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PubMed1984

Occurrence of Isóenzymes of Glutamine Synthetase in the Alga Chlorella kessleri.

Two forms (GS(1) and GS(2)) of glutamine synthetase have been isolated, separated by ion exchange chromatography, and partly characterized from cells of the green alga Chlorella kessleri. Both forms are present in cells grown autotrophically or heterotrophically on various nitrogen sources, but under all nutritional conditions GS(1) was found to be the major isoenzyme present (60-80%). The activity of both isoenzymes was greatest in cells grown under nitrogen-limiting conditions. Both isoenzymes have molecular weights in the range 340 to 350,000 daltons. GS(1) was found to have a greater thermostability than GS(2): GS(1) was stable at 30 degrees C while GS(2) lost 95% of its activity in 30 minutes. GS(1) was much less sensitive to thiol reactive reagents than GS(2).

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PubMed1984

Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica.

Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.

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PubMed1984

A New Type of Hexose Monophosphate Shunt in Chlorella sorokiniana: CO(2) Release from Differentially Labeled Glucose.

Using differentially labeled glucose as a substrate to probe the operation of the hexose monophosphate shunt (pentose cycle) in Chlorella sorokiniana, we found that the labeling patterns for the release of (14)CO(2) over the first 5 minutes are compatible with the operation of the recently described L-type pentose shunt. Experimentally, this L-type differs from the F-type or ;textbook' variety in that no radioactivity is obtained from C-2 labeled glucose, and the small amount derived from C-6 labeled glucose is due to a second pass of the glucose molecule (derived from the pentose cycle) through the pentose cycle.

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PubMed1984

Nitrogen Metabolism of the Marine Microalga Chlorella autotrophica.

The levels of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella autotrophica (clone 580) are strongly regulated by the nitrogen source and salt concentration of the medium. GS is present at high levels in NO(3) (-)-grown cells, and at maximum levels in nitrogen-starved cells. However, the levels of GS in these cells are somewhat decreased by increasing salinity. Cells growing on NH(4) (+) have high NADPH-GDH activity, the levels of which increase with increasing NH(4) (+) supply, while GS decreases to a very low level under these conditions. Salinity intensifies the induction of NADPH-GDH activity in NH(4) (+)-grown cells. The levels of NADH-GDH are low in this alga, but present under all growth conditions. Methionine sulfoximine (MSX) has little effect on growth and nitrogen assimilation of the alga in the presence of NH(4) (+).

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PubMed1984

Decline in Energy Reserves of Chlorella sorokiniana upon Exposure to Ozone.

Ozone exposure has been shown to increase the loss of K from Chlorella cells due to an increase in passive permeability and a depolarization of membrane potential. One factor which likely influences or can be influenced by these changes is the energy state of the cell. To study this relationship, various indicators of cell energy status were examined in the presence and absence of O(3).The active uptake of chloride and deoxyglucose is nearly completely inhibited by O(3) at a dose at which cellular death, measured by plating efficiency, is minimal. Glucose-stimulated respiration, dependent upon ATP/ADP balance, is depressed to a greater degree than endogenous respiration in ozonated cells. Total ATP and glucose-6-phosphate levels also decrease but not as rapidly, and labeled intermediates of glucose metabolism are lost.Thus, exposure to O(3) results in a depletion of the cell's energy reserves as substantiated by changes observed in processes which both utilize and generate ATP. This loss in energy reserves occurs at the same exposure level of O(3) as do the changes in passive transport properties. Thus, we cannot tell which occurs first; and the processes seem to be linked with respect to O(3) injury.

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PubMed1985

Characterization of glutamine synthetase isoforms from chlorella.

Ion-exchange chromatography of extracts derived from Chlorella sorokiniana mutant strain (oxygen resistant) yielded two separate activity peaks of glutamine synthetase (GS). GS(I) and GS(II) were purified 220- and 187-fold and have molecular weights of approximately 398,000 and 360,000, respectively. Both enzymes are composed of eight identical subunits with a subunit molecular weight of 47,000 for GS(I) and 43,000 for GS(II). The amino acid composition, catalytic, and immunological properties for both enzymes are similar.

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PubMed1985

Effect of External pH on the Internal pH of Chlorella saccharophila.

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[(14)C]oxazolidine-2,4-dione ([(14)C]DMO) or [(14)C]benzoic acid ([(14)C]BA) between the cells and the surrounding medium. [(14)C]DMO was used at external pH of 5.0 to 7.5 while [(14)C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.

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PubMed1985

Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris.

ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the V(max). Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half V(max) value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half V(max) value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn(2+) and Mg(2+) were highly sigmoidal. The activator concentration at half V(max) values for Mn(2+) and Mg(2+) were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.

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PubMed1985

Purification and Partial Characterization of Ribulose Bisphosphate Carboxylase Holoenzyme and Its Subunits from Chlorella sorokiniana and Use of Its Antigen Affinity-Purified Antibodies in Specific Immunoprecipitation and Immunoadsorption Procedures.

Chlorella sorokiniana ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified to homogeneity with yields of 35 to 40%. Molecular weights of the holoenzyme and its large subunit (LS) and small subunit (SS) were estimated to be 562,000, 55,000, and 15,800, respectively. Amino acid compositions of LS from C. sorokiniana and spinach were similar, whereas the compositions of their SS were very different. Antisera prepared against holoenzyme, LS, and SS were purified by antigen-affinity column chromatography. Purified anti-holoenzyme immunoglobulin G (IgG) and anti-LS IgG cross-reacted with holoenzyme and LS but not with SS. Anti-SS IgG reacted neither with holoenzyme nor with LS. Because purified anti-holoenzyme IgG or the anti-LS IgG inhibited RuBPCase activity, antibody preparations were titered by the amount of (35)S-labeled RuBPCase immunoprecipitated. Approximately 40% of the total RuBPCase activity in cell homogenates was tightly particulate-bound and was solubilized with 0.5% Nonidet P-40 without inhibition of enzyme activity. Direct-immunoprecipitation and indirect-immunoadsorption procedures, with affinity-purified anti-holoenzyme IgG, gave specific and quantitative recovery of (35)S-labeled RuBPCase from cell extracts containing Nonidet P-40. Affinity-purified anti-LS IgG and anti-SS IgG were used to immunoprecipitate either the LS or SS antigens synthesized in vitro in a mRNA-dependent in vitro translation assay system. Rocket immunoelectrophoresis was used to quantify as little as 50 nanograms of RuBPCase antigen in cell extracts.

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PubMed1985

Comparison of Patterns of Accumulation of Ribulose Bisphosphate Carboxylase Antigen and Catalytic Activity and Measurement of Antigen Half-Life during the Cell Cycle of Chlorella sorokiniana.

By use of specific immunochemical procedures, ribulose-1,5-bisphosphate carboxylase (RuBPCase), antigen and catalytic activity were shown to have coincident step-patterns of accumulation during the cell cycle of Chlorella sorokiniana. Pulse-chase studies, employing radioactive sulfate, were performed during the period of rapid accumulation of enzyme activity and during the period of constant enzyme activity in the cell cycle. No degradation of RuBPCase antigen could be detected during either of these cell cycle periods. Thus, the step-pattern of accumulation of RuBPCase activity resulted from periodic synthesis of an enzyme that was stable under steady-state cell cycle conditions. Although inhibition of protein synthesis by cycloheximide, at different times in the cell cycle in the light, resulted in rapid decay of RuBPCase activity, this loss in activity occurred without detectable loss in enzyme antigen. When synchronous cells were placed into the dark, to slow the rate of protein synthesis in the absence of cycloheximide, the levels of enzyme antigen and activity decreased by 30 and 50%, respectively, during the 10-hour dark period. Thus, in C. sorokiniana changes in RuBPCase activity do not necessarily reflect parallel changes in enzyme antigen, particularly when cell growth is perturbed by changes from steady-state cultural conditions.

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PubMed1986

A Circadian Rhythm in the Number of Daughter Cells in Synchronous Chlorella fusca var vacuolata.

Chlorella fusca var vacuolata cells were transferred to continuous darkness or weak light (0.07 watts per square meter) (both were called waiting time, WT) after a 12-hour light and 12-hour dark schedule. A daily dilution is performed at the end of the light/dark schedule, resulting in always the same average production of 18 autospores per mother cell. After 12 and 24 hours of WT in darkness, the production of autospores in a subsequent light/dark schedule was 50 and 100%, respectively. If the WT was performed in weak light (0.07 watts per square meter) the lowest production was obtained, independently of the length of WT. However, an interruption of this weak light by dark pulses (3 hours) increased the autospore production by an amount that depends upon the phase of the circadian rhythm, and varied up to 70% of the control (WT in permanent darkness). If the WT (total darkness) was interrupted by light pulses of 0.5 hour (white, same as used for growth), a phase response curve of productivity resulted. Pulses between the 12th and 18th hour of WT in darkness gave a 3-hour delay of maximum; later on pulses shifted the maximum autospore production 3 hours ahead.

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PubMed1986

Changes of Ribulose Bisphosphate Carboxylase/Oxygenase Content, Ribulose Bisphosphate Concentration, and Photosynthetic Activity during Adaptation of High-CO(2) Grown Cells to Low-CO(2) Conditions in Chlorella pyrenoidosa.

Changes of some photosynthetic properties of high-CO(2) grown cells of Chlorella pyrenoidosa during adaptation to low-CO(2) conditions have been investigated. The K(m) value of photosynthesis of the high-CO(2) grown cells for dissolved inorganic carbon was 3.3 millimolar and decreased to 25 to 30 micromolar within 4 hours after transferring to air. In the presence of saturating CO(2) concentrations the photosynthetic activity of the high-CO(2) grown cells was 1.5 times as high as that of the low-CO(2) grown cells. There was a significant rise of the photosynthetic activity during adaptation of the high-CO(2) grown cells to air, followed by a steady decrease. The activity of ribulose 1,5-bisphosphate carboxylase/oxygenase in both the high- and low-CO(2) grown cells was close to the photosynthetic activity of the cells. The concentration of ribulose 1,5-bisphosphate (RuBP) was higher in the low-CO(2) adapting and low-CO(2) grown cells than in the high-CO(2) grown cells regardless of the photosynthetic rate. This seems to be due to an increased RuBP regeneration activity during adaptation followed by maintenance of the new higher concentration. The RuBP level always exceeded the concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase RuBP binding sites in both the high- and low-CO(2) grown cells at any dissolved inorganic carbon concentration.

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PubMed1986

Evidence for Cyclic Electron Flow around Photosystem II in Chlorella pyrenoidosa.

Electron flow around photosystem II was investigated in Chlorella pyrenoidosa. Using a bare platinum O(2) electrode, simultaneous measuremnts were made of steady-state photosynthesis in continuous light, the yield of oxygen (Y(o(2) )) produced by a superimposed saturating xenon flash, and the change in fluorescence yield of a weak flash triggered before and 70 microseconds after the saturating flash. Throughout most of the continuous photosynthesis-irradiance curve, normalized O(2) flash yields (Y(o(2) )/Y(o(2)max)) and normalized variable fluorescence yields (Deltaphi/Deltaphi') were linearly correlated with a slope of 1.0. As photosynthetic rates reached light saturation, however, the variable fluorescence yields remained relatively constant while O(2) flash yields decreased. These results strongly suggest that there is a cyclic electron flow around photosystem II in unpoisoned intact cells at light saturation and supraoptimal light intensities.

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PubMed1986

Effect of Different Carbon Sources on the Ammonium Induction of Different Forms of NADP-Specific Glutamate Dehydrogenase in Chlorella sorokiniana Cells Cultured in the Light and Dark.

The ammonium induction of the chloroplast-localized NADP-specific glutamate dehydrogenase (NADP-GDH) was shown not to be a light-dependent process per se in Chlorella sorokiniana. In the dark without exogenous organic substrates, the cells synthesized low levels of fully active NADP-GDH, provided endogenous starch reserves had not been depleted. When cells were supplied with exogenous acetate, the rate of induction of NADP-GDH activity per milliliter of culture in the dark was equal to or slightly greater than the rate observed under photosynthetic conditions without an organic carbon source. Glucose supported only a low rate of induction of NADP-GDH activity in the dark. Both acetate and glucose inhibited induction of enzyme activity in the light. The NADP-GDH holoenzyme had at least 7 different electrophoretic forms. These forms differed in net charge and/or molecular weight. Their difference in molecular weight was due to the presence of 2 subunits with similar antigenic properties but different molecular weights (M(r) = 55,500 and 53,000; alpha-and beta-subunits, respectively). Depending upon the cultural conditions and length of the induction period, a wide variation was observed in the alpha:beta subunit ratio and in the numbers and sizes of the NADP-GDH holoenzymes.

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PubMed1986

Specific Polysome Immunoadsorption to Purify an Ammonium-Inducible Glutamate Dehydrogenase mRNA from Chlorella sorokiniana and Synthesis of Full Length Double-Stranded cDNA from the Purified mRNA.

A specific polysome immunoadsorption procedure, employing soluble rabbit anti-NADP-GDH IgG and sheep anti-rabbit IgG covalently-linked to an insoluble cellulose matrix, was used to immunoselect polysomes translating mRNA for a chloroplastic ammonium-inducible NADP-GDH in fully induced cells of Chlorella sorokiniana. The immunoselected polysomes were dissociated, and the NADP-GDH mRNA was recovered by oligo (dT)cellulose chromatography. The translatable NADP-GDH mRNA was estimated to be 0.07 and 90% of the total polysomal poly(A)(+)RNA before and after immunoselection of the polysomes, respectively. The immunoadsorption procedure resulted in an 83% recovery and 1,291-fold purification of translatable NADP-GDH mRNA. In vitro translation of the immunoselected poly(A)(+)RNA yielded a single radioactive protein (on sodium dodecyl sufate polyacrylamide gels) with a molecular weight of 58,500, i.e. size of the putative precursor-protein of the NADP-GDH subunit in the holoenzyme in fully induced cells. The purified NADP-GDH mRNA was used for synthesis of a high proportion of nearly full-length single-stranded cDNA and double-stranded cDNA molecules.

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PubMed1987

Purification and Partial Kinetic and Physical Characterization of Two Chloroplast-Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels.

Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either alpha- or beta-subunits with molecular weights of 55,500 or 53,000, respectively. The alpha-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the beta-isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar). Purification of isoenzymes was achieved by (NH(4))(2)SO(4) fractionation, gel-filtration, anion-exchange fast protein liquid chromatography, and affinity chromatography. The alpha- and beta-isoenzymes were separated by their differential binding to Type 4 nicotinamide adenine dinucleotide phosphate-Sepharose. Both isoenzymes bound to an antibody affinity column to which purified antibody (prepared against beta-isoenzyme) was covalently attached. Peptide mapping of the subunits showed them to have a high degree of sequence homology. Both subunits were synthesized in vitro from precursor protein(s) with a molecular weight of 58,500. Although the subunits have similar chemical, physical, and antigenic properties, their holoenzymes have strikingly different ammonium K(m) values. The ammonium K(m) of the beta-isoenzyme remained constant at approximately 75 millimolar, whereas this K(m) of the alpha-isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration.

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PubMed1987

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells.

The kinetics of accumulation (per milliliter of culture) of the alpha- and beta- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The beta-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the alpha-subunit holoenzyme(s). After 120 minutes, the alpha-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using (35)SO(4) and immunochemical procedures, the rate of synthesis of the alpha-subunit was shown to be greater than the beta-subunit during the first 80 minutes of induction. The alpha- and beta-subunits had different rates of degradation during the induction period (t((1/2)) = 50 versus 150 minutes, respectively) and during the deinduction period (t((1/2)) = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

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PubMed1987

Correction of Freezing Artifacts in the Application of Electron Paramagnetic Resonance for Quantification of the Reduction of Ferric Hydroxamate by Chlorella vulgaris.

Electron paramagnetic resonance has been used to quantitate reduction of ferric ion bound to hydroxamate siderophores by iron-stressed fungi (DJ Ecker et al. 1982 J Biol Chem 257:8623-8626) and by iron-stressed Chlorella vulgaris (ATCC 11468) (FCT Allnutt, WD Bonner 1984 J Plant Nutr 7: 427-435). Application of this technique to quantitation of bound ferric ion has been questioned due to the possibility of freezing artifacts. Dipole-dipole interaction which resulted in broadening of the g = 4.3 signal was observed. Quantitation was affected when peak to trough amplitude was applied to quantitate the ferric ion but could be corrected by the application of internal standards and maintenance of constant cell content in the samples.

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