Plasma membrane electron transport coupled to Na(+) extrusion in the halotolerant alga Dunaliella.

The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na(+)](in) at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na(+). In this work we examined the possibility that Na(+) export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na(+) extrusion was induced in Dunaliella cells by elevation of intracellular Na(+) with Na(+)-specific ionophores. Elevation of intracellular Na(+) was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na(+) by inhibition of Na(+) extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na(+) content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na(+) extrusion in Dunaliella plasma membrane. We propose that redox-driven Na(+) extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.

Light-harvesting complex II pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella.

Like higher plants, unicellular green algae of the genus Dunaliella respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light-inducible proteins (Elips) in plants. Earlier studies indicated that Cbr was associated with the light-harvesting complex of photosystem II (LHCII) and suggested it acted as a zeaxanthin-binding protein and fulfilled a photo-protective function (Levy et al. 1993, J. Biol. Chem. 268: 20892-20896). To characterize the protein-pigment subcomplexes containing Cbr in greater detail than attained so far, thylakoid membranes from Dunaliella salina grown in high light or normal light were solubilized with dodecyl maltoside and fractionated by isoelectric-focusing. Analysis of the resolved LHCII subcomplexes indicated preferred associations among the four LHCIIb polypeptides and between them and Cbr: subcomplexes including Cbr contained one or two of the more acidic of the four LHCIIb polypeptides as well as large amounts of lutein and zeaxanthin relative to chlorophyll a/b. After sucrose gradient centrifugation, Cbr free of LHCIIb polypeptides was detected together with released pigments; this Cbr possibly originated in subcomplexes dissociated in the course of the analysis. These results agree with the conclusion that Cbr is part of the network of LHCIIb protein-pigment complexes and suggest that the role played by Cbr involves the organization and/or stabilization of assemblies highly enriched in zeaxanthin and lutein. Such assemblies may function to protect PSII from photodamage due to overexcitation.

Differential responses to different light spectral ranges of violaxanthin de-epoxidation and accumulation of Cbr, an algal homologue of plant early light inducible proteins, in two strains of Dunaliella.

Unicellular green algae of the genus Dunaliella, similar to higher plants, respond to light stress by enhanced de-epoxidation of violaxanthin and accumulation of Cbr, a protein homologous to early light inducible proteins (Elips) in plants. These proteins belong to the superfamily of chlorophyll a/b binding proteins. Two Dunaliella strains, D. bardawil and D. salina, were compared for these two responses under light in the UVA, blue, green and red spectral ranges. In D. bardawil, the two stress responses were similarly induced under UVA, blue or red light and to a lesser extent under green light. In D. salina, a similar spectral range dependence was exhibited for violaxanthin de-epoxidation. However, Cbr accumulated only under UVA or blue light but not under green or red light. A strong synergistic effect of a low dose of blue light superimposed on red light resulted in Cbr accumulation. These results reveal strain-specific differences in spectral range requirements of the two light-stress responses. In the two strains, violaxanthin de-epoxidation is triggered under photosynthetically-active spectral ranges but at least in D. salina, Cbr accumulation appears to require a specific light signal additionally to a signal(s) generated by light stress.

Iron uptake by the halotolerant alga Dunaliella is mediated by a plasma membrane transferrin.

A 150-kDa transferrin-like protein (Ttf) is associated with the plasma membrane of the halotolerant unicellular alga Dunaliella salina (Fisher, M., Gokhman, I., Pick, U., and Zamir, A. (1997) J. Biol. Chem. 272, 1565-1570). The Ttf level rises with medium salinity or upon iron depletion. Evidence that Ttf is involved in iron uptake by Dunaliella is presented here. Algal iron uptake exhibits characteristics resembling those of animal transferrins: high specificity and affinity for Fe3+ ions, strict dependence on carbonate/bicarbonate ions, and very low activity in acidic pH. Reducing the level of Ttf by mild proteolysis of whole cells is accompanied by lowered uptake activity. Conversely, accumulation of high levels of Ttf is correlated with an enhancement of iron uptake. Kinetically, iron uptake consists of two steps: an energy-independent binding of iron to the cell surface and an energy-dependent internalization. Salinities as high as 3.5 M NaCl do not inhibit iron uptake or decrease the apparent affinity for Fe3+ ions, implying that Ttf activity is not affected by high salt. These results indicate that transferrins, hitherto identified only in animals, are present and function in iron transport also in plant systems.

Involvement of Thylakoid Overenergization in Tentoxin-Induced Chlorosis in Nicotiana spp.

The purpose of this work was to clarify the mechanism of tentoxin-induced chlorosis in Nicotiana spp. seedlings. We found that chlorosis does not correlate with the inhibition of chloroplast ATP synthesis in vivo, since it occurs at tentoxin concentrations far higher than that required for the inhibition of photophosphorylation measured in the same seedlings. However, tentoxin-induced chlorosis does correlate with in vivo overenergization of thylakoids. We show that tentoxin induces overenergization in intact plants and isolated thylakoids, probably via multiple interactions with ATP synthase. Furthermore, gramicidin D, a protonophore that relieves overenergization, also relieves chlorosis. Two lines of evidence suggest that reactive oxygen species may be involved in the process of chlorosis: ascorbate, a quencher of oxygen radicals, significantly protects against chlorosis, whereas transgenic Nicotiana spp. mutants overexpressing chloroplast superoxide dismutase are partially resistant to tentoxin-induced chlorosis. It is proposed that chlorosis in developing seedlings results from overenergization of thylakoids, which leads to the generation of oxygen radicals.

A structurally novel transferrin-like protein accumulates in the plasma membrane of the unicellular green alga Dunaliella salina grown in high salinities.

The alga Dunaliella salina is outstanding is its ability to withstand extremely high salinities. To uncover mechanisms underlying salt tolerance, a search was carried out for salt-induced proteins. The level of a plasma membrane 150-kDa protein, p150, was found to increase with rising external salinity (Sadka, A., Himmelhoch, S., and Zamir, A. (1991) Plant Physiol. 95, 822-831). Based on its cDNA-deduced sequence, p150 belongs to the transferrin family of proteins so far identified only in animals. This, to our best knowledge, is the first demonstration of a transferrin-like protein in a photosynthetic organism. Unlike animal transferrins, p150 contains three, rather than two, internal repeats and a COOH-terminal extension including an acidic amino acid cluster. In intact cells p150 is degraded by Pronase, indicating that the protein is extracellularly exposed. The relationship of p150 to iron uptake is supported by the induction of the protein in iron-deficient media and by its radioactive labeling in cells grown with 59Fe. Accumulation of p150 is transcriptionally regulated. It is proposed that p150 acts in iron uptake other than by receptor-mediated endocytosis and that its induction permits the cells to overcome a possible limitation in iron availability under high salinities.

Modification of Sulfhydryl Groups in the [gamma]-Subunit of Chloroplast-Coupling Factor 1 Affects the Proton Slip through the ATP Synthase.

A large proton leak not coupled to ATP synthesis (slip) occurs at alkaline pH through the chloroplast ATP synthase (Y. Evron, M. Avron [1990] Biochim Biophys Acta 1019: 115-120). The involvement of the ATP synthase [gamma]-subunit in the regulation of proton conductance was analyzed by measuring the effect of thiolalkylating agents on proton slip. Alkylation by N-ethylmaleimide of [gamma]-cysteine (Cys)-89, which is exposed upon energization of thylakoids, increases the slip only at alkaline pH. The slip is partially suppressed by low concentrations of adenine nucleotides and is completely eliminated by venturicidin, a blocker of the hydrophobic polypeptide complex of the chloroplast ATP synthase (CF0). Conversely, cross-linking of [gamma]-Cys-89 with [gamma]-Cys-322 renders the ATP synthase leaky to protons and insensitive to ATP also at neutral pH. The accessibility of [gamma]-Cys-89 to alkylation by fluorescein maleimide is completely suppressed by N,N-dicyclohexylcarbodiimide and by venturicidin, which block proton conductance through CF0 and increase the pH gradient. These results suggest that the [gamma]-subunit has a dominant role in proton gating through the ATP synthase and responds to changes in pH and ligands taking place on either side of the thylakoid membrane. It is proposed that the conformational changes that induce the proton slip and the exposure of [gamma]-Cys-89 reflect the conversion of the enzyme from a catalytically latent to an active state, and depend on the deprotonation of a stromal site at alkaline pH and on protonation of an intrathylakoid inner site upon energization. Therefore, conditions that induce the conformational activation also provide the driving force for ATP synthesis.

Primary structure and effect of pH on the expression of the plasma membrane H(+)-ATPase from Dunaliella acidophila and Dunaliella salina.

The plasma membrane H(+)-ATPase gene was cloned and sequenced from the extremely acidophilic green alga Dunaliella acidophila and from the extremely halotolerant Dunaliella salina. A special feature of the Dunaliella H(+)-ATPase is an extended C-terminal domain. The deduced amino acid sequences of the two proteins are 75% identical but differ in their C terminus. A hydrophilic loop within this domain in D. salina, which presumably faces the cell exterior, has a high ratio of acidic over basic amino acids, typical of halophilic proteins. The amount of the ATPase protein in plasma membranes and the level of its mRNA transcript in D. acidophila are far higher than in D. salina, suggesting that D. acidophila overexpresses the enzyme. A pH shift from 9.0 to 7.0 induces in D. salina a large increase in the level of the H(+)-ATPase mRNA and in the amount of the H(+)-ATPase protein. This suggests that the expression of the H(+)-ATPase in D. salina is pH-regulated at the transcriptional level. The implications of these findings are discussed with respect to the adaptive pressures imposed on these algal species by their exceptional environmental conditions.

A salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina.

The mechanisms allowing proliferation of the unicellular green alga Dunaliella salina in up to saturating NaCl concentrations are only partially understood at present. Previously, the level of a plasma membrane Mr 60,000 protein, p60, was found to increase with rising external salinities. Based on cDNA cloning and enzymatic assays, it is now shown that p60 is an internally duplicated carbonic anhydrase, with each repeat homologous to animal and Chlamydomonas reinhardtii carbonic anhydrases, but exceptional in the excess of acidic over basic residues. Increasing salinities, alkaline shift, or removal of bicarbonate induced in D. salina parallel increases in the levels of p60, its mRNA, and external carbonic anhydrase activity. Moreover, purified p60 exhibited carbonic anhydrase activity comparable to other carbonic anhydrases. A p60-enriched soluble preparation showed maximal carbonic anhydrase activity at approximately 1.0 M NaCl and retained considerable activity at higher salt concentrations. In contrast, a similar preparation from C. reinhardtii was approximately 90% inhibited in 0.6 M NaCl. These results identified p60 as a structurally novel carbonic anhydrase transcriptionally regulated by CO2 availability and exhibiting halophilic-like characteristics. This enzyme is potentially suited to optimize CO2 uptake by cells growing in hypersaline media.

Plasma Membrane Sterols Are Essential for Sensing Osmotic Changes in the Halotolerant Alga Dunaliella.

The halotolerant alga Dunaliella responds to hyperosmotic stress by synthesis of massive amounts of glycerol. The trigger for this osmotic response is the change in cell volume, but the mechanism that senses volume changes is not known. Preincubation of Dunaliella salina with tridemorph, a specific inhibitor of sterol biosynthesis, inhibits glycerol synthesis and volume recovery. The inhibition is associated with suppression of [14C]bicarbonate incorporation into sterols and is correlated with pronounced depletion of plasma membrane sterols. Incubation of sterol-depleted cells with cholesterol hemisuccinate restores the capacity for volume regulation in response to hyperosmotic stress. Tridemorph as well as lovastatin also inhibit volume changes that are induced by high light in Dunaliella bardawil, a species that responds to high light intensity by synthesis of large amounts of [beta]-carotene. These volume changes result from accumulation of glycerol and are associated with de novo synthesis of sterols. The major plasma membrane sterol in D. salina and the high-light-induced sterol in D. bardawil co-migrate with ergosterol on thin-layer chromatography and on reversed-phase high-performance liquid chromatography. These results suggest that the osmosensory mechanism in Dunaliella resides in the plasma membrane, and that sterols have an important role in sensing osmotic changes.

Isolation and Characterization of a Protein Associated with Carotene Globules in the Alga Dunaliella bardawil.

The halotolerant alga Dunaliella bardawil accumulates very large amounts of [beta]-carotene when exposed to high light intensity. The accumulated [beta]-carotene is concentrated in small, oily globules within the chloroplast and has been suggested to protect the alga against photodamage by high irradiation (A. Ben-Amotz, A. Katz, M. Avron [1982] J Phycol 18:529-537;A. Ben-Amotz, M. Avron [1983] Plant Physiol 72: 593-597; A. Ben-Amotz, A. Shaish, M. Avron [1989] Plant Physiol 91: 1040-1043). A 38-kD protein was identified and purified from [beta]-carotene globules and was designated carotene globule protein (Cgp). Induction of Cgp occurs in parallel with [beta]-carotene accumulation in D. bardawil grown under different inductive conditions. Cgp is overproduced in a constitutive mutant strain that overproduces [beta]-carotene and is not detected in Dunaliella salina, a species that does not accumulate [beta]-carotene. Cgp production was not suppressed by norflurazon, an inhibitor of [beta]-carotene synthesis that leads to accumulation of the carotenoid precursor phytoene. Immunogold-labeling analysis by electron microscopy demonstrates that the protein is localized at the periphery of the globules. Proteolytic cleavage by trypsin enhances the coalescence and destruction of the globules, in parallel with Cgp disappearance. It is suggested that the function of Cgp is to stabilize the structure of the globules within the chloroplast.

Reduction and transport of lipoic acid by human erythrocytes.

Reduction of exogenous lipoic acid to dihydrolipoate is known to occur in several mammalian cells and tissues. Dihydrolipoate is a potent radical scavenger, and may provide significant antioxidant protection. Because lipoic acid appears in the bloodstream after oral administration, we have examined the reduction of exogenous lipoate by human erythrocytes. Normal human erythrocytes reduced lipoate to dihydrolipoate only in the presence of glucose; deoxyglucose did not substitute for glucose, indicating that the reduction of lipoate requires glucose metabolism. Furthermore, the reduction was shown to be NADPH dependent. Erythrocytes isolated from a human subject with a genetic deficiency of glucose-6-phosphate dehydrogenase (and, therefore, deficient in the formation of NADPH) did not reduce lipoate. Dehydroepiandrosterone, a specific inhibitor of glucose-6-phosphate dehydrogenase, inhibited lipoate reduction. Our findings imply that some of the reduction of exogenous lipoic acid is catalysed by glutathione reductase, a flavoprotein dehydrogenase; mitomycin C, an inhibitor of FAD-dependent reductases, inhibited lipoate reduction by erythrocytes, and glutathione reductase purified from human erythrocytes was observed to reduce lipoic acid in a cell-free system. We further explored these findings with erythrocyte ghosts and liposomes. Our results indicate that a transport system exists for alpha-lipoic acid and dihydrolipoate; resealed erythrocyte ghosts, containing trapped lipoamide dehydrogenase and pyridine nucleotides, reduced externally added lipoate. By contrast, liposomes prepared with enzyme and pyridine nucleotides did not catalyze reduction of lipoate. This work indicates that uptake of exogenous lipoate and reduction to dihydrolipoate by normal human erythrocytes may contribute to oxidant protection in the human bloodstream.

Glutathione reductase and lipoamide dehydrogenase have opposite stereospecificities for alpha-lipoic acid enantiomers.

The reduction of exogenous alpha-lipoic acid to dihydrolipoate by mammalian cells and tissues confers additional antioxidant protection to the cell. Both (R+) and (S-) isomers of alpha-lipoic acid were analyzed as substrates with glutathione reductase from several sources and with mammalian lipoamide dehydrogenase. Mammalian glutathione reductase catalyzed faster reduction of (S)-lipoic acid (1.4-2.4-fold greater activity) than of (R)-lipoic acid, whereas lipoamide dehydrogenase had a very marked preference for (R)-lipoic acid (18-fold greater activity) over (S)-lipoic acid. Mammalian glutathione reductase showed better affinity for (S)-lipoic acid substrate; Km values were 3.5 mM for (S)-lipoic acid and and 7 mM for (R)-lipoic acid. Glutathione reductase from yeast reduced lipoic acid less efficiently than the mammalian enymes, had a Km for both stereoisomers of about 10 mM, and showed little stereospecificity. Although (S)-lipoic acid is not formed in nature, these findings indicate that exogenous (S)-lipoic acid may have a useful role as an antioxidant for mammalian systems.

A Salt-Induced 60-Kilodalton Plasma Membrane Protein Plays a Potential Role in the Extreme Halotolerance of the Alga Dunaliella.

The halotolerant alga Dunaliella salina grows in saline conditions as varied as 0.5 and 5 M NaCl, maintaining throughout this range a low intracellular ion concentration. To discover factors potentially involved in ionic homeostasis, we grew cells in media with different salinities or osmolarities and compared their protein profiles. The comparisons indicated that the amount of a 60-kD protein, p60, greatly increased with an increase in salinity and was moderately enhanced when NaCl was substituted with iso-osmotic glycerol. Cells transferred from low to high NaCl or from high glycerol to iso-osmotic NaCl media transiently ceased to grow, and resumption of growth coincided approximately with an increase in p60. The protein, extracted from a plasma membrane fraction, was purified to homogeneity. Anti-p60 antibodies cross-reacted with a 60-kD protein in Dunaliella bardawil. Immunoelectron microscopy of D. salina cell sections indicated that p60 was exclusively located in the plasma membrane. Its induction by salt, the correlation between its accumulation and growth resumption in high concentrations of salt, and its plasma membrane localization suggest the possibility that p60 could play a role in ionic homeostasis in conditions of high salinity, although different types of function could also be considered.

Activation of the Dunaliella acidophila plasma membrane H(+)-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site.

Trypsin treatment of purified H(+)-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H(+)-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H(+)-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.

Utilization of amiloride analogs for characterization and labeling of the plasma membrane Na+/H+ antiporter from Dunaliella salina.

The interactions of amiloride analogs with the Na+/H+ antiporter from plasma membrane of the halotolerant alga Dunaliella salina [Katz et al. (1989) Biochem. Biophys. Acta 983, 9-14] have been investigated. Analogs bearing hydrophobic substitutions at the guanidino moiety of amiloride, such as benzamil, are the most effective inhibitors of Na+ uptake in plasma membrane vesicles, whereas substituents of the 5-amino group are less effective inhibitors than amiloride. This order of specificity is opposite to that found for most Na+/H+ antiporters. The photoaffinity amiloride analog 2'-methoxy-5'-nitrobenzamil (NMBA), a competitive inhibitor with respect to Na+ with Ki = 10 microM, photolabels upon illumination two polypeptides of apparent MW 30 and 50 kDa in purified plasma membrane vesicles. Similar labeling is obtained by immunodetection with antiamiloride antibodies and by incorporation of [125I]NMBA. The specificity of the labeling was ascertained by competition with benzamil. Plasma membrane preparations from high-salt or ammonia-adapted cells, which have higher Na+/H+ antiporter activity [Katz et al. (1992) Plant Physiol. 100, 1224-1229], also show increased incorporation of NMBA into the 30- and 50-kDa polypeptides. It is suggested that: (1) the structure of the Na+ binding site of the D. salina Na+/H+ antiporter differs from that of most Na+/H+ antiporters and (2) the 50- and/or 30-kDa polypeptides are subunits of the plasma membrane antiporter of this alga.

Inhibition of the plasma-membrane H(+)-ATPase from Dunaliella adidophila by omeprazole.

The acid-activated sulfhydryl reagent omeprazole inhibits light-induced H+ secretion at pH 1 in cells of the halotolerant alga Dunaliella acidophila. Plasma-membrane vesicles, prepared from omeprazole-treated cells, have impaired vanadate-sensitive ATPase and ATP-induced H+ uptake activities. Omeprazole inhibits ATPase activity also in isolated plasma-membrane vesicles. The inhibition is enhanced at acidic pH and can be prevented by protonophores indicating that it is promoted by internal acidification of the vesicles. Mercaptoethanol partially reverses omeprazole inhibition. ADP does not afford protection against omeprazole but it does protect against inhibition by N-ethylmaleimide, indicating that these reagents modify different sulfhydryl groups. It is suggested that omeprazole blocks SH groups of the D. acidophila plasma-membrane H(+)-ATPase, which face the outer side of the cell.

Purification and Properties of a Plasma Membrane H+-ATPase from the Extremely Acidophilic Alga Dunaliella acidophila.

This paper describes partial purification and characterization of a vanadate-sensitive H+-ATPase from plasma membranes of Dunaliella acidophila, an extremely acidophilic unicellular alga (I. Sekler, H.U. Glaser, U. Pick [1991] J Membr Biol 121: 51-57). Purification is based on the insolubility in and stability of the enzyme in Triton X-100. The purified enzyme is highly enriched in a polypeptide of molecular mass 100 kD, which cross-reacts with antibodies against the plant plasma membrane H+-ATPase. Upon reconstitution into proteoliposomes, the enzyme catalyzes an ATP-dependent electrogenic H+ uptake. ATP hydrolysis is stimulated by lipids, is inhibited by vanadate, diethylstilbestrol, dicyclohexylcarbodiimide, erythrosine, and mercurials, and shows a sharp optimum at pH 6. Unusual properties of this enzyme, by comparison with plant plasma membrane H+-ATPases, are a higher affinity for ATP (Km = 40 [mu]M) and a larger stimulation by K+, which interacts with the enzyme from its cytoplasmic side. Comparative studies with cross-reacting antibodies, prepared against different domains of the plant H+-ATPase, suggest that the central hydrophilic domain containing the catalytic site is more conserved than the C- and N-terminal ends. The high abundance and stability of the plasma membrane H+-ATPase from D. acidophila make it an attractive model system for studies of the structure-function relations and regulation of this crucial enzyme.

Differential Reactivity of [beta]-Carotene Isomers from Dunaliella bardawil Toward Oxygen Radicals.

Dunaliella bardawil accumulates massive amounts of [beta]-carotene in two isoforms, a 9-cis and an all-trans stereoisomer, when grown under high irradiance, as a means to protect the cells against photoinhibition (A. Ben-Amotz, A. Shaish, M. Avron [1989] Plant Physiol 91: 1040-1043). The purpose of this work has been to find out if the mechanism of protection involves scavenging of reactive oxygen species. For this purpose high- and low-[beta]-carotene-containing cells were compared with respect to their sensitivity to several external oxidants [H2O2, methyl viologen, rose bengal, and 2,2[prime]-azobis(2-amidinopropane)HCl]. All oxidants induce a light-stimulated degradation of [beta]-carotene and of chlorophyll. The degradation of [beta]-carotene precedes that of chlorophyll, indicating that it is more reactive toward oxidants. The 9-cis [beta]-carotene is degraded faster than the all-trans stereoisomer when exposed to oxidants, both in intact cells and in isolated [beta]-carotene globules, indicating that it is a more effective scavenger of reactive oxygen species. Comparison of the sensitivity to different oxidants, between high- and low-[beta]-carotene-containing cells, reveals similar rates of chlorophyll and [beta]-carotene degradation in the two populations. Survival tests toward H2O2 and rose bengal show that high-[beta]-carotene cells have a similar sensitivity toward H2O2 but are more resistant toward rose bengal, a photoactivated generator of singlet oxygen, possibly due to masking of the latter by [beta]-carotene. These results suggest that the protection mechanism of massively accumulated [beta]-carotene in Dunaliella against photoinhibition is not due to scavenging of reactive oxygen species.

Modulation of Na/H Antiporter Activity by Extreme pH and Salt in the Halotolerant Alga Dunaliella salina.

The effect of different growth conditions on the activity of the Na(+)/H(+) antiporter in Dunaliella salina has been investigated. Adaptation of D. salina cells to ammonia at alkaline pH or to high NaCl concentrations is associated with a pronounced increase in the plasma membrane Na(+)/H(+) exchange activity. The enhanced activity is manifested both in vivo, by stimulation of Na(+) influx into intact cells in response to internal acidification, and in vitro, by a larger (22)Na accumulation in plasma membrane vesicles in response to an induced pH gradient. Kinetic analysis shows that the stimulation does not result from a change of the K(m) for Na(+) but from an increase in the V(max). In contrast, adaptation of cells to a high LiCl concentration (0.8 m) depresses the activity of the Na(+)/H(+) antiporter. Adaptation to ammonia is also associated with a large increase of three polypeptide bands in purified plasma membrane preparations, indicating that they may compose the antiporter polypeptides. These results suggest that adaptation to ammonia or to high salinity induces overproduction of the plasma membrane Na(+)/H(+) antiporter in Dunaliella.