Binding of bivalent cations by xanthan in aqueous solution

The interaction between xanthan and selected bivalent cations (Ca(2+), Mg(2+), Mn(2+), Fe(2+), Cu(2+), Zn(2+), Cd(2+) and Pb(2+)) was studied by means of conductometry, viscometry, and nuclear magnetic resonance spectroscopy. Xanthan from Xanthomonas campestris was studied in comparison with dextran from Leuconostoc mesenteroides. While dextran does not develop specific interactions with the bivalent cations, the analysis of the experimental data shows that xanthan chains (M(n) approximately 1.4x10(5) to 2.9x10(6)g/mol) reversibly bind Me(2+) species in aqueous solution at pH 6. Conductometric and viscometric titrations show that a single bivalent cation forms a complex which involves two disaccharide units of the main chain together with two side chains. Based on dipolar magnetic interactions between Mn(2+) and individual carbon positions of xanthan, a possible structure of a chelate-like complex is proposed which involves the pyruvate units at the terminal ends of the side chains as the main binding sites. According to the stoichiometric relation between cations and disaccharide units, a single bivalent cation is bound between the terminal ends of two side chains, leading to an intramolecular cross-link and a reduced hydrodynamic radius of the overall macromolecule. The results indicate that heavy metal ions (Cd(2+) and Pb(2+)) link stronger to the xanthan chain than lighter cations (Ca(2+) and Mg(2+)), a fact which may be of ecological relevance.     

A sulfated polysaccharide from the sarcoplasmic reticulum of sea cucumber smooth muscle is an endogenous inhibitor of the Ca(2+)-ATPase

Vesicles derived from the endoplasmic reticulum of sea cucumber smooth muscle retain a membrane bound Ca(2+)-ATPase that is able to transport Ca(2+) into the vesicles at the expense of ATP hydrolysis. In contrast with vesicles obtained from rabbit muscles, the activity of the Ca(2+)-dependent ATPase from sea cucumber is dependent on monovalent cations (K(+)>Na(+)>Li(+)). With the addition of highly sulfated polysaccharide to vesicle preparations from rabbit muscle, Ca(2+) uptake decreases sharply and becomes highly sensitive to monovalent cations, as observed with vesicles from sea cucumber muscle. These results led us to investigate the possible occurrence of a highly sulfated polysaccharide on vesicles from the endoplasmic reticulum of sea cucumber smooth muscle, acting as an "endogenous" Ca(2+)-ATPase inhibitor. In fact, vesicles derived from the invertebrate, but not from rabbit muscle, contain a highly sulfated polysaccharide. This compound inhibits Ca(2+) uptake in vesicles obtained from rabbit muscle and the inhibition is antagonized by monovalent cation. In addition, sea cucumber muscles contain high concentrations of another polysaccharide, which surrounds the muscle fibers, and was characterized as a fucosylated chondroitin sulfate. Possibly the occurrence of sulfated polysaccharides in the sea cucumber muscles is related with unique properties of the invertebrate body wall, which can rapidly and reversibly alter its mechanical properties, with change in length by more than 200%.     

Ionomycin, a carboxylic acid ionophore, transports Pb(2+) with high selectivity

Studies utilizing phospholipid vesicle loaded with chelator/indicators for polyvalent cations show that ionomycin transports divalent cations with the selectivity sequence Pb(2+) > Cd(2+) > Zn(2+) > Mn(2+) > Ca(2+) > Cu(2+) > Co(2+) > Ni(2+) > Sr(2+). The selectivity of this ionophore for Pb(2+) is in contrast to that observed for A23178 and 4-BrA23187, which transport Pb(2+) at efficiencies that are intermediate between those of other cations. When the selectivity difference of ionomycin for Pb(2+) versus Ca(2+) was calculated from relative rates of transport, with either cation present individually and all other conditions held constant, a value of approximately 450 was obtained. This rose to approximately 3200 when both cations were present and transported simultaneously. 1 microM Pb(2+) inhibited the transport of 1 mM Ca(2+) by approximately 50%, whereas the rate of Pb(2+) transport approached a maximum at a concentration of 10 microM Pb(2+) when 1 mM Ca(2+) was also present. Plots of log rate versus log ionomycin or log Pb(2+) concentration indicated that the transporting species is of 1:1 stoichiometry, ionophore to Pb(2+), but that complexes containing an additional Pb(2+) may occur. The species transporting Pb(2+) may include H.IPb.OH, wherein ionomycin is ionized once and the presence of OH(-) maintains charge neutrality. Ionomycin retained a high efficiency for Pb(2+) transport in A20 B lymphoma cells loaded with Indo-1. Both Pb(2+) entry and efflux were observed. Ionomycin should be considered primarily as an ionophore for Pb(2+), rather than Ca(2+), of possible value for the investigation and treatment of Pb(2+) intoxication.     

Functional dependence on calcineurin by variants of the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger Vcx1p

The Ca(2+)-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger Vcx1p. In yeast and plants, cation/H(+) exchangers are important in shaping cytosolic Ca(2+) levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca(2+), Mn(2+) and Cd(2+). Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca(2+) transporter and may also function in Cd(2+) transport, but cannot transport Mn(2+). Furthermore, direct Ca(2+) transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn(2+) tolerance. Here we demonstrate that this Mn(2+) tolerance is independent of the Ca(2+)/Mn(2+)-ATPase Pmr1p and results from Mn(2+)-specific vacuolar transport activity of Vcx1-M1p. This Mn(2+) transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn(2+) tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.     

Microbial polysaccharides with actual potential industrial applications

The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Baker's Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose. Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined. The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents. A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars. The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.     

Imaging of individual biopolymers and supramolecular assemblies using noncontact atomic force microscopy

A variety of biopolymers is imaged using noncontact atomic force microscopy. Samples are prepared by aerosol spray deposition of aqueous solutions on freshly cleaved mica followed by air drying. The distributions of contour lengths and chain or fibril thicknesses normal to the mica substrate can be measured for individual polymer molecules or molecular assemblies. In many cases it is possible to conclude that the structures imaged and quantitatively analyzed are representative of those present in solution and not artifacts of the deposition/dessication process. Imaging of linear and cyclic triple helices of the polysaccharide scleroglucan is demonstrated. Measurements of the triple helix thickness normal to the mica surface are analyzed, and successful measurements of the molecular weight distribution and mean molar mass are described. It is demonstrated that the extent of chain association in the polysaccharide xanthan can be modulated by the addition of low molecular weight salts. The contour length and chain thickness distributions in a xanthan fraction are presented. Increases in the extent of chain association with increasing polymer concentration are documented for the gelling polysaccharide gellan, and the formation of stiff fibrillar gellan aggregates in the presence of added low molecular salt is demonstrated. Images are presented of the polysaccharide κ-carrageenan in its disordered, and presumably single-stranded, state. Biopolymers other than polysaccharides can be imaged by the same technique; this is demonstrated with the fibrous protein collagen. In general it is shown that aerosol spray deposition of biopolymer samples can be used in conjunction with noncontact atomic force microscopy to provide a fast, reliable, and reproducible method for assessing the size and shape distributions of individual biological macromolecules and macromolecular assemblies in solution with a minimum of time and effort devoted to sample preparation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 133–146, 1997     

Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2

In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H(+) antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.     

An enzyme system hydrolysing the polysaccharides of Xanthomonas species

Enzymes hydrolysing the exopolysaccharides of Xanthomonas campestris and related species (xanthan) have been obtained from a Bacillus species isolated by enrichment culture. Growth on xanthan induced a number of enzymes acting on the xanthan molecule. These included one or more β-glucanohydrolases and β-glucosidases, together with mannosidases. The former activities were also present in cultures grown in the presence of laminaran or scleroglucan, but not in simple synthetic media with glucose as substrate. Partial purification of the enzymes active on glucans was achieved by ammonium sulphate precipitation and chromatography on DEAE-sepharose and CM-sepharose. The specificity of the β-glucosidase and the β-glucanohydrolase were investigated. Several β-glucans were hydrolysed to glucose and disaccharides, but there was no activity against β→ 6 linked polymers, cellulose azure or microcrystalline cellulose. Carboxymethylcellulose was hydrolysed, as were laminaran, scleroglucan and pachyman. Activity was greater against the β→ 4 linked glucans than against the β→ 3 linked glucans tested. As periodate-oxidized laminarin was also hydrolysed, it was concluded that the glucanohydrolase acted as an endo enzyme. The β-glucosidase had a pH optimum at about 8–2 and a temperature optimum at 45°C; it showed higher activity against o -nitrophenyl-D-glucopyranoside, cellobiose, trehalose and sophorose than against gentibiose.     

Selectivity of alkali cation influx across the plasma membrane of oat roots: cation specificity of the plasma membrane ATPase

Influx of alkali cations (Li(+), Na(+), K(+), Rb(+), Cs(+)) across plasma membranes of cells of excised roots of Avena sativa cv. Goodfield was selective, but different, in the absence and in the presence of 1 mm CaSO(4). Ca(2+) reduced the influx rates of all of the alkali cations-especially Na(+) and Li(+). Transport selectivity changed as the external concentrations of the alkali cations increased.Plasma membrane ATPase, purified from Avena sativa roots, was differentially stimulated by alkali cations. This specificity, however, was not altered by Ca(2+) or the external cation concentrations. A close correspondence existed between the relative influx rates of K(+), Rb(+), and Cs(+) and the relative stimulation of the ATPase by these cations. A similar correspondence did not occur for Na(+) and Li(+).Selective cation transport in oat roots could result, in part, from the specificity of the plasma membrane ATPase, but other factors such as specific carriers or porters or differential diffusion rates must also be involved.     

Differences between manganese and magnesium ions with regard to fatty acid biosynthesis, acetyl-coenzyme A carboxylase activity and malonyl-coenzyme A decarboxylation

Fatty acid-biosynthetic activity in rat liver cytosol fractions is much greater when the bivalent cation in the assay system is Mn(2+) than when it is Mg(2+). This difference between bivalent cations can be abolished if the cytosol fractions are preincubated with isocitrate and the bivalent cation for 30min before assay of fatty acid-biosynthetic activity. In a search for the biochemical basis of this phenomenon, the following differences between Mg(2+) and Mn(2+) were established: (1) Mn(2+) promotes acetyl-CoA carboxylase activity of the protomeric form of the enzyme under conditions in which Mg(2+) does not; (2) Mn(2+)+ATP have little inhibitory effect on the polymerization of acetyl-CoA carboxylase whereas Mg(2+)+ATP are markedly inhibitory; (3) under conditions in which utilization of malonyl-CoA in condensation reactions is prevented, the steady-state concentration of malonyl-CoA formed by a cytosol fraction is much greater with Mn(2+) than with Mg(2+). The role that each of these specific differences between Mn(2+) and Mg(2+) might play in causing liver cytosol preparations to have greater fatty acid-biosynthetic activity in the presence of Mn(2+) is discussed.     

Sorption of anionic polysaccharides by cellulose

The sorption of anionic polysaccharides pectin, alginate, and xanthan with cellulose were investigated in presence of calcium. Calcium sorption to cellulose was limited by the carboxyl group content in fibers. Atomic Absorption Spectroscopy (AAS) analysis was used to measure the calcium in cellulose fibers and chemical oxygen demand (COD) analysis reveals that the divalent ions calcium can bind the polysaccharide onto cellulose fibers. The amount of calcium and polysaccharide bound in Ca²⁺/polysaccharide modified cellulose fibers was 5.8-12.5 mM Ca²⁺/kg fibers and 1500-2400 mg polysaccharide/kg fibers, respectively. Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR) analysis confirmed the presence of polysaccharide on calcium containing cellulose fibers. The results of alizarin dyeing experiments at the end of polysaccharide sorption further confirmed the presence of calcium in Ca²⁺/polysaccharide modified cellulose fibers. The basic phenomenon of interaction of soluble ionic polysaccharide and cellulosic fibers in presence of divalent cations such as calcium is a key to understand biological functions and technological applications.     

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.     

A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance

The Arabidopsis AtMTP family of genes encode proteins of the cation diffusion facilitator (CDF) family, with several members having roles in metal tolerances. Four of the 11 proteins in the family form a distinct cluster on a phylogenetic tree and are closely related to ShMTP8, a CDF identified in the tropical legume Stylosanthes hamata that is implicated in the transport of Mn(2+) into the vacuole as a tolerance mechanism. Of these four genes, AtMTP11 was the most highly expressed member of the Arabidopsis subgroup. When AtMTP11 was expressed in Saccharomyces cerevisiae, it conferred Mn(2+) tolerance and transported Mn(2+) by a proton-antiport mechanism. A mutant of Arabidopsis with a disrupted AtMTP11 gene (mtp11) was found to have increased sensitivity to Mn(2+) but not to Cu(2+) or Zn(2+). At a non-toxic but sufficient Mn(2+) supply (basal), the mutant accumulated more Mn(2+) than the wild type, but did not show any obvious deleterious effects on growth. When grown with Mn(2+) supplies that ranged from basal to toxic, the mutant accumulated Mn(2+) concentrations in shoots similar to those in wild-type plants, despite showing symptoms of Mn(2+) toxicity. AtMTP11 fused to green fluorescent protein co-localized with a reporter specific for pre-vacuolar compartments. These findings provide evidence for Mn(2+)-specific transport activity by AtMTP11, and implicate the pre-vacuolar compartments in both Mn(2+) tolerance and Mn(2+) homeostasis mechanisms of Arabidopsis.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning alpha-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of "the cation" in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation相似文献   

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exopolysaccharide production by free and immobilized microbial cultures

The batch production of different exopolysaccharides (alginate, xanthan, pullulan, dextran) by free and immobilized microbial cultures was investigated. First, conventional free-cell cultures were performed to obtain control fermentation parameters and macromolecular characteristics of exopolysaccharides. Then microbial cultures were immobilized in composite agar layer/microporous membrane structures and tested for polysaccharide production. The immobilized-cell system proved unsuitable for xanthan and pullulan production. Owing to the fouling of the microporous membrane by the polysaccharide, dextran production by immobilized Leuconostoc mesenteroides also was inefficient. More promising results have been obtained with immobilized Azotobacter vinelandii cultures. The amount of alginate produced by immobilized A. vinelandii represented about 60% of that recovered from a free-cell culture, whereas the polysaccharide yield reached 35% instead of 9% for the free counterpart. These results are compared to the macromolecular characteristics of exopolysaccharides.     

Glutathione transport across intestinal brush-border membranes: effects of ions, pH, delta psi, and inhibitors

We characterized glutathione transport in brush-border membrane vesicles (BBMV) that were prepared from rabbit small intestine in which gamma-glutamyl transpeptidases (gamma-glutamyltransferases, EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent (AT125). Intact GSH transport was strongly increased by the presence of Na+, K+, LI+, Ca2+ and Mn2+ and, of all these, the Ca2+ activation effect was prevalent. This cation effect was selective and catalytic but not energetic; Vmax obtained in the presence of both Na+ and Ca2+ was about 6-times higher than it was in their absence, while Km did not change. Moreover, these cations almost completely eliminated GSH binding on the membrane surface. Na+ activation cannot be explained as a stimulation effect on the Na+-H+ antiport system, since a GSH proton-driven transport was excluded. We determined a pH optimum (7.5), while low or high extravesicular pH values diminished the GSH uptake rate. The Ca2+ effect on GSH transport, when an electrical potential difference was imposed across BBMV, was different from that of monovalent cations. Indeed, experiments performed by valinomycin-induced K+ diffusion potential or by anion substitution showed that the GSH transport system was an electroneutral process in the presence of Na+ or K+, but that it was electrogenic in the presence of Ca2+ or in the absence of extravesicular cations. These results suggest that GSH is also cotransported with these cations, without its accumulation inside vesicles. Moreover, since GSH is negatively charged, the effect of pH changes and of cation activation on GSH transport is arguably mediated by changes in the ionization state of certain groups as the carrier site and of GSH itself, indicating the electrostatic nature of GSH binding sites on the transporter. The high Ca2+ activation effect is perhaps also partly due to fluidity changes in the lipoproteic microenvironment of the GSH transporter. Moreover, this transport system has high affinity with GSH, given the low Km value (17 microM) and the fact that it was only inhibited by GSH S-derivatives and by GSH monoethyl ester, which probably share the same transport system.     

Ca2+-transport in sea urchin unfertilized eggs: regulation by endogenous sulfated polysaccharides and K+

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca(2+) by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K(+) leading to an enhancement of the Ca(2+) transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca(2+) at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K(+) but not by Li(+) and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K(+). These data suggest that the regulation of the SERCA pump by K(+) and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.