Configuration at C-24 of sterols from the liverwort Palavicinnia lyellii

The 4-desmethylsterol fraction of the liverwort Palavicinnia lyellii is composed of 36% 24β-methylcholest-5-en-3β-ol (dihydrobrassicasterol), 16% 24α-methylcholest-5-en-3β-ol (campesterol), 33% 24α-ethylcholest-5-en-3β-ol (sitosterol) and 15% 24ξ-ethylcholesta-5,22-dien-3β-ol.     

Nitrate uptake and extracellular alkalinization by the green alga Hydrodictyon reticulatum in blue and red light

Nitrate uptake and the medium alkalinization related to it were studied with nets of the coenocytic, giant cell, green alga Hydrodictyon reticulatum. A comparison of red, blue and white light irradiation showed no special control of nitrate uptake and of the corresponding alkalinization of the external medium by light quality, but rather a response as expected for the photosynthetic apparatus. In the dark, nitrate uptake rates amounted to one-fifth of those in saturating white light. This is in contrast to the chlorococcal microalga Monoraphidium braunii, where blue light specifically switched on nitrate uptake-dependent alkalinization and where uptake and reduction of nitrate strongly depended on blue light; the rates in pure red light and in the dark being very low. The stoichiometric ratio between nitrate taken up and extracellular alkalinization was close to 1 (0.86) in air with CO2 but close to 2 (1.84) in N2 for nitrate pre-loaded cells. In the absence of any carbon source, a high proportion of the absorbed and reduced nitrogen is released, most of it as ammonium which causes the excess alkalinization and some as nitrite, which lowers the ratio. Nitrite and ammonium release rates under anaerobic, CO2-free conditions were also independent of red or blue light and continued for several hours when the medium was buffered at pH 6. The data indicate that nitrate uptake, but less its reduction, is regulated differently in vacuolate, coenocytic algae from microalgae. In Hydrodictyon, nitrate uptake and reduction seems to be controlled by energy supply; in various microalgae, in addition, it is controlled specifically by blue light.     

Configuration at C-24 of sterols from the marine phanerogames Posidonia oceanica and Cymodocea nodosa

Sterols were extracted from two marine phanerogames, Posidonia oceanica and Cymodocea nodosa. The two plants contain 24α-ethyl sterols, while the 24α-methyl sterols are accompanied by 24β-epimers. The most abundant components are sitosterol, cholesterol and stigmasterol.     

Effects of amiloride on the transport of sodium and other ions in the alga Hydrodictyon reticulatum

The diuretic amiloride, an almost specific inhibitor of sodium transport in animal cells and tissues, appears to produce a number of effects in the alga Hydrodictyon reticulatum. At 1 mmol/l concentration it markedly reduces the influx of sodium ions (but not their active outflux), the influxes of potassium, chloride as well as of bicarbonate ions, and causes a profound decrease in the plasmalemma membrane potential. This plurality of inhibitory effects suggests that individual transport processes in the alga are mutually coupled.     

Steric effects at C-20 and C-24 on the metabolism of sterols by Tetrahymena pyriformis

Cultures of Tetrahymena pyriformis were incubated with various sterols and the extent of dehydrogenation at C-7 and C-22 was determined. The sterols incubated were desmosterol, 22-dehydrodesmosterol, 24-methyldesmosterol, 24 alpha-methylcholesterol (campesterol), 24-methylene-cholesterol, isohalosterol (26,27-bisnorcampesterol, also known as 24,24-dimethylchol-5-en-e beta-ol, a naturally occurring C26-sterol), and 20-isohalosterol. 20-Isohalosterol was not metabolized, while products with delta 7- and delta 22-bonds were formed from isohalosterol and all of the other sterols studied. This confirms an earlier conclusion, based on results with 20-isocholesterol and cholesterol, that inversion of the configuration from 20(R) to 20(S) completely prevents metabolism both in the nucleus and the side chain. On the other hand, changes in the electronics or stereochemistry at C-24 had a direct affect only on metabolism in the side chain. The presence of a methyl group at C-24 reduced the yield of metabolites with a delta 22-bond relative to those with a delta 7-bond producing an accumulation of 7-dehydro metabolite. A double bond at position-24 counteracted this steric effect, presumably by enhancing the rate of dehydrogenation, and a delta 24(28)-bond was more effect than was a delta 24(25)-bond.     

Bioremediation of cadmium-contaminated water systems using intact and alkaline-treated alga (Hydrodictyon reticulatum) naturally grown in an ecosystem

Cadmium can enter water, soil, and food chain in amounts harmful to human health by industrial wastes. The use of intact and NaOH-treated dried algal tissues (Hydrodictyon reticulatum), a major ecosystem bio-component, for Cd removal from aqueous solutions was characterized. Cadmium biosorption was found to be dependent on solution pH, bioadsorbent dose, the interaction between pH and dose, contact time, and initial Cd concentration. The experimental results indicated that the biosorption performance of alkaline-treated algal tissues was better than that of intact tissues. The maximum biosorption capacities were 7.40 and 12.74 mg g^?1 for intact and alkaline-treated bioadsorbents, respectively, at optimum operating conditions. Biosorption reaches equilibrium after 24 and 240 minutes of contact, respectively, for alkaline-treated and intact bioadsorbents. Cadmium biosorption was best fitted to Langmuir isotherm model (R² ≈ 0.99) and the kinetic study obeyed the pseudo-second-order kinetic model, which suggests chemisorption as the rate-limiting step in the biosorption process. Alkaline-treated algal tissues can be used as a new material of low-cost bioadsorbent for continuous flow rate treatment systems.     

Response of Chara globularis and Hydrodictyon reticulatum to lead pollution: their survival, bioaccumulation, and defense

The responses of the pioneer submerged macroalga (Chara globularis) and the rapidly spreading floating macroalga (Hydrodictyon reticulatum) to high levels of lead (40, 80, and 160 mg L⁻¹) at pH 7.14 were studied. Growth rate, Pb bioaccumulation, and physiological response of plants were measured after 5 and 15 days exposure. Both macroalgae efficiently postponed the deposition process of Pb from water column to soil. The Pb bioaccumulation in C. globularis was concentration- and time-dependent increase during the experiment and the maximum bioaccumulation activity was about 3,650 mg Pb kg⁻¹ DW in 160 mg L⁻¹ Pb at pH 7.14 after 15 days, whereas H. reticulatum showed saturable bioaccumulation in 5 days and the maximum was approximately 4,000 mg Pb kg⁻¹ DW; in addition, H. reticulatum exhibited higher tolerance to Pb pollution than C. globularis. The results also showed that the antioxidant defense systems of both tested macroalgae were overwhelmed under high Pb levels with superoxide radical and malondiadehyde levels increasing significantly. The antioxidant enzymes, superoxide dismutase, catalase, and guaiacol peroxidase activities were inhibited severely increasing Pb levels and exposure time. These results indicate that the pioneer species C. globularis would have difficulty growing in a habitat polluted by Pb >40 mg L⁻¹and the rapidly spreading H. reticulatum may not grow in an environment polluted by >80 mg L⁻¹ Pb. Because Pb levels in most water bodies are lower than 40 mg L⁻¹, both C. globularis and H. reticulatum can be considered for phytoremediation of Pb pollution.     

A fibrinolytic enzyme from a marine green alga, Codium latum

A fibrinolytic enzyme was isolated from a marine green alga, Codium latum, and designated C. latum protease (CLP). It also had fibrinogenolytic activity, hydrolyzing A alpha, B beta and gamma chains with preference in this order. As CLP hydrolyzed oxidized insulin B chain at position Arg22-Gly23, and the peptide map of lysozyme digested with CLP was similar to that with trypsin, CLP would be expected to have a high substrate specificity, similar to that of trypsin. Protease activity peaked at pH 10, and was completely inhibited by diisopropyl fluorophosphate (DFP). Therefore, we conclude that CLP is a trypsin-like serine protease.     

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.     

First crystal structure of Rubisco from a green alga, Chlamydomonas reinhardtii

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 A resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between beta strands A and B of the small subunit (betaA-betaB loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the betaA-betaB loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.     

Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

In the green alga Hydrodictyon reticulatum zoospores are arranged in a regular fashion to form an intricate hexagonal network during the asexual reproductive cycle. A monoclonal antibody which was raised against a homogenate of zoospores recognized a single poly‐peptide in zoospores with a molecular mass of 31 kDa. The antigenic polypeptide, which was designated Amy1, was localized within the cytoplasm of zoospores. The accumulation of Amy1 occurred concomitantly with the transition from multinuclear vegetative cells to mononuclear zoospores, and the degradation of Amy1 occurred concomitantly with the further development of zoospores. Amy1 was constantly expressed during the period of mononuclear zoospores. Thus, we conclude that Amy1 is a zoospore‐specific polypeptide. Using the anti‐Amy1 monoclonal antibody, we could easily distinguish between mononuclear zoospores and multinuclear vegetative net‐cells. This provides an important tool for analysing the molecular mechanisms involved in the hexagonal net formation by zoospores.     

Confirmation of the Relative Stereochemistry at C-23 and C-24 of Tautomycin

Degradation product including the moiety for C-19 through to C-25 of tautomycin were synthesized. This synthesis confirmed the relative stereochemistry at C-23 and C-24 of tautomycin.     

Complete amino acid sequence of plastocyanin from a green alga, Enteromorpha prolifera

The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.     

Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii

A second thioredoxin, Ch1, distinct from the one recently reported [Decottignies, P., Schmitter, J.M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch, Biochem. Biophys. 280, 112-121] has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m and f). Ch1 cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin-thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP-dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose-1,6-bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Ch1 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Ch1 consists of a polypeptide of 111 amino acids (11634 Da) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. Compared to thioredoxins from other sources, the algal thioredoxin Ch1 displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Ch1 may be an h-type thioredoxin.