THE EFFECT OF SODIUM CHLORIDE ON CARBONIC ANHYDRASE ACTIVITY IN MARINE MICROALGAE1

The effects of NaCl on the internal and external carbonic anhydrase (CA) activity of several marine microalgae were studied. Unlike freshwater microalgae in which CA activity is generally inhibited by NaCl, marine microalgae exhibited considerable species-dependent variation when exposed to NaCl. CA activity in Phaeodactylum tricornutum, a diatom, was inhibited, whereas it was activated in the coccolithophorid Pleurochrysis carterae. CA activity in the chlorophyte Dunaliella primolecta was not significantly affected by NaCl. In Dunaliella salina and Dunaliella parva, NaCl inhibited external CA without affecting the internal activity, whereas in Chlorella vulgaris C-133 and Dunaliella peircei only the internal CA was inhibited. Internal CA of Dunaliella tertiolecta was not affected by NaCl, but the external enzyme was significantly enhanced. Salt substitution experiments revealed that chloride (Cl^-) is the ion affecting CA activity; the effect of Cl^- can be replaced by bromide ion. Cl^- affects external CA activity while also affecting the apparent affinity for inorganic carbon during photosynthesis. Microalgae whose internal CA activity was enhanced by Cl^- showed higher intracellular Cl^- concentrations than those species that were inhibited.     

Sensitivity of the surface coat of the halotolerant green alga Dunaliella parva (Volvocales, Chlorophyceae) to lysozyme

The surface coat of Dunaliella parva Lerche was investigated using several techniques. Degradation by several cell lytic enzymes and ultrastructural observation revealed that D. parva has a specialized cell surface structure containing a glycoprotein that is sensitive not only to proteinases but also to lysozyme. This sensitivity was also demonstrated by electrophoresis of the cells and measurement of released glycerol after enzyme treatment. Immunochemical labeling indicated that the surface glycoprotein of D. parva is analogous to pepti-doglycan.     

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.     

Isolation of disproportionating enzyme from halotolerant microalga Dunaliella parva (Volvocales, Chlorophyceae)

Starch metabolism in Dunaliella parva Lerche is regulated by the osmolarity of the surrounding solute. Two isozymes showing amylolytic activity were obtained after purification by gel filtration chromatography. The isozymes show disproportionating activity (D‐enzyme) that is specific for malto‐oligosaccharides as substrate. Properties of the D‐enzyme in D. parva are similar to those in higher plants. The activity of the D‐enzyme is also found in various Dunaliella and Chlamydomonas, indicating that the D‐enzyme is also important in the starch metabolism in algae.     

THE GLYCEROL PERMEABILITY OF THE PLASMALEMMA OF THE HALOTOLERANT GREEN ALGA DUNALIELLA PARVA (VOLVOCALES)1

The glycerol permeability of the plasmalemma of the green alga Dunaliella parva Lerche was investigated by efflux studies with labelled glycerol, by enzymatic determination of glycerol leakage, and the determination of the reflection coefficient from osmotically induced volume changes (zero flow method). All results indicate that the plasmalemma of D. parva does not exhibit a special low permeability towards glycerol as would be expected from a glycerol accumulating alga. Rather, significant amounts of glycerol diffuse continuously into the medium following the glycerol concentration gradient between the cells and the medium. Efflux rates vary between 0.1 and 2 μmoles glycerol·mg^?1 chlorophyll·h^?1 depending on the external NaCl concentration. After one day up to 25% of the total glycerol of the algal suspension was found in the medium. Within 10 days this value can increase to 60%, depending on the growth constant of the culture. The reflection coefficient σ was determined to be 0.87, the permeability coefficient 2800 × 10^?11 m·sec^?1. To maintain a proper endogenous glycerol level corresponding to the external osmotic pressure, glycerol efflux in D. parva has to be balanced by a continuous synthesis of glycerol. D. parva follows the strategy of “glycerol efflux tolerance” instead of “glycerol efflux avoidance”. The alga has to pay the energetic costs of this strategy of tolerance.     

A novel bioreactor for immobilized phototrophs

A novel configuration of photobioreactor is described in which filaments of alginate containing immobilized cells of a leaky mutant of Dunaliella parva are wound round a central light well which is located within a glass outer chamber so that a liquid medium is caused to flow in the annular space between the outside chamber and the alginate filaments. Glycerol production by D. parva was maintained for 700 h and the highest concentration of glycerol attained was approx. 12 mg l⁻¹.     

ADAPTATION OF THE UNICELLULAR ALGA DUNALIELLA PARVA TO A SALINE ENVIRONMENT1

The holophilic alga Dunaliella parva produces glycerol as a major product of photosynthetic ¹⁴CO₂ incorporation and accumulates very large amounts of intracellular glycerol. A method was adopted for the determination of the cell water space based on the distribution of ¹⁴C sorbitol and ³H₂O between the cells and the medium. Using these measurements the internal concentration of glycerol was found to be isoomotic with that of the medium over a broad range of 0.6 to 2.1 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular water content immediately varied so as to keep an osmotic equilibrium between the cells and the medium. During the following 90 min under metabolic conditions, glycerol content changed until a new level was reached. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. Determination of the glycerol sensitivity of enzymic and photosynthetic reactions of cell-free preparations from D. parva showed a broad range of tolerance to high concentrations of glycerol. These results indicate that osmoregulation in Dunaliella depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration. A proposed scheme of glycerol synthesis in Dunaliella is suggested.     

The salt relations of marine and halophilic species of the unicellular green alga,Dunaliella

1. Comparisons were made of the effects of salt on the exponential growth rates of two unicellular algae,Dunaliella tertiolecta (marine) andDunaliella viridis (halophilic).
2. The algae contained glycerol in amounts which varied directly with the salt concentration of the growth media. The highest measured glycerol content ofD. tertiolecta was approximately equivalent to 1.4 molal and occurred in algae grown in 1.36 M sodium chloride. The highest glycerol content measured inD. viridis was approximately equivalent to 4.4 molal and occurred in algae grown in 4.25 M sodium chloride. Lower concentrations of free glucose, which varied inversely with extracellular salt concentration, were also detected.
3. It is inferred that Na⁺ is effectively excluded from the two algae. There was some evidence of a moderate uptake of K⁺.
4. Comparisons were made of erude preparations of the glucose-6-phosphate dehydrogenase and an NADP-specific glycerol dehydrogenase from each species and of the effects of salt and glycerol on the activities of these enzymes. It is concluded that the different salt tolerances of the two algae cannot be explained by generalized differences between their enzyme proteins.
5. Although intracellular glycerol must necessarily contribute to the osmotic status of the algae, its primary function in influencing their salt relations is considered to be that of a compatible solute, whereby glycerol maintains enzyme activity under conditions of high extracellular salt concentration and hence low (thermodynamic) water activity.

     

A mutant ofDunaliella parva CCAP 19/9 leaking large amounts of glycerol into the medium

A mutant of the halotolerant green algaDunaliella parva, which leaks large amounts of intracellular glycerol into the surrounding medium, was isolated. The mutant has potential applications in the commercial production of glycerol on a large scale since there is no need to extract glycerol from the cells. The mutant was compared with the wild type and it was found that, despite the leakage of glycerol, the mutant showed the same growth rate as the wild type. However, when the rates of oxygen evolution and uptake and intracellular starch content between mutant and wild type were compared at high salinity, considerable differences were found.     

The salt relations of Dunaliella

Dunaliella tertiolecta (marine) and D. viridis (halophilic) were each trained by serial transfer to growth at salt concentrations previously regarded as the other's domain. D. viridis then had a salt optimum at 1.0–1.5 M sodium chloride whereas that for D. tertiolecta was less than 0–2 M. Nevertheless D. tertiolecta grew faster than the halophil at all salt concentrations up to 3.5 M, the highest at which they were compared.Both species accumulate glycerol, which is necessary for growth at elevated salinities and which responds in its content to water activity (a _w ) rather than specifically to salt concentration. Variation in glycerol content is a metabolic process which occurs in the dark from accumulated starch as well as photosynthetically. Regulation of glycerol content by a _w does not require protein synthesis. The NADP-specific glycerol dehydrogenase of each of the algae is likely to be directly involved in the regulation of glycerol content. Kinetic studies, together with those described in an earlier publication, show that the enzyme has regulatory properties, and that both glycerol and dihydroxyacetone act as effectors as well as reactants. A mechanism of the reaction is tentatively proposed.     

Isolation, Characterization, and Partial Purification of a Reduced Nicotinamide Adenine Dinucleotide Phosphate-dependent Dihydroxyacetone Reductase from the Halophilic Alga Dunaliella parva

An NADP⁺-dependent dihydroxyacetone reductase, which catalyzes specifically the reduction of dihydroxyacetone to glycerol, has been isolated from the halophilic alga Dunaliella parva. The enzyme has been purified about 220-fold. It has a molecular weight of about 65,000 and is highly specific for NADPH. The pH optima for dihydroxyacetone reduction and for glycerol oxidation are 7.5 and 9.2, respectively. The enzyme has a very narrow substrate specificity and will not catalyze the reduction of glyceraldehyde or dihydroxyacetone phosphate. It is suggested that this enzyme functions physiologically as a dihydroxyacetone reductase in the path of glycerol synthesis and accumulation in Dunaliella.     

Glycerol Dehydrogenase from Cellulomonas sp. NT3060: Purification and Characterization

NAD⁺-dependent glycerol dehydrogenase from Cellulomonas sp. NT3060 was purified by a procedure of 10 steps involving crystallization. Dihydroxyacetone was identified as the oxidation product of glycerol with the enzyme. The purified enzyme did not lose activity on heating below 60°C. The enzyme oxidized other alcohols such as 1,2-propanediol, 2,3-butanediol and glycerol-α-monochlorohydrin, beside glycerol. The enzyme activity was inhibited by p-chloromercuribenzoate, Zn²⁺, Cu²⁺ and Cd²⁺. Oxidation of glyberol was activated by Na⁺ and reduction of dihydroxyacetone was activated by K⁺ at pH 7.5.     

Photosynthetic Activities of the Halophilic Alga Dunaliella parva

Dunaliella parva, a unicellular halophilic alga, was found to evolve oxygen photosynthetically only in the presence of a high osmolar concentration. Cell free preparations were obtained by placing the cells in a medium of low osmolarity. The fragments obtained showed a high photoreducing and photophosphorylating activity except for their inability to catalyze all ferredoxin dependent photoreactions. Placing the cells in a medium of intermediate osmolarity produced a “chloroplast” preparation which maintained some capacity for O₂ evolution and CO₂ fixation, while possessing the ability to catalyze the photoinduced reduction of ferricyanide. Enzymic and photosynthetic reactions of cell-free preparations from D. parva were inhibited, rather than stimulated, by the salt concentration optimal for growth. These results were interpreted as indicating the existence of a steep NaCl gradient in vivo between the medium and the cell compartments which are not permeable to salt.     

Effects of aluminum chloride on the kinetics of rat cortex synaptosomal ATP diphosphohydrolase 9EC 3.6.1.5)

Aluminum chloride (AlCl₃), a neurotoxic compound, inhibited ATP diphosphohydrolase activity of synaptosomes obtained from cerebral cortex of adult rats. The metal ion significantly inhibited ATPase and ADPase activities of the enzyme at all concentrations tested in vitro (0.01, 0.05, 0.5, 5 and 10 mM) in the presence of 1.5 mM calcium. When tested in the absence of Ca²⁺, and with increasing amounts of Al³⁺, enzyme activity remained below basal levels, suggesting that the trivalent cation Al³⁺ is not a substitute for the divalent cation Ca²⁺ in ATP-Ca²⁺ and ADP-Ca²⁺ complexes. The Al³⁺ inhibition was competitive with respect to Ca²⁺. The enzyme inhibition was reversed by the addition of deferoxamine (DFO). NaF significantly inhibited ATP diphosphohydrolase activity, and this inhibition was reversed by the addition of Ca²⁺ to the medium. Such inhibition was not potentiated by AlF₄, which is an inhibitor of cation-transport ATPases.     

The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva

Dunaliella parva, a green halophilic alga, was found to accumulate very large amounts of intracellular glycerol. Through measurements of the intracellular volume the internal concentration of glycerol was calculated and found to be around 2.1 m in cells cultured in 1.5 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular glycerol varied accordingly, reaching its new osmotic equilibrium after about 90 minutes. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. The above results indicate the existence of a new type of algal osmoregulation, in which the osmotic balance depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration.     

Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay

An enzyme assay was developed to determine the activities of methyl chloride dehalogenase and O-demethylase of the homoacetogen strain MC. The formation of methyl tetrahydrofolate from tetrahydrofolate and methyl chloride or from tetrahydrofolate and vanillate was coupled to the oxidation of methyl tetrahydrofolate to methylene tetrahydrofolate mediated by methylene tetrahydrofolate reductase purified from Peptostreptococcus productus (strain Marburg) and to the subsequent oxidation of methylene tetrahydrofolate to methenyl tetrahydrofolate catalyzed by methylene tetrahydrofolate dehydrogenase purified from the same organism. To drive the endergonic methyl tetrahydrofolate oxidation with NAD⁺ as an electron acceptor, the NADH formed in this reaction was reoxidized in the exergonic lactate dehydrogenase reaction. The formation of NADPH and methenyl tetrahydrofolate in the methylene tetrahydrofolate dehydrogenase reaction was followed photometrically at 350 nm; ε₃₅₀ was about 29.5 mM^–1cm^–1 (pH 6.5). Using the coupled enzyme assay, the cofactor requirements, the apparent kinetic parameters, the pH and temperature optima of both enzymes, and the effect of inhibitors were determined. The activity of methyl chloride dehalogenase and of O-demethylase was dependent on the presence of ATP; arsenate severely inhibited both enzyme activities in the absence of ATP. The coupled enzyme assay described allows purification and characterization of methyl chloride dehalogenase and O-demethylase and is also appropriate for the enzymatic determination of methyl tetrahydrofolate. Received: 2 August 1995 / Accepted: 28 September 1995     

Involvement of the Plasma Membrane ATPase in the Osmoregulatory Mechanism of the Alga Dunaliella salina

The unicellular halotolerant alga Dunaliella salina recovers normally from a hypertonic shock even when suspended in NaCl and buffer only. Furthermore, addition of Cu²⁺, valinomycin and KCl, or permeable ions such as methyltriphenylphosphonium or thiocyanate, do not affect the recovery. However, treatment with two specific inhibitors of the plasma membrane adenosine triphosphatase (ATPase), diethylstilbestrol, or vanadate, fully inhibit the recovery. The inhibition is manifested by the inability of the cells to both synthesize glycerol and return to their original volume. The inhibitions are nonlethal, reversible and equally effective in the dark or the light. Since the plasma membrane ATPase is the only enzyme known to be inhibited by both diethylstilbestrol and vanadate, it is concluded that its activity is essential for the recovery of Dunaliella from a hypertonic shock. Mechanisms by which the plasma membrane ATPase may participate in the activation of glycerol production in the algae are discussed.     

Determination of Volume of Dunaliella Cells by Lithium Dilution Measurements and Derivation of Internal Solute Concentrations

A method has been developed to measure the cell volume of theunicellular green alga Dunaliella parva 19/9 using Li⁺ measurementsonly. Concentrations of internal solutes can also be calculatedif they are assayed in the same samples as Li⁺. We found thatD. parva cells grown in 0.4 kmol m^–3 NaCl have an averageaqueous cell volume of 65.1 ?2.9 µm³, a K⁺ concentrationof 126?6 mol m^–3, a Na⁺ concentration of 11?11 mol m^–3and a glycerol concentration of 615?27 mol m–3 (n= 12).Algae grown in 1.5 kmol m^–3 NaCl have an average aqueouscell volume of 131 ?7.5 µm³, a K⁺ concentration of 109?4mol m^–3, a Na⁺ concentration of 10?39 mol m^–3 anda glycerol concentration of 1 425?59 mol m^–3 (n = 12).These results indicate that D. parva cells adapted to high salinitieshave larger cell volumes than those adapted to lower salinities.However, there is no evidence for a significant difference ininternal Na⁺ concentration, despite the almost 4-fold differencein the concentration of external NaCl. The intracellular glycerolconcentration alone accounts for 65% and 54%, respectively,of the osmotic balance in low and high salt grown cells. Key words: Dunaliella, cell volume, intracellular solutes     

Osmoregulatory isoform of dihydroxyacetone phosphate reductase from Dunaliella tertiolecta: purification and characterization

The osmoregulatory isoform of dihydroxyacetone phosphate (DHAP) reductase (Osm-DHAPR) is an enzyme unique to Dunaliella, photosynthetic unicellular green algae adapted to extreme environments. This is the first report of purification of an isoform of DHAP reductase from Dunaliella, specifically the osmoregulatory isoform that is involved in the synthesis of free glycerol for osmoregulation in extreme environments, such as high salinity. The Osm-DHAPR is cold labile, inactivated by ammonium sulfate, forms a strong complex with Rubisco, and is unstable in the absence of glycerol. These difficulties have been addressed, and a four-step procedure has been developed to purify the Osm-DHAPR from Dunaliella tertiolecta: precipitation of Rubisco by polyethylene glycol, followed by successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. Yield of the purified enzyme was 3.6%, with a specific activity of 938 micromol.min-1.mg-1 of protein and a subunit molecular mass of approximately 38 kDa. A maximum specific activity of 2580 micromol.min-1.mg-1 of protein could be achieved by assay with 150 mM NaCl. The Osm-DHAPR had little preference for NADH or NADPH, but it is highly specific for DHAP. Other metabolites of glycolysis, the tricarboxylic acid cycle, and the C3 reductive photosynthetic carbon cycle were not reduced by the enzyme. The purified enzyme was stimulated three-fold by 150 to 250 mM NaCl/KCl and by 25 mM MgCl2. Detergents, lipids, or long-chain acyl CoA derivatives, all of which inhibited the chloroplastic glyceride form of DHAP reductase, did not affect the activity of Osm-DHAPR. The Osm-DHAPR has different properties than the other chloroplastic isoform of DHAP reductase from plants and algae for glycerol phosphate formation and triglyceride synthesis.