In vitro activation of a galactosyl transferase involved in the osmotic regulation of ochromonas

Osmotic regulation in the flagellate Ochromonas malhamensis Pringsheim is mainly mediated by fluctuations in the pool size of α-galactosyl-(1→1)-glycerol (isofloridoside). A regulated key enzyme of isofloridoside metabolism is the galactosyl transferase producing isofloridoside phosphate. The activity of this enzyme in crude extracts can be increased 5- to 20-fold by incubation at pH 6. The activation occurs in a reaction with a Q₁₀ of 1.5 to 3 and is dependent on time and pH value. Inactivation of the activated form of the enzyme is also time-dependent, and is minimal at the pH value at which activation is optimal. The data suggest a regulation of the enzyme by chemical modification due to the action of auxiliary enzymes.     

Proteolytic activation of a galactosyl transferase involved in osmotic regulation

Osmotic regulation in the flagellate Ochromonas malhamensis Pringsheim is mainly mediated by changes in the pool size of α-galactosyl-(1 → 1)-glycerol (isofloridoside). Isofloridoside phosphate synthase, a regulated key enzyme responsible for the formation of isofloridoside phosphate, appears to exist as an inactive proenzyme which can be activated by incubation of crude cell extracts with endogenous or exogenous proteases.     

A method for determination of glyoxylate with pumpkin isocitrate lyase and its application to photosynthesizing Euglena gracilis Z

Isocitrate lyase (ICL) was highly purified from cotyledons of dark-grown pumpkin (Cucubita maxima Kuri Nankin) seedlings by rapid means, and a method was devised to determine glyoxylate using the enzyme in combination with yeast isocitrate dehydrogenase (NADP⁺). The enzymic properties of both enzymes were adequate for determination of varying concentrations of glyoxylate. The intracellular content of glyoxylate in Euglena gracilis Klebs strain Z Pringsheim growing photosynthetically in air was determined to be 2.0 nmol mg⁻¹ chlorophyll using this method.     

Aliphatic Chains of Esterified Lipids in Isolated Eyespots of Euglena gracilis var. bacillaris

Isolated eyespot granules of Euglena gracilis Klebs var. bacillaris Pringsheim contained approximately 6% lipids (based on protein). Separation of the lipid extracts by thin layer chromatography revealed four major fractions: wax esters, triacylglycerols, free fatty acids, and phospholipids. Methanolysis of each fraction yielded between 27 and 29 different fatty acids ranging from 12:0 to 22:6. Acetates of the fatty alcohols of the wax fraction consisted of 11:0 to 18:0 carbon chains, with 14:0 being the major component; unsaturated alcohols were not detected.     

A Role for Zinc in the Structural Integrity of the Cytoplasmic Ribosomes of Euglena gacilis

Zinc deficiency in dark-grown Euglena gracilis Klebs, Z strain Pringsheim, results in the disappearance of cytoplasmic ribosomes. In contrast, ribosomes in zinc-sufficient Euglena are conserved, do not undergo turnover, and can be demonstrated at any stage of growth. The zinc content of ribosomes from zinc-deficient Euglena just prior to ribosomal disappearance is 300 to 380 micrograms of zinc per gram rRNA as compared to 650 to 1280 micrograms of zinc per gram rRNA in ribosomes from zinc-sufficient cells. Ribosomal disappearance is believed to involve a generalized disintegration process related to the lower content of zinc in the ribosomes. Reappearance of ribosomes requires the addition of zinc. It is proposed that adequate zinc may be essential for normal tertiary and quaternary structure of the cytoplasmic ribosomes of Euglena.     

Heterotrophic Carbon Dioxide Fixation Products of Euglena: EFFECTS OF AMMONIUM

The metabolic products of heterotrophic (dark) CO₂ fixation by Euglena gracilis Klebs strain Z Pringsheim were separated and identified. They consisted of amino acids, phosphorylated compounds, tricarboxylic acid cycle intermediates, and nucleotides. Exposure of the cells to NH₄⁺ after a period of NH₄⁺ deprivation stimulated heterotrophic CO₂ fixation almost 4-fold, modifying the spectrum of the fixation products. In particular, the NH₄⁺ treatment stimulated fixation of CO₂ into glutamine, glycine, alanine, and serine.     

Volume regulation in poterioochromonas: involvement of calmodulin in the ca-stimulated activation of isofloridoside-phosphate synthase

In Poterioochromonas malhamensis Peterfi (syn. Ochromonas malhamensis Pringsheim) osmotically induced shrinkage is reversed by an accumulation of isofloridoside. Addition of Ca²⁺ ions to homogenates from standard volume cells initiates an enzyme system for the activation of isofloridoside-phosphate synthase. This process is stimulated in the presence of Ca²⁺ by calmodulin, isolated from the same alga or from bovine brain, and requires the presence of membranes. The stimulation observed when Ca²⁺ is added without exogenous calmodulin is inhibited by the calmodulin-binding substance R 24571. These results show that the effect of Ca²⁺ is mediated by calmodulin. The Ca²⁺/calmodulin-dependent activation is enhanced when fluoride or molybdate ions are present in the homogenization buffer. This might indicate the involvement of a phosphorylated compound in the activation mechanism.     

Large Scale Isolation and Purification of Eyespot Granules from Euglena gracilis var. bacillaris

Large volumes of eyespot granules were isolated from homogenates of Euglena gracilis Klebs var. bacillaris Pringsheim by flotation centrifugation in a Beckman Ti-15 zonal rotor, and were further purified by centrifugation in a swinging bucket rotor. Examination with the electron microscope showed the eyespot granules to be free from other cellular material. Freezing had no apparent effect on the structure or on the absorption properties of the eyespots. Absorption spectra of pure fractions of eyespot granules free of chloroplast contamination showed the previously reported curves in the range of 360 to 520 nanometers, as well as a peak at 660 to 675 nanometers. The procedure for the large scale isolation of eyespot granules from Euglena gracilis is compared with other methods which have employed conventional centrifugation, and the significance of the use of zonal rotors for isolating large quantities of pure eyespot granules is discussed.     

Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 × 10⁻⁶ molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and ⁵⁹Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX.     

Mobilisation of NADPH-glutamate dehydrogenase mRNA in regreening cultures of Euglena gracilis

Exposure of dark-grown resting Euglena gracilis Klebs strain Z Pringsheim to light results in a transient increase in the specific activity of NADPH-glutamate dehydrogenase. NADPH-glutamate dehydrogenase antibody was used to detect NADPH glutamate dehydrogenase resulting from the translation of total polyadenylated RNA and polysomal RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in cells at all stages of development and present on polysomes from dark-grown and regreening cells but not on polysomes from dark-grown resting cells. These results indicate that the light-induced increase in NADPH-glutamate dehydrogenase in dark-grown resting cells represent an increase in the rate enzyme synthesis resulting from the mobilisation of NADPH-glutamate dehydrogenase mRNA onto polysomes.     

AXENIC CULTURE OF UTRICULARIA

Pringsheim , Ernest G., and Olga Pringsheim . (Pflanzenphysiologisches Inst., Göttingen, Bundesrepublik Deutschland.) Axenic culture of Utricularia. Amer. Jour. Bot. 49(8): 898–901. Illus. 1962.—Utricularia exoleta, a small tropical floating carnivorous plant, was freed of other organisms by hypochlorite treatment. An inorganic nutrient solution was composed which produced good vegetative growth but flowering only if organic compounds, as contained in beef-extract, were added aseptically.     

Ribulose Diphosphate Carboxylase Synthesis in Euglena: III. Serological Relationships of the Intact Enzyme and its Subunits

Ribulose 1,5-diphosphate carboxylase was isolated from Euglena gracilis Klebs strain Z Pringsheim, Chlorella fusca var. vacuolata, and Chlamydobotrys stellata, and the subunits from each enzyme were separated and purified by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulfate. Rabbit antibody was elicited against purified Euglena ribulose 1,5-diphosphate carboxylase whole enzyme and the isolated large and small subunits. Euglena ribulose 1,5-diphosphate carboxylase showed partial immunological identity on Ouchterlony gels with the Chlorella and Chlamydobotrys carboxylases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates between antibody to the Euglena large subunit and the isolated large subunits of the Chlorella and Chlamydobotrys enzymes showed this was due to determinants on the large subunit. There was no serological affinity between the small subunits of the Euglena, Chlorella, and Chlamydobotrys carboxylases, and NH₂-terminal amino acid analyses provided further evidence of variability in the structure of the small subunits.     

Book Reviews

Book reviewed in this article:
Kröber, H ., Erfahrungen mit Phytophthora de Bary und Pythium Pringsheim. [Experiences with Phytophthora de Bary and Pythium Pringsheim].
J. A. Roberts and G. A. Tucker (Eds.) , Ethylene and Plant development.     

Pythium root rot of crucifers

A destructive root disease of cabbage (Brassica oleracea L. var. capitata) and cauliflower (Brassica oleracea L. var. botrytis) incited by a species of Pythium Pringsheim is described as occurring in Varanasi, U.P. The pathogen was isolated on potato dextrose and corn meal agar. Pathogenicity and host range of the disease were studied. Cultural characters, morphology and developmental stages and life cycle of the fungus indicated its identity with Pythium middletonii Sparrow.     

Analysis and Characterization of 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU)-resistant Euglena: I. Growth, Metabolic and Ultrastructural Modifications during Adaptation to Different Doses of DCMU

Cultures of Euglena gracilis Klebs strain Z Pringsheim were grown photoorganotrophically in the presence of different concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) in the range of 0.05 to 250 micromolar. Cultures were serially transferred and various metabolic parameters were followed for 10 weeks. A process of adaptation occurred which was divided operationally into three phases. A phase of ultrastructural disorganization occurred, succeeded by a recovery phase; their intensity and duration were functions of the dose of DCMU. A stable adaptation phase then ensued. This phase was observed in all cultures except that exposed to the highest DCMU concentration. Adapted cells from all of the DCMU cultures contained twice the protein and half the paramylon of the control cells and thus utilized the carbon source to accumulate cellular reserves with only half the efficiency of controls. DCMU affected cellular metabolism as well as photosynthesis.     

Towards a Revision of the Systematics of the Genus Chlamydomonas (Chlorophyta)

Strains of Chlamydomonas with cross-reacting sporangium wall autolysins also correspond in morphology, reproduction, development, and physiological properties. We therefore undertook a revision of the systematics of the genus based on this correlation. Isolates of Chlamydomonas available in culture collections were re-examined microscopically, preferably with respect to their reproduction and development. In particular all authentic clones, on which earlier species diagnoses had been founded, were included. Their detectable autolysins were tested crosswise in bioassays. We intend to complete and extend species diagnoses in order to separate species more precisely. - At first 7 strains with a common sporangium autolysin have been examined, four of which are the authentic cultures of the species C. aggregate Deason et Bold, C. akinetos Deason et Bold, C. applanata Pringsheim, and C. humicola Lucksch. They have been brought together under the name of the species first described, C. applanata Pringsheim, due to their apparent similarities in morphology and development. An amended species diagnosis is presented. The significance of authentic cultures as important sources of further information is discussed.     

Photoreactivating enzyme from Euglena and the control of its intracellular level

Photoreactivating (PR) enzyme activity has already been demonstrated by us in cell-free extracts of Euglena gracilis var. bacillaris Pringsheim using the Hemophilus transformation assay. This activity can also be detected in extracts using a direct non-biological assay for the photorepair of thymine dimers in DNA. PR enzyme is found in extracts of both wild-type cells and cells of an aplastidic mutant, W₃BUL, lacking detectable chloroplast DNA, indicating that the PR enzyme is neither coded nor translated exclusively in the chloroplast, but is probably coded in the nucleus and translated in the cytoplasm. Growing cultures of wild-type cells manifest a large increase in PR enzyme activity in vitro upon entering stationary phase. This correlates with the increased photoreactivability of chloroplast inheritance in vivo in stationary phase cells, previously found for Euglena, and suggests that a substantial part of the newly synthesized PR enzyme is available to repair plastid DNA. When dark-grown nondividing wild-type cells are exposed to light, there is a large increase in the specific activity of PR enzyme measured in vitro. This increase is prevented by cycloheximide but not by chloramphenicol or streptomycin, indicating that the enzyme is synthesized on 87s cytoplasmic ribosomes rather than 68s chloroplast ribosomes. Wavelengths of light effective for PR of chloroplast DNA in vivo are also effective for the light induction of PR enzyme. A brief illumination (45 min) of dark-grown nondividing wild-type cells triggers the synthesis of PR enzyme which continues in the absence of light. Growing cultures of W₃BUL also exhibit a preferential synthesis of PR enzyme in the staionary phase of growth, but the specific activity in vitro is consistently ten times higher than that of wild-type. Dark-grown non-dividing cultures of W₃BUL also show a cycloheximide-sensitive light induction of PR enzyme synthesis which, however, is dependent on the continued presence of light. The light induction of PR enzyme synthesis can be regarded as the induction of an enzyme by one of its substrates.     

Synthesis and Release of Cyclic Adenosine 3':5'-Monophosphate by Ochromonas malhamensis

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3′:5′-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [³²P]cAMP was purified from cultures supplied [³²P]phosphate. The compound was identified as [³²P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a ³²P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a ³²P compound with the chromatographic behavior of 5′-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [³H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step.     

Events surrounding the early development of euglena chloroplasts: v. Control of paramylum degradation

The degradation of the storage carbohydrate, paramylum, is induced by light in wild-type Euglena gracilis Klebs var. bacillaris Pringsheim and in a mutant, W₃BUL, which lacks detectable plastid DNA. Treatment of wild type with cycloheximide in the dark produces 60% as much paramylum breakdown as light, whereas treatment with levulinic acid in the dark yields a slightly greater response than light. Both cycloheximide and levulinic acid produce a greater paramylum breakdown in the light than they do in the dark. Treatment of W₃BUL with levulinic acid in darkness produces a larger paramylum degradation than light, with values similar to wild type in the light. Treatment of W₃BUL with cycloheximide induces paramylum degradation in darkness, and as with wild type, light is slightly stimulatory in the presence of both cycloheximide or levulinic acid. Streptomycin brings about only a very small amount of paramylum breakdown in the dark and only slightly inhibits breakdown in the light. Thus paramylum breakdown induced by light does not require the synthesis of proteins on cytoplasmic or plastid ribosomes. A model which explains these results postulates the existence of a protein which inhibits paramylum breakdown. When the synthesis of this protein is prevented either by light, cycloheximide, or by levulinic acid acting as a regulatory analog of delta amino levulinic acid, paramylum breakdown takes place. Because levulinic acid is a better inducer than light in W₃BUL, W₃BUL may not be able to form as much delta amino levulinic acid in light as wild type. The small amount of induction by streptomycin is viewed as a secondary regulatory effect attributable to interference with plastid protein synthesis which affects regulatory signals from the plastid to the rest of the cell.     

Development and Repair of Photosystem II Activity in Normal and Chloramphenicol-treated Euglena gracilis Cells

Photosystem II activity, development, and organization were studied in membranes from dark-adapted Euglena gracilis Klebs var. Z Pringsheim cells during a modulated greening process of light-dark-light cycles. The results obtained from measurements of overlapping partial photosystem II (PSII) reactions (fluorescence induction parameters, quantum yield, flash yield, and maximal rate of H₂O → 2,6-dichlorophenolindophenol [DCIP] and 1,5-diphenylcarbazide [DPC] → DCIP reactions) during these cycles indicate the formation of active PSII units in the dark. The necessity for proteins from the chloroplast translational machinery for this formation is evidenced by the inhibition of synthesis of the PSII units in chloramphenicol-treated cells. The effect of this drug, both during the dark and second light periods, can be summarized as follows: (a) disruption of the electron transfer connection to the plastoquinone pool, or decrease in the pool size; (b) loss of excitation energy transfer efficiency in the second light period; (c) impairment of the O₂ evolution appratus, as shown by comparison of the efficiency of DCP and H₂O as electron donors.

These conclusions are based on the use of a previously developed method of measurement and analysis of data (Cahen et al. 1976 Plant Physiol 58: 257-267).