Rates of Turnover and Net Accretion of Calcium and the Role of Calcium Binding Polysaccharides During Calcification in the Calcareous Alga Halimeda opuntia (L.)

Calcification in the calcareous alga Halimeda opuntia involves Calcium exchange with the seawater. Calcium influx and-outflux is nearly the same and approximately 300 μg Ca/g alga/min. Calcium net accretion is 3.6 ± 1.1 μg Ca/g alga/min. Calcium outflux alga-seawater is higher in darkness than in light. Calcium exchange in live algae is 3–6 times faster than in dead algae. Calcium is lost from the seawater at two distinctly different rates and is taken up by watersoluble polysaccharides in the algal mucilage and by the skeleton. These two Calcium pools represent 0.1 and 99.9% of the algal Calcium respectively. Between 0.8–2.0% of the skeletal Calcium is exchangeable. The analysis of the ⁴⁵Ca specific activity curves suggests that there are more than 2 Calcium pools in the alga. The polysaccharides constitute a small but rapidly exchanging Calcium pool which communicates with the seawater and at least 2 other pools. The Calcium binding polysaccharides are involved in the Calcium metabolism of the alga and may play a role in algal calcification.     

COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca²⁺-binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO₃ crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO₃ in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.     

COCCOLITH-ASSOCIATED POLYSACCHARIDES FROM CELLS OF EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler, a unicellular calcifying alga, consist of calcite closely associated with an acidic, Ca²⁺-binding polysaccharide. This polysaccharide is thought to play a regulatory role in coccolith synthesis by interfering with CaCO₃ crystallization. Here we show that the polysaccharides from three different strains, A 92, L and 92 D, all inhibit the precipitation of CaCO₃ in vitro to the same extent. The monosaccharide compositions of the A 92 and L polysaccharide are similar. The 92 D material, however, deviates from the other two: it contains significantly lower amounts of methylated sugars and ribose, and elevated levels of rhamnose and galactose. It also contains antigenic determinants not detected in the A 92 and L polysaccharides. In contrast to the latter two macromolecules the 92 D polysaccharide migrates as two bands upon polyacrylamide gel electrophoresis, possibly resulting from complexing with small amounts of protein. The coccolith polysaccharide from L cells, cultured at an elevated growth rate, also migrates as two bands. This phenomenon is due to an increase in molecular size distribution. The results suggest that certain properties of the molecule may be subject to variation without interfering with its function.     

FIXED CARBON PARTITIONING IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)

The main products of carbon fixation in the red algae are sulfated cell-wall polysaccharides, floridean starch, and low molecular weight (LMW) carbohydrates, mainly floridoside. In the red microalga Porphyridium sp., sulfated polysaccharide—cell bound and soluble—comprises up to 70% of the algal biomass. The purpose of this study was to elucidate the partitioning of fixed carbon in Porphyridium sp. toward the different products of carbon fixation. Using pulse-chase technique with [¹⁴C]bicarbonate, we followed ¹⁴C flow into the major compounds, namely, cell-wall polysaccharide, floridoside, starch, and protein, under various environmental conditions (i.e. carbon dioxide enrichment and nitrate starvation). ¹³C-NMR and gas chromatography analysis showed the main LMW product in Porphyridium sp. to be floridoside. After the short [¹⁴C]bicarbonate pulse (20 min), 42%–53% of total ¹⁴C uptake was initially found in floridoside. The appearance of ¹⁴C in the soluble polysaccharide was evident immediately at the end of the 20-min [¹⁴C]bicarbonate pulse. The specific radioactivity in the floridoside fraction declined by 80% after the 48-h chase, this decline being accompanied by increased labeling of starch and the soluble polysaccharide. In cells exposed to high CO₂ concentration, larger amounts of ¹⁴C (about twice as much) were channeled into starch and soluble polysaccharide than in cells under low CO₂ concentration. The most significant increase (1500%) in labeling during chase was found in the soluble polysaccharide of the nitrate-deprived cultures. It therefore seems likely that the large amounts of carbon incorporated by Porphyridium sp. cells into floridoside were subsequently used for the synthesis of macromolecular components. The data thus support the premise that floridoside serves as a dynamic carbon pool, which channels the fixed carbon toward polysaccharides and other end products according to the ambient conditions.     

Nitrogen Effect on Water-Soluble Polysaccharide Accumulation in <Emphasis Type="Italic">Streblonema</Emphasis> sp. (Ectocarpales,Phaeophyceae)

The water-soluble polysaccharides of brown algae attract the increasing attention of researchers as an important class of polymeric materials of biotechnological interest. The sole source for production of these polysaccharides has been large brown seaweeds such as members of Laminariales and Fucales. A new source of water-soluble polysaccharides is suggested here: it is a filamentous brown alga Streblonema sp., which can be cultivated under controlled conditions in photobioreactors that allow obtaining algal biomass with reproducible content and quality of polysaccharides. The accumulation of water-soluble polysaccharides can be stimulated by macronutrient limitation. In response to nitrogen deficiency, Streblonema sp. accumulated water-soluble polysaccharides (WSPs) rich in laminaran. WSP accumulation started after 3–4 days following nitrate depletion and reached a plateau at around day 7. Polysaccharide accumulation was related to cellular nitrogen content. The critical internal N level that triggered the onset of polysaccharide accumulation was 2.3% dry weight (DW); at a cellular N concentration less than 1.4% DW, the polysaccharide synthesis stopped. Upon nitrate re-supply, mobilization of WSP occurred after 3 days. These results suggest that a two-stage cultivation process could be used to obtain large algal biomass with high water-soluble polysaccharide production: a first cultivation stage using nitrate-supplemented medium to accumulate algal biomass followed by a second cultivation stage in a nitrate-free medium for 3 to 7 days to enhance polysaccharide content in the alga.     

CELL-WALL FORMATION DURING THE CELL CYCLE OF PORPHYRIDIUM SP. (RHODOPHYTA)

The cell wall of the red microalgae Porphyridium sp. (UTEX 637) comprises a complex amorphous polysaccharide (6–7 × 10⁶ Da). The polysaccharide is made up of xylose, glucose, and galactose as the main sugars, as well as some minor sugars, protein, sulfate, and glucuronic acid, the latter two conferring a negative charge on the polysaccharide. In this study, we used synchronized cultures as one of the ways of unraveling the mechanism of biosynthesis of this complex polysaccharide by following cell-wall formation during the cell cycle. Synchronization of Porphyridium sp. was achieved with an alternating light:dark regime of 12:12 h LD and dilution of the culture at the end of the cycle. Under these conditions, cell duplication occurred between the 12th and 14th hours of the cycle. The following order of building toward formation of the final polysaccharide appeared to take place: Intermediate polysaccharides with molecular masses ranging from 0.5 × 10⁶ to 2 × 10⁶ Da appeared in succession during hours 2–6 of the cycle, and the full-sized polysaccharide was detected by the 8th hour. At the beginning of the cycle, xylose was the predominant sugar. Sulfur peaked at hours 2–4; glucose, galactose, and glucuronic acid at hours 8–12; and the minor sugars at hours 12–14. Upon incubation of low molecular mass polymer (0.5 × 10⁶ Da) collected from the 4th hour with cellular crude extract from cells of the 6th hour of the cycle, two intermediates were formed (0.8 × 10⁶ Da and 2 × 10⁶ Da). We suggest that the 0.5 × 10⁶ Da polymer intermediate, which is composed mainly of xylose, is the first polymer secreted into the medium, where it is further polymerized enzymatically to produce the 2 × 10⁶ Da polymer via an intermediate 0.8 × 10⁶ Da polymer. Later, the full-size polysaccharide is produced.     

ALCIAN BLUE: A QUANTITATIVE AQUEOUS ASSAY FOR ALGAL ACID AND SULFATED POLYSACCHARIDES1

Alcian Blue, a cationic copper phthalocyanine dye, complexes with the anionic carboxyl and half-ester sulfate groups of acidic algal polysaccharide in aqueous solution to form an insoluble precipitate. The quantity of dye removed from solution is proportional to the quantity of polyanion in solution, and this principle forms the basis for the quantitative determination of acid and/or sulfated algal polysaccharides. The assay is linear between 0 and 100 μg/ml agar, alginic acid, carrageenan, pectin and Porphyridium aerugineum Geit. polysaccharide. In addition, the technique is used to determine the anion density of acid polysaccharides on a molar or weight equivalency basis.     

Binding of DL-[3H]-alpha-Amino-3-Hydroxy-5-Methyl-Isoxazole-4-Propionic Acid (AMPA) to Rat Cortex Membranes Reveals Two Sites or Affinity States

Abstract

A method for measuring [³H]-AMPA binding in rat cortex membranes is described. Specific binding was saturable and accounted for 95% of total binding at 5 nM of [³H]-AMPA. Non linear curve fitting of [³H]-AMPA saturation isotherms suggested the presence of two binding sites: the high affinity site showed a pKd of 8.26 ± 0.07 (Kd = 5.49 nM) and a Bmax of 0.19 ± 0.03 pmol/mg protein, whereas the low affinity site indicated a pKd of 7.28 ± 0.05 (Kd = 52 nM) and a Bmax of 1.30 ± 0.23 pmol/mg protein. The pharmacological profile of [³H]-AMPA binding has been determined by studying a series of compounds in binding displacement experiments: Quisqualate was the most potent inhibitor of [³H]-AMPA binding (IC₅₀ = 9.7 nM), followed by AMPA (19 nM), CNQX, DNQX and L-Glutamate (272–373 nM). Kainate was a moderate displacer (6.2 μM); Ibotenic acid and glycine were very weak inhibitors (74 and 92 μM, respectively). CPP, GAMS and L-Aspartic acid showed IC₅₀-values of over 400 μM and MK-801, DL-AP5 and NMDA were almost inactive at the maximal concentration used in our experiments.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Composition analysis and material characterization of an emulsifying extracellular polysaccharide (EPS) produced by Bacillus megaterium RB-05: a hydrodynamic sediment-attached isolate of freshwater origin

Aims: This work was aimed to isolate, purify and characterize an extracellular polysaccharide (EPS) produced by a freshwater dynamic sediment‐attached micro‐organism, Bacillus megaterium RB‐05, and study its emulsifying potential in different hydrocarbon media. Methods and Results: Bacillus megaterium RB‐05 was found to produce EPSs in glucose mineral salts medium, and maximum yield (0·864 g l^?1) was achieved after 24‐h incubation. The recovery rates of the polysaccharide material by ion‐exchange and gel filtration chromatography were around 67 and 93%, respectively. As evident from HPLC and FT‐IR analyses, the polysaccharide was found to be a heteropolymer‐containing glucose, galactose, mannose, arabinose, fucose and N‐acetyl glucosamine. Different oligosaccharide combinations namely hexose₃, hexose₄, hexose₅deoxyhexose₁ and hexose₅deoxyhexose₁pentose₃ were obtained after partial hydrolysis of the polymer using MALDI‐ToF‐MS. The polysaccharide with an average molecular weight of 170 kDa and thermal stability up to 180°C showed pseudoplastic rheology and significant emulsifying activity in hydrocarbon media. Conclusions: Isolated polysaccharide was found to be of high molecular weight and thermally stable. The purified EPS fraction was composed of hexose, pentose and deoxyhexose sugar residues, which is a rare combination for bacterial polysaccharides. Emulsifying property was either better or comparable to that of other commercially available natural gums and polysaccharides. Significance and Impact of the Study: This is probably one of the few reports about characterizing an emulsifying EPS produced by a freshwater sediment‐attached bacterium. The results of this study contribute to understand the influence of chemical composition and material properties of a new microbial polysaccharide on its application in industrial biotechnology. Furthermore, this work reconfirms freshwater dynamic sediment as a potential habitat for bioprospecting extracellular polymer–producing bacteria. This study will improve our knowledge on the exploitation of a nonconventional renewable resource, which also seems to be ecologically significant.     

Cosmid library of the marine macroalga Bryopsis maxima (Bryopsidales, Ulvophyceae)

The purpose of this study was to construct a cosmid library from chromosomal DNA of a marine macroalga, Bryopsis maxima Okamura ex Segawa (Bryopsidales, Ulvophyceae), in a rapid, simple and inexpensive manner. In the DNA purification, polysaccharides were removed by covalently binding them to resin particles containing free boric acid groups. The DNA yield was 20 μg g^?1 of B. maxima fresh weight. This DNA was 100–200 kb in length, and its A₂₆₀/A₂₈₀ and A₂₃₀/A₂₆₀ ratios were 1.8 and 0.4, respectively. It was of sufficient quality for molecular research. The cloning procedures were carried out in the following steps: controlled partial shearing of purified DNA through a microsyringe, optimal size separation of the DNA by biased sinusoidal field gel electrophoresis, ligation of the DNA to the cosmid vector in the gel, and in vitro packaging into the lambda phage. The library consisted of 2.0 × 10³ independent clones with an average insert size of 40 kb. The fragment amplified by polymerase chain reaction in the library was hybridized with a DNA fragment (328 bp) encoding B. maxima glutamate dehydrogenase under high‐stringency conditions by Southern blot analysis, thus demonstrating that the library contained B. maxima chromosomal DNA. This cosmid library is the first to be constructed for any species of marine macroalgae.     

Ecological notes on the species of Phacus Dujardin (Euglenophyta) from the central region of Portugal

Twenty-one species, belonging to the genus Phacus, were identified during the study of samples from the central region of Portugal collected in lentic systems. The abundance of each taxon was determined. Water samples were taken for determination, by means of standard methods, of physicochemical parameters (water temperature, pH, organic matter (K₂Cr₂O₇), conductivity, alkalinity, nitrogen as N(NH₄⁺), N(NO₂^–) and N(NO₃^–), orthophosphate P(PO₄^3–) and metals in a total of 35 parameters). Some species were found more frequently, namely Phacus agilis Skuja, Ph. aenigmaticus Drez., Ph. caudatus Hübn., Ph. gigas Da Cunha, Ph. triqueter (Ehr.) Duj., Ph. longicauda (Ehr.) Duj. and Ph. tortus (Lemm.) Skv. Eighteen taxa were found in the sampling sites characterized by the following variation intervals of the environmental parameters: water temperature: 11.4–21.6 °C; pH: 6.2–7.5; dichromate oxidability: 10–59 mg l^–1; conductivity: 145–779 μS cm^–1; nitrogen as NO₃^–: n.d.–2.852 mg l^–1; orthophosphate: n.d.–0.892 mg l^–1; chloride: 14.2–109.3 mg l^–1; sodium: 10.3–47.5 mg l^–1 and total iron: 135–6446 μg l^–1. In this work, information concerning the environmental conditions that preceded the occurrence of these species as well as results of the cytological and morphologic studies (with bright field microscopy as a resource) is presented and discussed.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

Calcium deficiency in potato (Solanum tuberosum ssp. tuberosum) leaves and its effects on the pectic composition of the apoplastic fluid

Potato plants (Solanum tuberosum ssp. tuberosum cv. Adelheid), multiplied in vitro, were cultivated in growth chambers on nutrient solution at calcium regimes of 1000, 90, 60 or 30 μM Ca. An absolute Ca deficiency, particularly at the low Ca‐supply levels of 30 and 60 μM Ca, manifested itself initially in the form of marginal necrosis in younger, but not in the youngest, leaves of the potato plants. Further symptoms were rolling of the leaf lamina, browning of veins and roots, and finally necrosis also of the youngest leaves. Only in an advanced stage of Ca deficiency, the meristem of the shoots died. Ca‐deficiency symptoms could be expected at a Ca content in the leaves of less than 5 mg Ca (g dry weight)^?1. However, there was no close negative correlation between the extent of leaf damage and the total Ca content of the leaves. In order to obtain information about the Ca concentration in the apoplast fluid of the leaves, apoplastic washing fluid was extracted by an infiltration‐centrifugation technique. A low Ca supply reduced the Ca concentration both in the apoplast fluid of the leaves and in the cell walls. Up to 60% more diffusible pectin fragments were then found in the apoplast of younger leaves, as compared to the control supplied with an optimum Ca level of 1000 μM. The amount of diffusible pectins accounted for 1–2% of the total pectin content of younger potato leaves. The size of the existing pectin fragments varied depending on the Ca supply. Compared with an optimum Ca supply of 1000 μM, fewer monomers and up to 7 times more diffusible pectin fragments with a degree of polymerization 9–20 were present at the low Ca‐supply level (30 μM). In addition, polygalacturonase activity in tissue homogenates increased remarkably with Ca deficiency. Thus it appears that one major effect of Ca deficiency was a stimulation of the activity of polygalacturonase, which could control the breakdown of pectic polysaccharides in the cell wall. Whether the release of potentially biologically active pectic fragments in cell walls might be involved in the occurrence of Ca‐deficiency symptoms is discussed.     

Inter-specific differences in photosynthetic carbon uptake, photosynthate partitioning and extracellular organic carbon release by deep-water characean algae

1. The effect of light intensity on photosynthesis and the fate of newly fixed organic carbon was compared for three characean algae collected at the same depth (10 m) but differing in their depth distributions. For each species we determined photosynthesis–irradiance (P–E) responses, the partitioning of newly fixed carbon into four intracellular pools (low molecular‐weight compounds, polysaccharides, lipids and proteins) and the extracellular organic carbon (EOC) release at a range of photon flux densities (PFD) 0–60 μmol m^–2 s^–1. 2. The P–E responses differed between the three species, with the light compensation point (E_c) and dark respiration rate highest in the shallowest species (Chara fibrosa), intermediate in the mid‐range species (C. globularis) and lowest in the deepest species (C. corallina). Photosynthetic efficiency (α) and photosynthesis: respiration ratios were lowest in C. fibrosa and highest in C. corallina. 3. In all three species, the low molecular weight pool was the principal photosynthetic product (>60% of fixed C) at 3 μmol m^–2 s^–1 PFD, but its proportional contribution decreased rapidly with increasing irradiance. Polysaccharide rose to become the major product (>35% of fixed C) at saturating PFD (35 μmol m^–2 s^–1). 4. Protein synthesis was saturated at 5 μmol m^–2 s^–1 in all species and was consistently a lower proportion of the fixed carbon in C. corallina than the other species. The fraction incorporated in the lipid pool increased slightly with irradiance but was always less than 10% of fixed C, while the proportion lost as EOC was unaffected by light, being significantly higher in C. fibrosa than the other species. 5. A kinetic experiment with C. fibrosa at 35 μmol m^–2 s^–1 PFD revealed a continued increase in net polysaccharide, protein and lipid synthesis during a 22.5‐h light period, whereas the net size of the low molecular weight pool remained constant. In a subsequent dark period, protein and lipid synthesis continued at the expense of the polysaccharide and low‐molecular‐weight pools. The EOC release rose to a constant low release in the light, then peaked slightly immediately after the dark–light transition before returning to the same rate as in the light. Extrapolating these data over 24 h suggests that the proportion of fixed carbon lost as EOC may be as high as 10% in this species. 6. The interspecific differences in carbon acquisition between the three species reflected their depth distributions, with the deeper species having more efficient photosynthetic metabolism, lower P:R ratios and less EOC release, although no apparent differences in internal partitioning of photosynthate.     

Phytic acid: divalent cation interactions: III. A calorimetric and titrimetric study of the pH dependence of copper(II) binding

The interaction of the cupric ion with phytic acid as a function of pH has been studied by potentiometric and thermal titration and by the determination of ligand binding. As has been found for the reaction of zinc and calcium cations with phytate, the presence of the Cu(II) ion results in a displacement of the titration curves to more acid values. Evaluation of the parameters that describe such changes in ionization behavior by curve-fit analysis showed that as the Cu(II):phytate mol ratio was increased from one to eight, the pK' values of the ionizable group sets of phytic acid (ranging from 1.59 to 9.79) were consolidated into just two sets with curve-fit (CP) values ca. 1.5 and 3.7. Marked pH hysteresis effects are seen in such systems because of the pronounced acid strength of the Cu(II):aqua ion and the Cu(II) ligand aqua ion complex. The combined heat of binding and precipitation (plus solvation changes, etc.) of Cu(II) to phytate is endothermic (21.8–22.2 kcal mol⁻¹). This is similar in magnitude to that reported for the binding of either Zn(II) or Ca(II) to phytate. In the titration of Cu(NO₃)₂ with KOH, presumably to form Cu(OH)₂, ΔH° was exothermic (−12.5 kcal mol⁻¹). From measurements of free Cu(II) cation concentration in the presence of phytate the binding reaction was found to be stoichiometric with 6 mols Cu(II) bound at pH 6. Binding occurs within the pH range 2–6. An apparent necessary requirement for binding is the availability of the oxo dianion structure formed from the second dissociation step of a phosphoryl group. Curve-fit analysis of the binding data as a function of pH showed that a group or group set with CP value ca. 4 governs the binding reaction(s) at all mol ratios of Cu(II) to phytate examined. It is suggested that the binding of cupric ions to phytate may occur to the equatorial rather than the axial configuration as suggested for Ca(II) binding. A space-filling molecular model to illustrate this has been constructed. Soluble Cu(II):phytate complexes are formed within the pH range from 2 to ca. 3.4. This is supported by the results of difference absorption spectrometry.     

NITRATE UPTAKE BY LAMINARIA LONGICRURIS (PHAEOPHYCEAE)1,2

Laminaria longicrucis De la Pylaie took up exogenous nitrate under both summer and winter conditions. During July and August no NO₃^- was detected in the ambient water or in algal tissues although it was present in both in February. Discs (2.3 cm diam.) of thin blade tissue were incubated with NO₃^- at four temperatures, with and without illumination. Similar values Jor NO₃^- uptake were found for both summer and winter collected plants when measured in light at 0 C. An apparent K of 4–6 μM was recorded for both types of plants; the V_max ranged from 7 to 10 μmol h^-1 g^-1 dry wt measured in ca. 1800 μW cm^-2 of cool-white fluorescent light. Uptake rates at 5 C were 66%, and at 0 C 30% of those for controls run at 15 C. The alga scavenged NO₃- from solutions <0.5 μM. Ammonia did not inhibit NO₃- uptake. Antibiotic pretreatment reduced NO₃- uptake by a maximum of 12%. Nitrite uptake was inhibited in proportion to the concentration of NO₃- in the medium.     

The effect of methylmercury (II) binding on the conformation of poly(L-glutamic acid) and poly(L-lysine: a Raman spectroscopic study

The binding of the methylmercury cation CH₃Hg⁺ by poly(L -glutamic acid) (PGA) and by poly(L -lysine) (PLL) has been investigated by Raman spectroscopy. Coordination on the side-chain COO^? and NH groups of these polypeptides gave characteristic ligand–Hg stretching modes at ca. 505 and 450 cm^?1, respectively. Precipitation generally occurred upon formation of the complexes and changes of conformation were common. The solid complex obtained from PGA at pH 4.6 was found to have a mostly disordered conformation, which differed from the respective α-helical and β-sheet structures of the dissolved and precipitated uncomplexed polypeptide in the same conditions. An α-helical structure was generally adopted by the complex formed with PLL, even in pH and temperature conditions where the free polypeptide normally exists in another conformation. The addition of a stronger complexing agent, glutathione, to the PLL/CH₃Hg⁺ complex caused a migration of the bound cations and a restoration of the polypeptide to its original state.     

SIMULTANEOUS ASSIMILATION OF AMMONIUM AND NITRATE BY GELIDIUM NUDIFRONS (GELIDIALES: RHODOPHYTA)1

Simultaneous assimilation of NH₄ and NO₃ by Gelidium nudifrons Gardner was observed in culture experiments of 4 possible combinations of NH₄ and NO₃. The combinations tested were those in which the concentration of both N sources were in the range of 3.0–4.0 μg-atN · l^?1; both in the range of 0.5–1.0 μg-atN · l^?1; one in the 3.0–4.0 μg-atN · l^?1 range and the other in the 0.5–1.0 μg-atN · l^?1 range; and, visa versa. The data suggest that the pools of both NH₄ and NO₃ are simultaneously available for algal assimilation.     

The extracellular polysaccharides of Rhizobium japonicum: Compositional studies

The extracellular polysaccharides of seven strains of Rhizobium japonicum were investigated by using a gas-chromatographic scheme developed for determination of the various sugars present. These polysaccharides were more heterogeneous in their composition than those of any other species of Rhizobium yet examined. Five strains (1809, 110, 123, 127, and 709) produced polysaccharides containing the same constituents, although in varying relative amounts: glucose (36–44%), galactose (7–25%), mannose (18–20%), 4-O-methylgalactose (5–13%), galacturonic acid (12–16%), and acetyl groups (4–8%). The sugars of the polysaccharide of strain 1809 were all of the d series. These are the first bacterial polysaccharides reported to contain 4-O-methylgalactose and the first Rhizobium polysaccharides in which galacturonic acid has been found. In contrast to this, the polysaccharide of strain 129 consisted of glucose (7%), galactose (51%), mannose (5%), xylose (5%), glucuronic acid (5%), and pyruvic acid (2%). The polysaccharide of strain 711 contained glucose (34%), galactose (13%), mannose (27%), and pyruvic acid (6%).