Bacteriocuprein superoxide dismutases in pseudomonads.

Two new instances of the rare bacteriocuprein form of superoxide dismutase have been discovered in Pseudomonas diminuta and P. maltophilia. Each species contains a manganese superoxide dismutase as well. Eight other strains of Pseudomonas and Xanthomonas spp. lacked bacteriocupreins and contained either a manganese or an iron superoxide dismutase. Native molecular weights and isoelectric points were determined for all these bacterial dismutases. A monospecific polyclonal antibody was prepared against the bacteriocuprein from Photobacterium leiognathi; it was not cross-reactive with the bacteriocuprein from either Pseudomonas strain. Bacteriocupreins have previously been identified in only two procaryotes, P. leiognathi and Caulobacter crescentus. The discovery of the Pseudomonas bacteriocupreins reveals a broader distribution, raising the possibility that bacteriocupreins are a continuous line of descent among procaryotes and not isolated evolutionary occurrences, as previous data suggested.     

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.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

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.     

Antimutagenicity in Euglena gracilis

The genotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, α-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis. The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100 and TA102, were used. The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102.     

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.     

Peroxidative Activity in Euglena gracilis

Cell-free homogenates of Euglena gracilis contain very low levels of catalase activity as compared to higher plants and some other algae. Purified Euglena cytochrome c acts catalytically as a peroxidase. The observed catalytic activity of cytochrome c in extracts from heterotrophically grown cells was more than enough to account for the observed rates of hydrogen peroxide destruction. The peroxidative activity of Euglena cytochrome c was completely inhibited by 20 mm 3-amino-1,2,4-triazole.     

Thiamin uptake in Euglena gracilis

Thiamin uptake has been investigated in Euglena gracilis Z. This protozoon possessed an active transport system for thiamin with a Km value of 17 nM and a Vmax value of 7.8 pmol per 10(6) cells per min. Thiamin uptake was dependent on pH and temperature, but not on exogenous glucose as an energy source. Oxythiamin and pyrithiamin were competitive inhibitors with Ki values of 33 nM and 15 nM, respectively. Thiamin monophosphate, thiamin pyrophosphate, thiamin triphosphate, heteropyrithiamin, quinolinothiamin, thiamin chloride and amprolium inhibited uptake. Inhibition of thiamin uptake by various metabolic inhibitors and anaerobiosis suggest that thiamin uptake requires an energy source generated by respiration and glycolysis.     

Extrachromosomal circular nuclear rDNA in Euglena gracilis.

The presence of extrachromosomal nuclear ribosomal DNA (rDNA) in the unicellular alga Euglena gracilis has been established. This rDNA is circular. Each circle is 3.8 micron long and contains one rDNA unit. Oligomers are rare. Extrachromosomal rDNA is present in large amounts during the exponential phase of growth and appears less abundant during the stationary phase. It was found in all wild-type and mutant strains of Euglena examined. Our estimations suggest that rDNA in Euglena is mainly extrachromosomal. Research of extrachromosomal rDNA in spinach and Petunia was negative.     

Circadian rhythm of gravitaxis in Euglena gracilis.

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavioral reactions. One pronounced reaction is the orientation with respect to gravity. In synchronized cultures with no cell growth a distinct circadian rhythm of negative gravitactic orientation could be observed. The main maximum of sensitivity was detected 5 h after the beginning of the subjective day, the main minimum 5 h before the beginning of the subjective day. Transferring synchronized cultures to continuous light resulted in an almost instantaneous loss of rhythmicity. In contrast, after transfer to permanent darkness cells exhibited a circadian rhythm with a progressive shortening of the period for more than 5 days. These findings are in contrast to the circadian rhythm of phototaxis in Euglena, where a free-running period of 24 h was observed. Parallel measurements of negative gravitactic orientation, velocity, cell shape as well as cAMP concentration in synchronized cultures revealed a circadian rhythm of all reactions. The results are discussed with regard to the possible role of cell shape and cAMP in gravitactic orientation.     

Studies on polyamine biosynthesis in Euglena gracilis.

Euglene gracilis (strain Z) was found to contain five polyamines which could be separated by high-pressure cation-exchange chromatography. 1,3-Diaminopropane, putrescine, norspermidine (N-(3-aminopropyl)-1,3-diaminopropane), spermidine and norspermine (N,N'-bis(aminopropyl)-1,3-diaminopropane) were identified. Biosynthesis of putrescine in E. gracilis proceeds through decarboxylation of L-ornithine, no arginine decarboxylase (EC 4.1.1.19) activity could be detected. The properties of the enzymes ornithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50) in this alga were found to be similar to those of the enzymes isolated from animal tissues or yeast cells. A bioxynthetic scheme is proposed which relates the different polyamines occurring in E. gracilis.     

Physical characterization of gravitaxis in Euglena gracilis.

Gravitaxis in unicellular microorganisms like Euglena gracilis has been known for more than 100 years. The current model explains this phenomenon on the basis of a specific density difference between cell body and surrounding medium. In order to test the feasibility of the current model in terms of physical considerations the specific density of different Euglena gracilis cultures was determined. Depending on the culture conditions the specific density was in a range between 1.046 g mL-1 and 1.054 g mL-1. Size and gravitaxis measurements were performed in parallel, which allowed to relate the force applied to the lower membrane to the kinetic properties of gravitactic reorientation. A linear relationship between force and gravitaxis kinetics was found. A comparison between estimated activation energy of the proposed stretch-sensitive ion channels and energy supplied by the displacement of the lower membrane by the sedimentation of the cell body revealed that a focusing, an amplification and/or an integration period over time must be involved in the gravitactic signal transduction chain. Analysis of stimulus-response curves revealed an integration period of about 5 seconds before a gravitactic reorientation starts. The kinetics of gravitaxis at 1 x gn, and 0.12 x gn, was found to be similar. A hypothesis is presented that explains this finding on the basis of a combination of an integration period and an all-or-none reaction during gravitactic reorientation.     

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.     

Inhibition of superoxide dismutases by azide.

Iron-containing Superoxide dismutases are more sensitive to inhibition by azide than are the corresponding manganese containing enzymes, while the copper-zinc Superoxide dismutases are least sensitive. Thus, at pH 7.8, 10 mm azide inhibited Cu-Zn, Mn, and Fe enzymes by ~10%, ~30% and ~73%, respectively. Stated differently, the concentrations of N₃^? required to cause 50% inhibition of the Cu-Zn, Mn, and Fe enzymes was ~32 mm, ~20 mm and ~4 mm, respectively. These inhibitions by azide, which were imposed and reversed rapidly, appear to provide a useful criterion for distinguishing among the classes of these enzymes. Sensitivity towards inhibition by N₃^?can be applied to the Superoxide dismutases in crude extracts, for the purpose of deciding to which class they belong.     

Isolation and properties of gamma-tocopherol methyltransferase in Euglena gracilis.

Gamma-Tocopherol methyltransferase (EC2.1.1.-), which catalyzes the conversion of gamma-tocopherol into alpha-tocopherol, was present in a cell homogenate of Euglena gracilis. The enzyme was loosely bonded to the outer membrane of chloroplasts and solubilized from chloroplast membranes by a detergent, followed by partial purification in a three-step procedure. The methyltransferase showed a pH optimum of 7.5 and a temperature optimum of 35 degrees C and had an M(r) of 150,000. The activity was about 1.4-fold higher with gamma-tocopherol than with beta-tocopherol as substrate. The enzyme was specific for S-adenosylmethionine as a methyl donor, with a Km value of 50 microM. The addition of homogentisate, L-tyrosine and L-phenylalanine into a suspension of Euglena cells increased the relative pool sizes of alpha- and gamma-tocopherol, but not those of beta- and delta-tocopherol. The contents of alpha- and gamma-tocopherol in a chloroplast fraction of Euglena were always higher than those of any other fraction after any period of incubation with homogentisate. Based on the results of the present experiments, we propose a biosynthetic pathway of alpha-tocopherol in Euglena gracilis.