Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplasts completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W₁₀BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.     

Circadian Variations in the Affinities of Nad Kinase and Nadp Phosphatase for their Substrates, Nad+ and Nadp+ in Dividing and Nondividing Cells of the Achlorophyllous Zc Mutant of Euglena Gracilis Klebs (Strain Z)

-We have previously shown that NAD kinase and NADP phosphatase activities display circadian rhythms, in the soluble (SN) and membrane-bound (P) fractions of crude extracts of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis (which displays circadian rhythmicity of cell division). We determined if changes in the affinity of NADP phosphatase and NAD kinase for their substrates, NADP⁺ and NAD⁺, were occurring by calculating the ratios 100(velocity found in Km conditions/velocity found in saturating conditions). The rationale was that if the affinity remained unchanged according to circadian time (CI), these values should always equal 50, independently of any changes in enzyme quantity; values greater than 50 should indicate increases in enzyme affinity, and values less than 50 decreases in affinity. Our results indicated that these values calculated for NADP phosphatase exhibited a complex pattern of rhythmicity, while those for NAD kinase displayed circadian variations strongly correlated with the rhythms in enzyme activity. The curves showed troughs at CT 00-04 both in dividing and nondividing cells and peaks at CT 18-20 or at CT 08-14 in cells sampled, respectively, from a dividing or a stationary culture. Such variations are indicative of changes in the kinetic properties of the enzyme, which may reflect modifications in its affinity either for effectors (such as Ca2⁺-calmodulin) or for its substrate, NAD⁺. This may be due to (i) the expression of different isoenzymes at different CTs; (ii) different posttranslational modifications of the enzyme; or (iii) concentrations of effectors varying in a circadian manner.     

Responses of the photosynthetic flagellate,Euglena gracilis,to hypergravity

Motility and orientation has been studied in the unicellular photosynthetic flagellate, Euglena gracilis, using real time image analysis capable of tracking up to 200 cells simultaneously in the slow rotating centrifuge microscope (NIZEMI) which allows one to observe the cells' swimming behavior during centrifugation accelerations between 1 g and 5 g. At 1 g the cells show a weak negative gravitaxis, which increases significantly at higher accelerations up to about 3 g. Though most cells were capable of swimming even against an acceleration of 4.5 g, the degree of gravitaxis decreased and some of the cells were passively moved downward by the acceleration force; this is true for most cells at 5 g. The velocity of cells swimming against 1 g is about 10% lower than that of cells swimming in other directions. The velocity decreases even more drastically in cells swimming against higher acceleration forces than those at 1 g. The degree of gravitactic orientation drastically decreases after short exposure to artificial UV radiation which indicates that gravitaxis may be due to an active physiological perception rather than a physical effect such as an asymmetry of the center of gravity within the cell. Offprint requests to: D.-P. Häder     

Substance P and enkephalin in transected axons of medulla and spinal cord

The accumulation of transported materials in cut axons is demonstrated by the light and electron microscopic immunocytochemical localization of substance P and enkephalin in the caudal medulla and cervical spinal cord of adult rat. Two days following unilateral knife-cuts in the caudal medulla or spinal (C2-C3) levels, substance P and enkephalin-like immunoreactivity (SPLI and ELI) are detected in lesioned axons located rostral and caudal to the transection. Rostrally, SPLI and ELI are detected in the lateral reticular region and ventrolateral fasciculus corresponding to the location of previously identified bulbospinal pathways. Caudally, previously unidentified, propriospinal pathways showing SPLI are detected in the dorsal columns and in the dorsolateral fasciculus. In contrast, ELI is found caudal to the transection only in the reticular region of the medulla. For both peptides, immunoreactivity is present throughout axons containing numerous large, dense core, and small clear vesicles. These results support the concept of both particulate and soluble modes of transport for substance P and enkephalin within axons of the central nervous system.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN-STARVED EUGLENA GRACILIS1

Growth of Euglena gracilis Z Pringsheim under photoheterotrophic conditions in a nitrogen-deprived medium resulted in progressive loss of chloroplastic material until total bleaching of the cells occurred. Biochemical analysis and ultrastructural observation of the first stages of the starvation process demonstrated an early lag phase (from 0 to 9 h) in which cells increased in size, followed by a period of cell division, apparently supported by the mobilization of some chloroplastic proteins such as the photosynthetic CO₂-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. The degradation of the enzyme started after 9 h of starvation and was preceded by a transient concentration of this protein in pyrenoidal structures. Protein nitrogen and photosynthetic pigments as well as number of chloroplasts per cell decreased during proliferation through mere distribution among daughter cells. However, after 24 h, when cell division had almost ceased, there was a slow but steady decline of photosynthetic pigments. This was paralleled by observable ultrastructural changes including progressive loss of chloroplast structure and accumulation of paramylon granules and lipid globules in the cytoplasm. These findings reinforce the role of chloroplastic materials as a nitrogen source during starvation of E. gracilis in a carbon-rich medium. The excess of ribulose-1,5-bisphosphate carboxylase/oxygenase acts as a first reservoir that, once exhausted, is superseded by the generalized disassembly of the photosynthetic structures, if the adverse environment persists more than 24 h.     

Simulation of phototaxis in the flagellateEuglena gracilis

A three-dimensional model of the flagellateEuglena gracilis was developed to simulate phototaxis and movement in space. The simulation of the phototactic behavior was compared with thein vivo behavior in order to determine the mechanism of orientation with respect to light. Phototactic behavior with respect to one light source, can be explained by the shading hypothesis as well as by a dichroic orientation of the absorbing vectors of the photoreceptor pigments. In contrast, the behavior of the cells when exposed to two perpendicular light beams is not compatible with the shading hypothesis. Likewise, the phototactic orientation of stigmaless cells cannot be accounted for on the basis of the shading hypothesis. In contrast, simulations andin vivo observations of the behavior under polarized light strongly indicate the validity of the dichroic orientation of the photoreceptor pigments.     

Algal carotenoids 52; secondary carotenoids of algae 3; carotenoids in a natural bloom of Euglena sanguinea

Quantitative carotenoid analysis of a natural bloom of Euglena sanguinea Ehrenberg revealed the presence of β,β-carotene (1% of total carotenoids), monoesters of adonirubin (3%), diesters of (3S, 3′R)-adonixanthin (13%), diesters of (3S, 3′S)-astaxanthin (75%), 19-monoester of (3R, 3′R, 6R)-loroxanthin (1%), (3R, 3′R)-diatoxanthin (6%), diadinoxanthin (1%) and neoxanthin (traces). The carotenoid content amounted to 0.7% of the dry wt. Methods employed included TLC, HPLC, VIS, MS, CD and H NMR (400 and 500 MHz). The high content of ketocarotenoids is characteristic of secondary carotenoids produced under stressed growth conditions. Previously secondary carotenoids were associated with green algae (Chlorophyceae), but have now been encountered in Euglenophyceae.     

EFFECTS OF VARIATION IN TEMPERATURE. I. ON THE BIOCHEMICAL COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

The influence of temperature on the biochemical composition of eight species of marine phytoplankton was investigated. Thalassiosira pseudonana Hasle and Heim-dal, Phaeodactylum tricornutum Bohlin and, Pavlova lutheri Droop (three of eight species studied) had minimum values of carbon and nitrogen quotas at intermediate temperatures resulting in a broad U-shaped response in quotas over the temperature range of 10 to 25°C. Protein per cell also had minimum values at intermediate temperatures for six species. For T. pseudonana, P. tricornutum, and P. lutheri, patterns of variation in carbon, nitrogen, and protein quotas as a function of temperature were similar. Over all species, lipid and carbohydrate per cell showed no consistent trends with temperature. Only chlorophyll a quotas and the carbon: chlorophyll a ratios (θ) showed consistent trends across all species. Chlorophyll a quotas were always lower at 10°C than at 25°C. Carbon: chlorophyll a ratios (θ) were always higher at 10°C than at 25°C. We suggest that although θ consistently increases at lower temperatures, the relationship between temperature and θ ranges from linear to exponential and is species specific. Accordingly, the interspecific variance in θ that results from species showing a range of possible responses to temperature increases as temperature declines and reaches a maximum at low temperatures. High photon flux densities appear to increase the potential interspecific variance in the carbon: chlorophyll a ratio and therefore exacerbate these trends.