The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Differential binding of lectins IL-2 and CSL to candida albicans and cancer cells

The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor ss, IL-2 associates the latter which the CD3/TCR complex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al. , 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R beta by ++p56(lck) , the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2- dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous "amplifier of activation signals" (Zanetta et al. , 1995, Biochem. J., 311, 629-636).      

Isolation of the domain containing the molybdenum, iron-sulfur I, and iron-sulfur II centers of chicken liver xanthine dehydrogenase.

Chicken liver xanthine dehydrogenase, like other xanthine-oxidizing enzymes, is a dimer of Mr = 150,000 subunits. Each subunit contains one molybdenum, one FAD, and two distinct Fe2S2 centers. Treatment with a number of proteases shows that the native enzyme subunit is cleaved at three distinct sites. However, the cleavage products can be separated only under denaturing conditions. Prolonged treatment with subtilisin at pH 10.1 has permitted the isolation of an Mr = 65,000 catalytically active fragment that is devoid of FAD but which contains the molybdenum and both types of iron-sulfur center. A model of the domain structure of the native enzyme is proposed.     

Oxidation--reduction potentials of turkey liver xanthine dehydrogenase and the origins of oxidase and dehydrogenase behaviour in molybdenum-containing hydroxylases.

Redox potentials for the various centres in the enzyme xanthine dehydrogenase (EC 1.2.1.37) from turkey liver determined by potentiometric titration in the presence of mediator dyes, with low-temperature electron-paramagnetic-resonance spectroscopy. Values at 25 degrees C in pyrophosphate buffer, pH 8.2, are: Mo(VI)/Mo(V)(Rapid),-350 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -362 +/- 20mV; Fe-S Iox./Fe-S Ired., -295 +/- 15mV; Fe-S IIox./Fe-S IIred., -292 +/- 15mV; FAD/FADH,-359+-20mV; FADH/FADH2, -366 +/- 20mV. This value of the FADH/FADH2 potential, which is 130mV lower than the corresponding one for milk xanthine oxidase [Cammack, Barber & Bray (1976) Biochem. J. 157, 469-478], accounts for many of the differences between the two enzymes. When allowance is made for some interference by desulpho enzyme, then differences in the enzymes' behaviour in titration with xanthine [Barber, Bray, Lowe & Coughlan (1976) Biochem. J. 153, 297-307] are accounted for by the potentials. Increases in the molybdenum potentials of the enzymes caused by the binding of uric acid are discussed. Though the potential of uric acid/xanthine (-440mV) is favourable for full reduction of the dehydrogenase, nevertheless, during turnover, for kinetic reasons, only FADH and very little FADH2 is produced from it. Since only FADH2 is expected to react with O2, lack of oxidase activity by the dehydrogenase is explained. Reactivity of the two enzymes with NAD+ as electron acceptor is discussed in relation to the potentials.     

Isolation and characterization of an alkaline phosphatase from pea thylakoids

Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.     

The role of glycinebetaine in the protection of spinach thylakoids against freezing stress

The quaternary ammonium compound glycinebetaine has been tested for cryoprotective properties, using isolated spinach thylakoids as a model membrane system. The effect of a 3-h,-20°C freezing regime on different photosynthetic parameters was measured. These parameters were the light-stimulated pH formation and dark pH decay, light-stimulated proton uptake, electron flow through photosystem II, photosystem I and total linear electron flow, and pyocyanine-mediated cyclic photophosphorylation. It was shown that below 100 mM glycinebetaine was superior as a cryoprotectant to sucrose on a molar, a molal and an activity basis. At higher concentrations, glycinebetaine was less efficient in preventing inactivation of thylakoids during freezing than sucrose. These observations are discussed in relation to the permeability of biomembranes to glycinebetaine and the colligative theory of cryoprotection. It is concluded that colligative protection is modified by direct interaction between cryoprotectant and membranes.Abbreviations Asc ascorbate - cyt f cytochrome f - DAD 2,3,5,6-tetramethyl--phenylenediamine - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DCPIP 2,6-dichlorophenolidophenol - DBMIB 2,5-dibromo-3-methyl-6-isopropyl--benzoquinone - DNP-INT 1,3-dinitrophenylether of iodonitrothymol - FeCy ferricyanide - MV methylviologen (1,1-dimethyl-4-4-bipyridinium-dichloride) - PQ plastoquinone - PS I photosystem I - PS II photosystem II     

Stereoselective reduction of ketones by <Emphasis Type="Italic">Daucus carota</Emphasis> hairy root cultures

Reduction of acetophenone by Daucus carota hairy root cultures afforded (S)-phenylethanol in high yield (96%) and excellent enantiomeric excess (ee ≥ 98%). Aromatic ketones, keto esters, and a simple aliphatic ketone were reduced with good stereoselectivity (ee = 62–98%) and moderate to high chemical yields (25–90%).