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[Structure: see text] The synthesis of bivalent mannosides by the grafting of alpha-D-mannopyranoside onto monosaccharide acceptors and conjugation to terephthalic acid or phenylenediamine is described. Computational methods were used to predict accessible orientations and distances between the mannose units.
[reaction: see text] Mannose 6-phosphate mimics locked into the alpha-configuration and bearing hydrolase-resistant phosphate surrogates were synthesized and evaluated for binding affinity to the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R). Affinity increases as the phosphate surrogate is varied in the order malonyl ether < malonate < phosphonate. An alkene cross-metathesis approach to sought-after bivalent M6P-bearing ligands is also described. These compounds were designed to map onto biantennary sectors of high-mannose-type oligosaccharides carried by glycoprotein M6P/IGF2R ligands.
[reaction: see text] Bivalent carbohydrate substrates for metathesis were synthesized from glucuronic acid and phenylene-1,4-diamine. The substrate secondary structure depends on whether secondary or tertiary amides are present, and this influences the course of the metathesis reaction leading to novel multivalent scaffolding. Molecular modeling suggests that a very rigid macrocyclic scaffold has potential for the development of alpha-helix peptidomimetics.
A biantennary GM3-saccharide (sialyllactoside) derivative (4) was constructed using allylmalonic acid as a bivalent linker, both carboxylic acids of which were condensed with 3-aminopropyl lactoside (2) prior to enzymatic sialylation with a fusion enzyme. While ozonolysis of its allyl group generated a saccharide having a terminal aldehyde (6), we were unable to couple 6 directly to protein by reductive amination. However, extension of the spacer by means of introducing a maleimide group to 6 through its aldehyde group to give 7 enabled the latter to be successfully coupled to thiolated proteins. The average ratios of saccharide to protein were observed to be 35 in KLH conjugate (13) and 9-12 in HSA conjugates (14 and 15). The antisera obtained by immunizing mice with the biantennary sialyllactoside-KLH conjugate (13) together with MPL adjuvant were analyzed by ELISA. Using several structurally related saccharide-HSA conjugates as screening antigens, it was concluded that anti-sialyllactoside antibodies, both IgG and IgM, were effectively raised. This was further supported by competitive inhibition experiments using lactoside (1), sialyllactoside (8) and biantennary sialyllactoside (4) as inhibitors.
We have studied Cd2+-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd2+] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 microM, Cd2+ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 microM, Cd2+ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO3 or NH4NO3 medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd2+ effects on mitochondria treated in sequence with 100 microM of Ca2+, Sr2+, Mn2+ or Ba2+(Me2+) and 7.5 microM RR, as well as the alterations in Cd2+ action on the uptake of 137Cs+ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca2+ or RR. The evidence obtained indicate that Ca2+ exhibits a synergestic action on all Cd2+ effects examined, whereas Sr2+ and Mn2+, conversely, are antagonistic. In the presence of RR, the Cd2+ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd2+ conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd2+-stimulated swelling and 137Cs+ (analog of K+) uptake in the acetate media. For the first time, we have shown that Cd2+-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.
The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form "miniantibodies" with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.
Cooperative interactions between metal ions and bivalent tryptophan side chain are identified in water by fluorescence quenching of a designed novel receptor, EDTA-bis(L-tryptophan methyl ester), supplemented by a circular dichroism study; results revealed that the receptor is capable of distinguishing a variety of metal ions on the basis of their abilities in quenching tryptophan fluorescence and the relative magnitude of these interactions is Cu2+ approximately Fe2+ >> Co2+ > Ni2+ >> Mn2+ > Zn2+ > La3+ > Al3+.
The crystal structure of a bivalent glycocluster containing aromatic amides reveals that alkylation of secondary amides alters amide configuration and thus carbohydrate presentation. This also facilitates non covalent interactions (azide-azide, carbonyl-pyranose and aromatic-pyranose) and thus carbohydrate-carbohydrate stacking.
1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).
1. The effects of 19 bivalent cations on the activity, stability and capacity to hydrolyse endogenous arginine of axolotl liver arginase were studied. 2. It was found that Fe(2+), Co(2+), Ni(2+) and Zn(2+), as well as Mn(2+), stabilize the enzyme and render it able to hydrolyse endogenous arginine. 3. By using different concentrations of Co(2+) and Ni(2+) it was possible to dissociate the effect of each of the metal ions on the activity and stability of arginase from that on its capacity to hydrolyse endogenous arginine. 4. The effects of Mn(2+), Co(2+) and Ni(2+) on the activity and stability of arginase in the homogenate were also observed with purer preparations of the enzyme. 5. It is suggested that the capacity to hydrolyse endogenous arginine is a consequence of the integration of arginase with the arginine-formation sites.
1. The molecular weight of the calcium-binding protein of rabbit white skeletal muscle was estimated to be 18500 by sedimentation equilibrium and electrophoresis in sodium dodecyl sulphate. 2. Addition of 2 Ca(2+) ions per molecule produced reversible changes in the u.v.-absorption spectrum that are interpreted as arising from conformational changes in the structure of the protein. 3. Cd(2+) was almost as effective as Ca(2+) in producing the spectral changes. Other bivalent metal ions, particularly Mg(2+), were less effective. 4. Binding of Ca(2+) by the calcium-binding protein produced an increase in mobility to the anode on electrophoresis in 6m-urea at pH8.6. The Ca(2+)-saturated form of the protein was more retarded on gel filtration than the Ca(2+)-free form. 5. In the presence of Ca(2+) the calcium-binding protein formed an equimolar complex with the inhibitory protein. This complex was stable in 8m-urea and in the pH range 7.0-8.6. 6. An isotope-dilution method for the measurement of the content of calcium-binding protein in whole muscle is described. In rabbit psoas muscle the ratio of actin monomers to molecules of calcium-binding protein was approx. 7:1. Similar values were obtained for red skeletal and cardiac muscle. 7. Evidence is presented indicating that in the rabbit the inhibitory protein of the troponin complex of red skeletal and cardiac muscles is different from the inhibitory protein of white skeletal muscle.
The Ca2+-sensitive ATPase (adenosine triphosphatase) of human erythrocyte membranes is activated, not only by Ca2+ ions, but also by a series of other bivalent metal ions including Sr2+, Ba2+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+ and Pb2+. The degree of activation is dependent on the radius of the ion rather than on its nature, in contrast with the dissociation constant of the enzyme--metal ion complex.
The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.
1. Ca2+ (0.4-16 mM) had no detectable action on the agglutination of hen erythrocytes by Sendai virus. 2. Pretreatment of the cells with Ca2+ (0.1-8 mM) in the presence of the bivalent cation ionophore A23187 led, however, to a significant decrease in the subsequent agglutination of the cells by the virus. 3. It thus appears that the entry of Ca2+ into the interior of these cells decreases cellular agglutination by Sendai virus; possible interpretations of this phenomenon are discussed in terms of the movement of intramembranous particles. 4. With a small number of virions, maximum cell fusion by Sendai virus occurred in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate]. 5. Virus-induced cell fusion was significantly decreased by Ca2+, even at a concentration of 0.2 mM; it is suggested that this may result from diminished interactions between virus particles and erythrocyte membranes.
Incubation of hen erythrocytes with Ca2+ and the bivalent-cation ionophore A23187 induced slight cell fusion in 1 h at 37 degrees C, and extensive fusion during a subsequent 15 min at 47 degrees C. Redistributions of intramembranous particles were observed, possibly involving interactions between Ca2+ and phospholipids, which are discussed in relation to molecular mechanimss of cell fusion.
The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.
The half-saturation constants for binding of the bivalent cations (Mg2+, Ni2+, Co2+, Fe2+ and Mn2+) to ribulose bisphosphate carboxylase/oxygenase from Glycine max and Rhodospirillum rubrum were measured. The values obtained were dependent on the enzyme and the cation present, but were the same for both oxygenase and carboxylase activities. Ribulose bisphosphate rather than its cation complex was the true substrate. The kinetic parameters Vmax.(CO2), Vmax.(O2), Km(CO2), Km(O2), and K1(O2) were determined for both enzymes and each cation activator. The evolutionary and mechanistic implications of these data are discussed.
The bivalent-cation requirements of two enzymes involved in phosphatidylinositol synthesis were defined for pig lymphocyte membranes using a citric acid buffer. CTP:phosphatidic acid cytidylyltransferase (EC 2.7.7.41) is activated by free Mn2+ concentrations above 20nM and by free Mg2+ concentrations above 10 microM. When activated by Mg2+, the enzyme is weakly inhibited by Ca2+ (Ki greater than 250 microM), but Ca2+ has no effect when Mn2+ is used to stimulate CDP-diacylglycerol synthesis. The synthesis of phosphatidylinositol from phosphatidic acid is also stimulated by Mn2+ and Mg2+ concentrations similar to those above and is inhibited by free Ca2+ concentrations above 500nM, probably by its action on CDP-diacylglycerol:inositol 3-phosphatidyltransferase (EC 2.7.8.11). Taken together, these studies suggest that under physiological conditions phosphatidylinositol synthesis is activated by Mg2+ and it is possible that it is further regulated by the free concentrations of Ca2+ and/or Mn2+.