Chemical methods for the analysis of sulphated galactans from red algae.

Methods are reported that facilitate the structural characterization of complex sulphated galactans of the red algae. Two procedures have been developed for the production of alditol acetates from carrageenans and agaroids. Both procedures generate 3,6-anhydrogalactitol acetate from the easily destroyed 3,6-anhydrogalactosyl residues in near quantitative yield. The "double hydrolysis-reduction" method involves preliminary hydrolysis under conditions sufficient to cleave all of the 3,6-anhydrogalactosidic bonds, but mild enough to avoid significant further degradation. The "reductive hydrolysis" method uses the acid-stable 4-methylmorpholine-borane to reduce the 3,6-anhydrogalactose end groups as they are released during acid hydrolysis. An alditol acetate sample can be prepared from a polysaccharide in a single tube, ready for g.l.c. analysis, in less than 2.5 h, i.e. more quickly than by any previous procedure. Problems associated with incomplete methylation of sulphated carrageenans and agaroids by the Hakomori procedure have been overcome by first converting the sulphated polysaccharide into its triethylammonium salt form. The reductive hydrolysis method is effective for the production of partially methylated alditol acetates from the methylated polysaccharides, enabling the rapid determination of the substitution pattern of these polysaccharides. These improved analytical methods have been applied successfully to kappa-, iota-, and lambda-carrageenans, as well as some agars.

Relationship of the flavodoxin isoforms from Porphyra umbilicalis.

The macroalga Porphyra umbilicalis contained two flavodoxins in approximately 5:1 ratio and differing in Mr by ca 1000. The N-terminal sequences of the isoforms were identical and there was strong immunochemical identity. Peptide mapping gave similar fragments which differed in Mr by constant amount for the two isoforms. The flavodoxins may therefore differ only at the C-terminus, possibly as a consequence of in vivo processing since inclusion of protease inhibitors during extraction had no effect on the ratio of the isoforms.

A new polyhalogenated monoterpene from the red alga Plocamium cartilagineum.

From a sample of the red alga Plocamium cartilagineum, four polyhalogenated monoterpenes [1-4] were isolated and characterized with (3R*, 4R*)-1-bromo-7-chloromethyl-3,4-dichloro-3-methyl-1E,5E,7-octatrie ne [1] being a new compound. Also reported are antifungal, antibacterial, and molluscicidal activities, as well as the brine shrimp toxicity of these metabolites.

Isolation of allophycocyanin B from Rhodella violacea results in a model of the core from hemidiscoidal phycobilisomes of rhodophyceae.

Two 'trimeric' allophycocyanin complexes could be isolated from the hemidiscoidal phycobilisomes of Rhodella violacea. AP = (alpha *AP alpha 2AP beta 2AP beta *AP) and APB = (alpha *AP alpha AP alpha APB beta 2AP beta *AP). Lc13.5APB. The isolation was performed by combined methods of gradient centrifugation, hydroxylapatite chromatography and 'native' polyacrylamide gel electrophoresis. AP showed the well-known spectral characteristics of allophycocyanin without linker polypeptide. APB is characterized by its long wavelength absorbing shoulder (675 nm) and fluorescence emission (682 nm), respectively. The existence of two low molecular linker polypeptides Lc12.5 and Lc13.5APB in the phycobilisomes of Rhodella violacea, their stoichiometric calculations and the localization of Lc13.5APB in allophycocyanin B facilitated the construction of a model of the phycobilisome core.

Crystallization and preliminary X-ray diffraction studies of C-phycocyanin from a red alga, Porphyra tenera.

C-Phycocyanin from a red alga, Porphyra tenera, has been crystallized by the vapor-diffusion procedure. Both orthorhombic and hexagonal forms were obtained from ammonium sulfate solutions, whereas only the orthohombic form was selectively grown from sodium citrate solutions. The orthorhombic crystals are more suitable for further crystallographic work; their space group is P2(1)2(1)2(1), with unit-cell dimensions of a = 105, b = 121, and c = 184 A. The asymmetric unit comprises two (alpha beta)3 trimer molecules of C-phycocyanin. These crystals diffract X-rays up to about 3 A resolution.

Gracilaria tikvahiae agglutinin. Partial purification and preliminary characterization of its carbohydrate specificity.

A potent agglutinin of rabbit and sheep red blood cells, obtained from the red alga Gracilaria tikvahiae, was purified by ammonium sulfate fractionation, ion exchange, gel filtration, and hydroxylapatite chromatography. Human A and B blood group erythrocytes were also agglutinated, whereas human O blood group erythrocytes were not agglutinated. The hemagglutination titer was not significantly affected by the addition of EDTA or the divalent cations Ca2+, Mg2+, or Mn2+. The carbohydrate specificity was characterized by hemagglutination inhibition using various monosaccharides, glycoproteins, and glycopeptides. The results suggested that the agglutinin has affinity for N-acetylneuraminic acid as well as glycoconjugates containing N-acetylneuraminic acid.

Isolation and characterization of a new hemagglutinin from the red alga Gracilaria bursa-pastoris.

A new agglutinin has been isolated from the red alga Gracilaria bursa-pastoris by affinity chromatography on a yeast mannan-Cellulofine column. This agglutinin was isolated as a monomeric glycoprotein with a relatively low molecular weight. It had an isoelectric point of 4.7 and contained large amounts of Gly, Asx and Glx. It agglutinated trypsin-treated rabbit erythrocytes at the low concentration of 30 ng/ml. The activity was inhibited only by glycoproteins bearing N-glycans. This agglutinin also showed mitogenic activity for mouse splenic lymphocytes.

Discovery of the mammalian insulin release modulator, hepoxilin B3, from the tropical red algae Platysiphonia miniata and Cottoniella filamentosa.

The insulin release enhancer, 10-hydroxy-11,12-trans-epoxy-5(Z),8(Z),14(Z)-icosatrienoic acid (hepoxilin B3), is a 12-lipoxygenase metabolite of arachidonic acid that has been found in various mammalian tissues. Although lipoxygenase pathways are well documented in terrestrial plants, this lipoxygenase product has never been isolated from the plant kingdom. Herein, we report the first isolation of this lipoxygenase product, hepoxilin B3, from two plants, the tropical red marine algae Platysiphonia miniata (C. Agardh) Børgesen and Cottoniella filamentosa Børgesen. Furthermore, through application of two-dimensional NMR methodology to the structural description of this algal natural product, we demonstrate the tremendous power of this technique in this chemical class.

Identification of localized redox states in plant-type two-iron ferredoxins using the nuclear Overhauser effect.

The homonuclear Overhauser effect (NOE), in conjunction with nonselective spin-lattice relaxation measurements, has been employed to assign the contact-shifted resonances for the reduced form of two typical plant-type two-iron ferredoxins from the algae Spirulina platensis and Porphyra umbilicalis. These results demonstrate that the NOE should have broad general applicability for the assignments and electronic structural elucidation of diverse subclasses of paramagnetic iron-sulfur cluster proteins. NOE connectivities were detected only among sets of resonance exhibiting characteristically different deviations from Curie behavior, providing strong support for the applicability of the spin Hamiltonian formulation for the NMR properties of the antiferromagnetically coupled iron clusters [Dunham, W. R., Palmer, G., Sands, R. H., & Bearden, A. J. (1971) Biochim. Biophys. Acta 253, 373-384; Banci, L., Bertini, I., & Luchinat, C. (1989) Struct. Bonding (in press)]. The geminal beta-methylene protons for the two cysteines bound to the iron(II) center were clearly identified, as well as the C alpha H and one C beta H for each of the cysteines bound to the iron(III). The identification of the iron bound to cysteines 41 and 46 as the iron(II) in the reduced protein was effected on the basis of dipolar contacts between the bound cysteines, as predicted by crystal coordinates of S. platensis Fd [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. (Tokyo) 90, 1763-1773].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid sequence of ferredoxin isolated from Cyanidium caldarium strain RK-1.

Ferredoxin was isolated from ther eukaryotic alga Cyanidium caldarium strain RK-1 and its amino acid sequence was determined. The ferredoxin is composed of 97 amino acid residues, and its molecular weight is 10,599 excluding the iron-sulfur cluster. The amino acid sequence differs from that of C. caldarium strain 1355/1, by 24 amino acid substitutions plus one deletion at the amino terminus.

The amino acid sequence of a flavodoxin from the eukaryotic red alga Chondrus crispus.

The amino acid sequence of the constitutive flavodoxin from the red alga Chondrus crispus was determined from the analyses of peptide fragments derived by enzymic digestions of the carboxymethylated protein. This is the first sequence reported for a flavodoxin from a eukaryote. The protein is composed of 173 amino acid residues and is a member of the longer-chain group of flavodoxins. The extent of sequence homology to the three other flavodoxins in the group for which sequences are available is in the range 36-39%, with the most strongly conserved regions being those implicated in binding of the FMN, the redox-active prosthetic group. Nevertheless, Chondrus crispus flavodoxin stands apart in a number of respects, in particular the possession of an unusually high content of proline, with these residues distributed more or less regularly along the peptide chain.

Isolation and characterization of an antitumor active agar-type polysaccharide of Gracilaria dominguensis.

Cold water extraction of the red alga Gracilaria dominguensis, followed by cetyltrimethylammonium bromide fractionation, gave a highly sulfated, agar-type polysaccharide which inhibited the transplantation of Ehrlich ascites carcinoma in mice. The structure of the polysaccharide has been investigated by methylation analysis, and 1H- and 13C-n.m.r. spectroscopy, and was shown to be mainly composed of alternating (1----3)-linked beta-D-galactopyranosyl 6-sulfate and (1----4)-linked 3,6-anhydro-alpha-L-galactopyranosyl residues.

Three new and bioactive icosanoids from the temperate red marine alga Farlowia mollis.

Three new dihydroxyicosanoids, 12(R),13(R)-dihydroxyicosa-5(Z),8(Z),10(E),14(Z)-tetraenoic acid, 12(R),13(R)-dihydroxyicosa-5(Z),8(Z),10(E),14(Z),17(Z)-pentaeno ic acid and 10(R*),11(R*)-dihydroxyoctadeca-6(Z),8(E),12(Z)-trienoic acid, have been isolated from a previously unstudied temperate red marine alga, Farlowia mollis (Cryptonemiales, Rhodophyta). The structures of these new metabolites have been deduced from detailed nuclear magnetic resonance and mass spectrometry analyses on stabilized diacetate-methyl esters and stereochemistry deduced by 1H NMR couplings and CD analysis of a dibenzoate derivative. Collectively, these new natural products modulate fMLP-induced superoxide anion generation in human neutrophils, inhibit the conversion of arachidonic acid to lipoxygenase products by human neutrophils, and inhibit the functioning of the dog kidney Na+/K+ ATPase.

Crystallization and preliminary X-ray diffraction studies of oxidized flavodoxin from Chondrus crispus, a red alga.

Crystals of the oxidized form of flavodoxin from a red alga, Chondrus crispus, have been grown in ammonium sulfate solution by the dialysis method. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), with unit cell dimensions of a = 63.6, b = 48.8, and c = 56.8 A. The asymmetric unit contains one molecule of flavodoxin. The crystals diffract X-rays to about 2.0 A resolution and are stable to X-ray beams. The diffraction patterns changed significantly upon soaking the crystal in a solution of a platinum complex. The major heavy-atom sites in the platinum derivative crystal have been identified from the difference Patterson function calculated at 4 A resolution.

Discovery of 12-(S)-hydroxy-5,8,10,14-icosatetraenoic acid [12-(S)-HETE] in the tropical red alga Platysiphonia miniata.

The potent mammalian immunohormone, 12-(S)-hydroxy-5,8,10,14-icosatetraenoic acid (12-(S)-HETE), is a 12-lipoxygenase metabolite of arachidonic acid that is widely distributed in animal tissues. In humans, it is produced and secreted by platelet cells and elicits both chemotactic and degranulatory responses in target neutrophils. As widely as 12-lipoxygenase activity and one of its major products, 12-(S)-HETE, have been found in animal tissues, it has never been found in plants. Herein, we report the first isolation of the 12-lipoxygenase product, 12-(S)-HETE, from a plant, the tropical marine alga Platysiphonia miniata (C. Agardh) Børgesen.

A method for RNA isolation from marine macro-algae.

Sulfated, carboxylic polysaccharides and polyphenols found in marine macro-algae interfere with RNA isolation from these plants and inhibit RNA activities in vitro. Methods based on differential precipitation of RNA or carbohydrates in high salts were used to eliminate the acidic carbohydrates. To protect RNA from inactivation by oxidized polyphenols, strong reducing reagents were used to prevent polyphenol oxidation. RNA was successfully isolated from Macro-cystis pyrifera (brown alga), Porphyra schizophylla (red alga), and Enteromorpha intestinalis (green alga). mRNA isolated from the total RNA was shown to be translationally active.