 15, 1989)     

Synchronization of Division in Vitamin B12-Starved Euglena gracilis

SYNOPSIS Vitamin B₁₂ deficiency arrests cell division in Euglena gracilis. B₁₂ starvation for short periods made it possible to induce synchronous growth by addition of the vitamin. Culture conditions were established to optimize replenishment synchrony. The DNA content of E. gracilis in steady state culture and vitamin B₁₂ deficiency culture was measured by flow cytofluorometry and was consistent with colorimetric determinations. The cell volume and DNA distributions of E. gracilis in synchronous culture were analyzed and the sequential changes during the division cycle were computed. Synchronous culture permits more definitive studies of shifts in cell volume and DNA distributions, in which the biochemical events required for cell division are presumably synchronized.     

Factors affecting pH-dependent photo-inhibition of division in Euglena gracilis

Visible light of moderate intensity (1200 ft-cd) can severely inhibit cell division of a non-photosynthetic mutant of Euglena gracilis when growth is supported by butanol, ethanol, or fumarate as sole carbon source. The degree of inhibition is pH dependent, being greatest at pH 4 to 5. A wide variety of other carbon sources permitted growth in the light without inhibition.     

Sequential protein-dependent steps in the cell cycle initiation and completion of division in vitamin B12 replenishedEuglena gracilis

Summary InEuglena gracilis Z, vitamin B₁₂ deficiency arrests cell divisions in S/G 2 phase. After the addition of vitamin B₁₂ to blocked cells, nuclear and cellular divisions begin to be induced between the 3rd and the 4th and between the 4th and the 5th hour respectively; the cell population is doubled after the 11th hour.Addition of cycloheximide either with vitamin B₁₂ or 2 to 6 hours later inhibits the resumption of divisions and blocks cell development in different stages between G 2, karyokinesis and cytokinesis. These results suggest that as a prerequisite protein-dependent steps are required at precise times during the reversibility of blocked cell divisions: at least sequential syntheses of protein concern a) formation of the mitotic spindle and replication of the pellicle; b) completion of the nuclear division; c) progression of the cleavage furrow.     

Influence of culture pH on photo-inhibition of division in Euglena gracilis

In the dark, growth rates of Euglena gracilis were independent of culture pH between the limits of three to eight. Visible light of moderate intensity inhibited growth rates, with the degree of inhibition being markedly pH dependent. The most severe inhibition was observed at pH 4 to 5, with little or no inhibition at pH 3 or above pH 6.8 at the light intensity used (400 foot-candles).     

Compartmentation of uracil in Euglena gracilis.

Compartmentation of uracil in the flagellate Euglena gracilis was studied by tracer-kinetic experiments. Lag times in the equilibration of exogenously given and intracellularly present uracil before linear labeling of catabolic and anabolic products was determined to estimate the size of its metabolically active pool. This pool operates in the incorporation and degradation of uracil. There were the same lag times in forming both final products when measured in parallel and when measured after preloading with pyrimidines, in different cell strains, and under various environmental conditions. The amount of the metabolically active uracil pool, estimated as 11 pmol/10(7) heterotrophically growing cells, decreased to almost zero during light-induced RNA synthesis and could be changed by preloading with uracil or thymine. Besides this metabolic pool, cells may contain large amounts of uracil in a membrane-enclosed storage compartment (up to 12 nmol/10(7) cells). This is metabolically inert, but may be mobilized by nitrogen-carbon starvation. The role of uracil compartmentation in this metabolically flexible organism is discussed.     

Occurrence of norspermine in Euglena gracilis.

A series of fluorescent probes which locate a graded series of depths from the surface to the centre of the lipid bilayer have been used to measure the fluidity gradient in liposomes and natural membranes. In dioleoyl phosphatidylcholine liposomes and in cells which have a high content of unsaturated phospholipids, a region of high microviscosity is detected near the cis double bond/s. The significance of this phenomenon is discussed in terms of the penetration and lateral movement of membrane protein.     

Heteroxanthin in Euglena gracilis

Summary 1. From a large scale preparation of Euglena gracilis, strain Z, besides the acetylenic pigments diatoxanthin and diadinoxanthin and the allene neoxanthin, an additional acetylenic xanthophyll has been isolated. 2. Mass and IR spectra and chemical reactions showed typical patterns of heteroxanthin from Vaucheria. 3. The pigment was transformed into diadinochrome-isomers with acidified acetone. 4. A partial synthesis of heteroxanthin from diadinoxanthin by LiAlH₄-reduction is described, confirming the structure proposed by Strain. 5. The identity of heteroxanthin with the trollein—like pigment described for Euglena is discussed.