Comparative studies of carbohydrate-binding proteins from Xenopus laevis skin and eggs. Sugar-binding specificities and affinity purification

Salt and detergent extracts of acetone-dried powder of Xenopus laevis skin and eggs were fractionated on sugar-Sepharose columns, to which lactose, melibiose, galactose, rhamnose and mannose had been covalently linked, by successive elution with chelating reagent and specific sugars, resulting in separation of the different Ca2(+)-dependent and Ca2(+)-independent carbohydrate-binding proteins. The skin of X. laevis contains a salt-extractable Ca2(+)-dependent lactose-binding lectin of 30 kilodalton (kDa) and the eggs a similar lectin of 43 kDa, but they both lack Ca2(+)-dependent galactose-binding lectins. The 30 kDa lactose-binding lectin which agglutinates human A erythrocytes was isolated by successive affinity chromatography on two linked sugar-Sepharose columns, i.e., a galactose-Sepharose-lactose-Sepharose (GL) column system. Since the 30 kDa lectin was not recovered in the Ca2(+)-dependent lactose-binding protein fraction from the GL column system under the dithiothreitol (DDT)-free conditions, it was concluded that the lectin requires the presence of DTT and calcium for binding to the lactose-Sepharose column.     

Reactivity of Cobalt-Fullerene Complexes towards Deuterium

The adsorption of molecular deuterium (D₂) onto charged cobalt-fullerene-complexes Co_nC₆₀⁺ (n=1–8) is measured experimentally in a few-collision reaction cell. The reactivity is strongly size-dependent, hinting at clustering of the transition metal atoms on the fullerenes. Formation and desorption rate constants are obtained from the pressure-dependent deuterogenation curves. DFT calculations indeed find that this transition metal clustering is energetically more favorable than decorating the fullerene. For n=1, D₂ is predicted to bind molecularly and for n=2 dissociative and molecular configurations are quasi-isoenergetic. For n=3–8, dissociation of D₂ is thermodynamically preferred. However, reaching the ground state configuration with dissociated deuterium on the timescale of the experiment may be hindered by dissociation barriers.     

Reactivity of polyhydroxyalkyl-heterocycles towards Lewis acids

We report the reactivity of different polyhydroxyalkyl-heterocycles towards ceric ammonium nitrate (CAN) and ferric chloride. The behaviour of 2-methyl-5-(tetritol-1-yl)-pyrroles and -furans is different towards CAN oxidation. Pyrroles afford 2,5-diformylheterocycles, while furans give access to 1,4-dicarbonyl compounds containing three stereogenic centres. Ferric chloride promotes an intramolecular C-arylation reaction on O-benzylated-polyhydroxyalkyl furans, yielding an isochroman moiety, which is the basic skeleton of a variety of natural products.     

Reactivity of tri-n-bytylphosphate towards solvated electrons

Decomposition yields of tri-n-butylphosphate in methanol solutions saturated with Ar or N₂O were determined. On the basis of difference between yields in Ar and N₂O saturated solutions the rate constant k_{/e s} ^– _+TBP/=5.0×10⁶ dm³.mol^–1.s^–1 was calculated.     

Melanopsins: Localization and Phototransduction in Xenopus laevis Melanophores

Xenopus laevis melanophores express two melanopsins, Opn4x and Opn4m. We identified Opn4x immunoreactivity throughout the melanophore cytoplasm and in the cell membrane. The strongest immunopositivity for Opn4m was observed in the nuclear region, and no labeling was seen in the cell membrane. This immunodistribution suggests Opn4x as the functional photopigment. In X. laevis melanophores, light triggers pigment dispersion and clock gene induction at blue wavelength, which maximally activates melanopsins. Although light stimulation activates phospholipase C and increases intracellular calcium and cGMP, this nucleotide does not participate in photo‐induced melanin dispersion. Nevertheless, the guanylyl cyclase activator YC‐1 stimulates Per1 expression, similar to blue light pulse, and the use of pharmacological inhibitors indicates the participation of the phosphoinositide cascade. Since cAMP levels does not change after blue light stimulation, the cAMP/PKA pathway most probably is not involved in blue light induction of Per in X. laevis melanophores. Given the localization of melanopsins and our pharmacological data, the light‐induced clock gene expression seems to be mediated by Opn4x through phosphoinositide cascade and rise in cGMP, thus leading to the reset of the biological clock in our model.     

Assay for sialidase using erythrocytes and peroxidase-labeled peanut lectin

A rapid and sensitive sialidase assay method based on peroxidase-labeled peanut lectin (PNA) binding to desialylated erythrocyte is described. Formalinized sheep erythrocytes were used both as a stable substrate for sialidase and as a target for the lectin. In the case of sialidases from Vibrio cholerae and Arthrobacter ureafaciens, a linear relationship was observed between the amount of peroxidase-labeled PNA bound to erythrocytes and the enzyme amount. Binding of the lectin to sialidase-treated erythrocytes was completely prevented in the presence of 25 mM lactose and galactose. The method is particularly useful as a selective assay for sialidase which is active towards gangliosides or sialoglycoproteins, because a mammalian sialidase which is preferentially active towards sialooligosaccharides and sialoglycopeptides is not able to remove sialic acid from erythrocytes.     

Reactivity of cationic decamethylmetallocene complexes towards ketones

Reaction of decamethylmetallocene cations [Cp∗₂M]⁺ (M = Sc, Ti, V) with acetone and benzophenone resulted in the formation of the corresponding acetone adducts [Cp∗₂M(OCMe₂)_n]⁺ (M = Sc, n = 2; M = Ti, n = 1; M = V, n = 1) and benzophenone adducts [Cp∗₂M(OCPh)]⁺. The stoichiometry of these adducts is determined by both the electronic configuration of the metal center as well as steric pressure imparted by the large Cp∗-ligands. In addition, the M-O-C angle is controlled by the number of free valence orbitals of the Cp∗₂M unit.     

Reactivity of di-n-butyl-dicyclopentadienylzirconium towards amido stabilized stannylenes

Reaction of Sn[(N(C₆H₃iPr₂-2,6)(SiMe₃)]₂ and [{Sn(N(C₆H₃iPr₂-2,6)(SiMe₃)(μ-Cl)₂] with di-n-butyl-dicyclopentadienylzirconium yielded the trimetallic a carbene-like complex {[(N(C₆H₃iPr₂-2,6)(SiMe₃)](n-Bu)Sn}₂Cp₂Zr. The oxidation of {[(N(C₆H₃iPr₂-2,6)(SiMe₃)](n-Bu)Sn}₂Cp₂Zr by oxygen gives the five-membered dioxadistannazirconacyclic complex {[(N(C₆H₃iPr₂-2,6)(SiMe₃)](n-Bu)Sn}₂O₂Cp₂Zr.     

Reactivity of NBS towards N-allyl glycinyl derivatives

The reaction of precursors 3 with NBS in radical conditions does not yield any azetidine resulting from a 4-exo radical cyclisation with bromine atom transfer, or any brominated product at the glycinyl position. Instead, dibrominated adducts 4 and aminals 5 have been formed in good yields. We have investigated the mechanism of this puzzling transformation and shown that the homolytic abstraction of a hydrogen atom by a Br^• radical does not take place at the capto-dative position but at the allylic one. The resulting α-bromo derivative 8 would then evolve into an iminium intermediate with concomitant production of bromine and of a succinimidyl anion, which explains the formation of products 4 and 5. To cite this article: C. Koerber et al., C.R. Chimie 6 (2003).     

Purification and Partial Characterization of (6-4) Photoproduct DNA Photolyase from Xenopus laevis

Abstract— The (6-4) photoproduct DNA photolyase was detected in two vertebrate animals Crotalus atrox (rattlesnake) and Xenopus laevis (South African clawed toad). The enzyme was extensively purified from X. laevis and characterized. The highly purified enzyme is fluorescent with an excitation maximum at 420-440 nm and emission maximum at 460-480 nm. The photorepair action spectrum matches the fluoresoence excitation spectrum with a 430 nm maximum.     

Reactivity of Carbenes and Related Compounds towards Molecular Nitrogen

The thermal N₂ exchange of a number of ¹⁵N-labelled diazo compounds was studied in solution. The compounds involved were 3-diazo-1-methylindolin-2-one ( 1 ), 9-diazofluorene ( 2 ), 5-diazo-1,3-cyclopentadiene-1,2,3,4-tetracarbonitrile ( 3 ), 2-diazo-2H-imidazole-4,5-dicarbonitrile ( 4 ), 4-diazocyclohexa-2,5-dienone ( 5 ), and the conjugate acids of 4 and 5 , namely 4,5-dicyano-1H-imidazole-2-diazonium ion ( 6 ) and 4-hydroxybenzenediazonium ion ( 7 ). Only 1 , 4 , 6 , and 7 exchange their diazo group with ‘external’ molecular N₂. The results are explained on the hypothesis that only organic species which have an empty σ orbital and which are effective in π electron back-donation are able to react with N₂. Thus, reaction with carbenes is likely to occur only if the carbene is in the ¹A₂ singlet state and if its electrophilicity is high.     

Different Reactivity of As4 towards Disilenes and Silylenes

The activation of yellow arsenic is possible with the silylene [PhC(NtBu)₂SiN(SiMe₃)₂] ( 1 ) and the disilene [(Me₃Si)₂N(η¹-Me₅C₅)Si=Si(η¹-Me₅C₅)N(SiMe₃)₂] ( 3 ). The reaction of As₄ with 1 leads to the unprecedented As₁₀ cage compound [(LSiN(SiMe₃)₂)₃As₁₀] ( 2 ; L=PhC(NtBu)₂) with an As₇ nortricyclane core stabilized by arsasilene moieties containing silicon(II)bis(trimethylsilyl)amide substituents. In contrast, the compound [Cp*{(SiMe₃)₂N}SiAs]₂ ( 4 ) containing a butterfly-like diarsadisilabicyclo[1.1.0]butane unit is formed by the reaction of As₄ with the disilene 3 . Both compounds were characterized by single-crystal X-ray diffraction analysis, NMR spectroscopy, and mass spectrometry. The reaction outcomes demonstrate the different reaction behavior of yellow arsenic (As₄) compared to white phosphorus (P₄) in the reactions with the corresponding silylenes and disilenes.     

Transketolase from human erythrocytes. Purification and properties

Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.     

Reactivity of 1,2-cyclic sulfite xylosides towards nucleophiles

1,2-Cyclic sulfite xylosides offer facile access to 1,2-oxazolines upon reaction with aromatic and alkyl nitriles under Lewis or Brönsted acid conditions. Additionally, hydrophobic ionic liquids facilitate acid-catalysed formations of such oxazolines and C- and O-linked xylosides, providing means to carry out fast reactions at room temperature, and this in yields comparable to reactions conducted in xylene at high temperature for extended reaction time.     

Unusual Reactivity of Sodium Tetramesityltetraphosphanediide towards Cyclohexyl Isocyanide

The reaction of [Na₂(thf)₄(P₄Mes₄)] (Mes=2,4,6‐Me₃C₆H₂) with cyclohexyl isocyanide (2:5) resulted in the formation of the heterocyclic N‐(tetramesityltetraphosphacyclopentylidene)cyclohexylamine [cyclo‐{P₄Mes₄C(NCy)}] ( 2 ) (30–35 %), the unusual 1,3,5‐triphospha‐1,4‐pentadiene ( 3 ) (40–45 %), and small amounts of the dimeric iminomethylidenephosphane cyclo‐{PMesC(NCy)}₂ ( 4 ). With catalytic amounts of AgBF₄, 2 was the major product. The reaction of 2 with [CuBr(SMe₂)] (1:1) produced bromido‐bridged dimeric Cu^I complex 5 . Molecular structures of compounds 3 , 4 , and 5 were obtained.     

Reactivity of benzoic acid esters towards cumylperoxy radicals

The alkoxy groups of aromatic carboxylic acid esters are attacked by cumylperoxy radicals. The rate constants for the abstraction of a hydrogen atom by cumylperoxy radicals are: for methyl benzoate k₂=(4.28±0.8)×10¹⁰ exp (–20.5±1.5/RT) and for ethyl benzoate k₂=(9.41±0.8)×10¹⁰ exp(–18.5±1.5/RT) l/mol sec. The strengths of the primary and the secondary C–H bonds of these compounds have been estimated.

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, . k₂=(4,28±0,8)·10¹⁰ exp (–20,5±1,5/RT) k₂=(9,41±0,8)·10¹⁰ exp (–18,5±1,5/RT) /·. C–H .

     

Reactivity of Gold Complexes towards Elementary Organometallic Reactions

For a while, the reactivity of gold complexes was largely dominated by their Lewis acid behavior. In contrast to the other transition metals, the elementary steps of organometallic chemistry—oxidative addition, reductive elimination, transmetallation, migratory insertion—have scarcely been studied in the case of gold or even remained unprecedented until recently. However, within the last few years, the ability of gold complexes to undergo these fundamental reactions has been unambiguously demonstrated, and the reactivity of gold complexes was shown to extend well beyond π‐activation. In this Review, the main achievements described in this area are presented in a historical context. Particular emphasis is set on mechanistic studies and structure determination of key intermediates. The electronic and structural parameters delineating the reactivity of gold complexes are discussed, as well as the remaining challenges.     

Reactivity of tert-butoxyl radicals towards substituted indole derivatives

Tert-butoxyl radicals react with indole and methyl substituted derivatives by hydrogen abstraction. For those compounds which are unsubstituted at the N-atom, hydrogen abstraction takes place almost exclusively at the N? H bond. The reactivity of these compounds correlates with their donor electron capacity, pointing to significant contribution of charge transfer to the transition state stability. Substitution at the N atom considerably decreases the reactivity.