Isolation and characterization of wheat peroxidase isoenzyme B1

Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p-coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn²⁺ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o- and p- diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H₂O₂ from oxygen. Mn²⁺ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product.     

Structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site

Langerin mediates the carbohydrate-dependent uptake of pathogens by Langerhans cells in the first step of antigen presentation to the adaptive immune system. Langerin binds to an unusually diverse number of endogenous and pathogenic cell surface carbohydrates, including mannose-containing O-specific polysaccharides derived from bacterial lipopolysaccharides identified here by probing a microarray of bacterial polysaccharides. Crystal structures of the carbohydrate-recognition domain from human langerin bound to a series of oligomannose compounds, the blood group B antigen, and a fragment of β-glucan reveal binding to mannose, fucose, and glucose residues by Ca²⁺ coordination of vicinal hydroxyl groups with similar stereochemistry. Oligomannose compounds bind through a single mannose residue, with no other mannose residues contacting the protein directly. There is no evidence for a second Ca²⁺-independent binding site. Likewise, a β-glucan fragment, Glcβ1-3Glcβ1-3Glc, binds to langerin through the interaction of a single glucose residue with the Ca²⁺ site. The fucose moiety of the blood group B trisaccharide Galα1-3(Fucα1-2)Gal also binds to the Ca²⁺ site, and selective binding to this glycan compared to other fucose-containing oligosaccharides results from additional favorable interactions of the nonreducing terminal galactose, as well as of the fucose residue. Surprisingly, the equatorial 3-OH group and the axial 4-OH group of the galactose residue in 6SO₄-Galβ1-4GlcNAc also coordinate Ca²⁺, a heretofore unobserved mode of galactose binding in a C-type carbohydrate-recognition domain bearing the Glu-Pro-Asn signature motif characteristic of mannose binding sites. Salt bridges between the sulfate group and two lysine residues appear to compensate for the nonoptimal binding of galactose at this site.     

Common Polymorphisms in Human Langerin Change Specificity for Glycan Ligands

Langerin, a C-type lectin on Langerhans cells, mediates carbohydrate-dependent uptake of pathogens in the first step of antigen presentation to the adaptive immune system. Langerin binds a diverse range of carbohydrates including high mannose structures, fucosylated blood group antigens, and glycans with terminal 6-sulfated galactose. Mutagenesis and quantitative binding assays indicate that salt bridges between the sulfate group and two lysine residues compensate for the nonoptimal binding of galactose at the primary Ca²⁺ site. A commonly occurring single nucleotide polymorphism (SNP) in human langerin results in change of one of these lysine residues, Lys-313, to isoleucine. Glycan array screening reveals that this amino acid change abolishes binding to oligosaccharides with terminal 6SO₄-Gal and enhances binding to oligosaccharides with terminal GlcNAc residues. Structural analysis shows that enhanced binding to GlcNAc may result from Ile-313 packing against the N-acetyl group. The K313I polymorphism is tightly linked to another SNP that results in the change N288D, which reduces affinity for glycan ligands by destabilizing the Ca²⁺-binding site. Langerin with Asp-288 and Ile-313 shows no binding to 6SO₄-Gal-terminated glycans and increased binding to GlcNAc-terminated structures, but overall decreased binding to glycans. Altered langerin function in individuals with the linked N288D and K313I polymorphisms may affect susceptibility to infection by microorganisms.     

High seed Mn content does not affect germination of in vitro produced Centaurium pulchellum seeds

The effect of high Mn²⁺ content on Centaurium pulchellum seed germination has been investigated. Seeds containing extremely high Mn²⁺ content were produced by culturing single-node flowering explants for 2 months in the MS-media, supplemented with Mn in concentrations ranging from 1 to 10,000 μM. Although the seeds displayed the capacity to accumulate high amount of Mn, their germination was undisturbed. EPR spectroscopy was used to measure the ratio of free (aqueous) Mn to bound Mn and it was found that over 97% of total Mn was in the bound form. With elevating the external Mn supply, seed Mn concentration also increased, but the proportion of free Mn²⁺ fraction decreased from 3% in the control (1 μM Mn) to 0.35% and 0.15% in high Mn supply (1000 μM and 10,000 μM, respectively). These results suggest that an elevation of internal Mn concentration in seeds is associated with increased Mn binding pools, hence Mn remains bound during germination. Consequently, the action of potentially harmful Mn²⁺ ions, which may generate ROS and affect seed viability, is alleviated.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins.     

A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2

Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1–3/4GlcNAcβ1–2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca²⁺ in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.     

Interconversion and translocation of free sugars during galactomannan utilization in germinating guar (Cyamopsis tetragonoloba) seed

With the onset of the degradation of galactomannan, the galactose and mannose levels increased in the endosperm. The hydrolysis of galactomannan was more or less complete within the first 3 days of germination. In the cotyledons, sucrose was the predominant free sugar during the period of rapid galactomannan hydrolysis and reducing sugars (glucose + fructose) were present in only 10–20% proportion. The level of soluble acid invertase activity was in the order of embryonic axis > endosperm > cotyledons. On the basis of (a) absence of galactose and mannose, (b) high proportion of sucrose, (c) very fast conversion of [¹⁴C]glucose and [¹⁴C]mannose to [¹⁴C]sucrose and (d) very low levels of both soluble and bound invertases in cotyledons, we conclude that there is an active synthesis of sucrose in this tissue where disaccharide seems to be least hydrolysed during the period of galactomannan mobilization. A rapid hydrolysis of galactomannan in endosperm during early germination resulted in the synthesis of some starch, as a temporary reserve, in cotyledons. When the cotyledons entered the phase of first leaf formation, cotyledonary sucrose was hydrolysed giving rise to invert sugars. In the embryonic axis, the increase in the ratio of reducing sugars to sucrose coupled with a higher level of invertase, compared with sucrose-UDP glucosyl transferase, indicated that free sugars from the cotyledons are translocated to the embryonic axis as sucrose.     

Glycosylation and posttranslational maturation of glycoproteins in embryonal carcinomas: identification of two distinct pools of high-mannose glycans

Embryonal carcinomas and early embryonic cells assemble a family of unusually large and complex carbohydrates. These glycans contain large amounts of the sugars galactose and N-acetylglucosamine and are decorated with fucose, sulfate, and sialic acids. We show that, by their sensitivity to inhibition by tunicamycin and by their resistance to cleavage by alkaline hydrolysis, in teratocarcinoma stem cells the expression of these glycans is on asparagine-linked cores. These glycans are part of the large spectrum of glycans that are assembled on mannose cores derived from a common, lipid-linked precursor glycan. We examined the fate of this precursor glycan after its transfer to protein and found that there are two distinct pools of protein-linked, high-mannose glycans, which can be distinguished on the basis of their rate of processing. One pool is processed rapidly to provide a wide spectrum of complex-type glycans. This processing occurs efficiently with little evidence of intermediate structures. The other, larger pool remains unprocessed, beyond glucose removal, at a time when complex-type glycans cease to accumulate. In contrast, high-mannose glycans are relatively minor components of the glycans labeled during long-term, continuous labeling, and in this situation they are processed to provide a spectrum of trimmed glycans.     

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.     

The stimulus-secretion coupling of glucose-induced insulin release. Does glycolysis control calcium transport in the B-cell?

1. The metabolism of glucose and the exchangeable Ca2+ pool were measured in rat pancreatic islets, in order to assess the possible significance of glycolysis in the process of glucose-induced insulin release. 2. At high glucose concentration (16.7 mM), glucose was metabolized at the following rate (pmol of glucose residue/h per islet +/- S.E.M.): 131 +/- 11 for glucose uptake, 129+/-8 for glucose utilization, as judged by the conversion of [5-3H]glucose into 3H2O,60+/-2 for lactate output and 25+/-2 for glucose oxidation. 3. The secretory pattern usually correlated with the metabolic data. For instance, the ability of different sugars (glucose, mannose, fructose, galactose, D-glyceraldehyde) to stimulate lactate output closely paralleled their relative insulinotropic capacity. A disparity between metabolic and secretory responses was, however, encountered in the presence of dibutyryl cyclic AMP and theophylline. 4. Despite this contrasting behaviour, the size of the Ca2+- exchangeable pool (net uptake of 45Ca2+) was invariably proportional to the rate of lactate output under all experimental conditions examined. It is concluded that glycolysis usually exerts a tight control on the rate constant for Ca2+ transport across the B-cell membrane.     

Isolation and properties of hexokinase from loranthus leaves

Hexokinase was partially purified from the leaves of Dendrophthoe falcata. The optimum pH for the enzyme was 8.5. The enzyme was sensitive to p-CMB and the inhibition could be reversed by 2-mercaptoethanol. The optimum temperature was 40° and energy of activation 6900 cal/mol. The enzyme had an absolute requirement for a divalent metal ion. Although Mg²⁺ was the preferred metal, it could be partially replaced by Mn²⁺ and Ca²⁺. ATP was the most effective phosphoryl donor. Glucose was the best substrate, the K_m values of 0.14 and 0.26 mM were obtained at saturated and sub-saturated ATP concentration. Phosphorylation coefficients show the following order of reactivity of sugars: glucose mannose 2-deoxy D-glucose fructose glucosamine galactose ribose. The K_m value for ATP was 0.16 mM, which increased to 0.35 mM in the presence of 0.5 mM ADP. ADP and 5′-AMP were competitive inhibitors with respect to ATP, and K_i values were 0.4 and 1.2 mM respectively.     

Isolation and characterization of a new class of acidic glycans implicated in sea urchin embryonal cell adhesion

Three major glycan fractions of 580 kDa (g580), 150 kDa (g150), and 2 kDa (g2) were isolated and purified from Lytechinus pictus sea urchin embryos at the mesenchyme blastula stage by gel filtration and high pressure liquid chromatography. Chemical analysis, by gas chromatography, revealed that g580 is highly sulfated and rich in N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, and fucose. The g150 fraction is less acidic than g580 and contains high amounts of amino sugars, xylose, and mannose. The g2 fraction is neutral, rich in N-acetylglucosamine, mannose, and galactose. The g580 and g150 fractions are resistant to glycosaminoglycan-degrading enzymes, indicating that they are distinct from the glycosaminoglycans. The g580 fraction resembles, with respect to chemical composition, a previously characterized 200 kDa sponge adhesion glycan (g200). The binding of the monoclonal antibody Block 2, which recognizes a repetitive epitope on g200, as well as of the anti-g580 polyclonal antibodies to both g580 and g200 indicated that these two glycans share similar antigenic determinants. The Fab fragments of the Block 2 antibody, which previously have been shown to inhibit cell adhesion in sponges, also blocked the reaggregation of dissociated sea urchin mesenchyme blastula cells. These results indicate that g580 carries a carbohydrate epitope, similar to the sponge adhesion epitope of g200, which is involved in sea urchin embryonal cell adhesion.     

Diversity of the Carbohydrate Composition of the Antigenic Polysaccharides of Proteobacteria of the Genera Pseudoalteromonasand Marinomonas

The sugar analysis of the glycans of the type strains of marine proteobacteria of the genera Pseudoalteromonasand Marinomonas—Pseudoalteromonas atlanticaIAM12927^T, P. aurantiaNCIMB 2033^T, P. citreaATCC 29719^T, P. elyakoviiKMM 162^T, P. espejianaATCC 29659^T, P. piscicidaNCIMB 645^T, P. tetraodonisIAM 14160^T, Marinomonas communisATCC 27118^T, and M. vagaATCC 27119^T—showed that they contain glucose, galactose, galactosamine, glucosamine, fucose, rhamnose, mannose, heptose, 2-keto-3-deoxyoctonate (KDO), uronic acids, colitose (3,6-dideoxy-L-xylo-hexose), and 6-deoxy-L-talose. The carbohydrate composition of the antigenic polysaccharides (PSs) of P. elyakoviiKMM 162^Tand P. espejianaATCC 29659^Tdepended on the type and the concentration of carbohydrate substrates in the nutrient media. The molar proportion between rhamnose, glucose, and galactose (ca. 1 : 0.3 : 2) in the PS of P. elyakoviiKMM 162^Twas almost the same in the media lacking carbohydrates or containing glucose or galactose at a concentration of 1 g/l. At the same time, the molar proportion between fucose, glucose, galactose, galactosamine, and glucosamine (ca. 1 : 1 : 1 : 2 : 0.5) in the PS of P. espejianaATCC 29659^Tdepended on the presence and the concentration of carbohydrate substrates in the medium. A high concentration of glucose in the medium (30 g/l) brought about a rise in the content of glucose in PSs (9-fold for the PS of P. elyakoviiKMM 162^Tand 4.6-fold for the PS of P. espejianaATCC 29659^T) and led to a decrease in the content of other carbohydrates. The cultivation of these two strains at a lactose concentration of 30 g/l resulted in their PSs containing glucose and galactose in about equal proportions (ca. 1 : 1 in the case of P. espejianaATCC 29659^Tand ca. 2.1 : 1.7 in the case of P. elyakoviiKMM 162^T).     

Comparison of industrial yeast strains for fermentation of spent sulphite pulping liquor fortified with wood hydrolysate

Ethanol production from spent sulphite pulping liquor (SSL) was compared for four different yeasts. A second strain of S. cerevisiae as well as a 2-deoxyglucose-resistant strain formed through protoplast fusions between S. uvarum and S. diastaticus produced up to 27% more ethanol from SSL fortified with hydrolysis sugars than was produced by S. cerevisiae. The incremental improvement in ethanol yield appeared to vary with the degree of fortification, ranging from 5.8% for unfortified SSL, to 27% for the highest level of fortification tested. Decreasing fermentation rates were observed for SSL fortified with glucose, mannose and galactose, respectively. Sugar uptake rates in SSL fortified with glucose, galactose and mannose were 6.8, 2.8 and 2.0 g L⁻¹ h⁻¹, respectively. However, when these sugars were fermented along with a glucose cosubstrate, the rate at which the combined glucose/mannose medium was fermented was nearly identical to that of the glucose control. Received 18 April 1996/ Accepted in revised form 27 August 1996     

Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations

In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at <0.2 mM, and supplementing medium with copper sulfate, were found to be effective in reducing lactate accumulation. Among them, copper sulfate supplementation was found to be critical for process optimization when glucose was in excess. When copper sulfate was supplemented in the new process, two-fold increase in cell density (66.5 ± 8.4 × 10⁶ cells/mL) and titer (11.9 ± 0.6 g/L) was achieved. Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.     

Transient expression of sialylated glycans during glycoprotein processing by embryonal carcinomas

Embryonal carcinoma and early embryonic cells express unusually large and complex carbohydrates on their surfaces that are lost during differentiation. These carbohydrates are composed of alternating galactose and N-acetylglucosamine residues and have either linear or branched architectures. Compared to the glycans expressed by many differentiated cells these glycans are poorly sialylated. However, metabolic studies reveal that there is a transient expression of sialylated glycans during the processing of glycoproteins by embryonal carcinomas. After a short pulse with mannose the major complex-type glycan is a biantennary glycan with two sialic acids. During subsequent chase periods this glycan species is replaced by unsialylated glycans that have elongated branches composed of alternating galactose and N-acetylglucosamine residues.     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

Effect of Low Levels of Calcium on Exudation of Sugars and Sugar Derivatives from Intact Peanut Roots under Axenic Conditions

The effects of 10, 20, 35 and 50 mg of Ca²⁺ per liter on the qualitative and quantitative exudation of sugars from roots of 5-week-old peanut plants, Arachis hypogaea L., grown axenically in nutrient solutions, were measured. Nutrient solutions in which plants had been growing were collected at weekly intervals for 4 weeks, sugars in them were measured by gasliquid chromatography of the trimethylsilyl derivatives. Arabinose, ribose, xylose, fructose, mannose, glucose, galactose, mannitol, galacturonic acid, inositol, sucrose, and five unknowns were found. Qualitative and quantitative differences in exudates were correlated with age of the plants and calcium level. Four times more sugar was exuded at 10 mg than at 50 mg of Ca²⁺ per liter but no significant differences in growth were observed. Ion efflux measurements suggested that low levels of Ca²⁺ increased root cell membrane permeability.     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.