Search

Rh glycoproteins are members of the ammonium transporter (Amt)/methylamine permease (Mep)/Rh family facilitating movement of NH3 across plasma membranes. Homology models constructed on the basis of the experimental structures of Escherichia coli AmtB and Nitrosomonas europaea Rh50 indicated a channel structure for human RhA (RhAG), RhB (RhBG), and RhC (RhCG) glycoproteins in which external and internal vestibules are linked by a pore containing two strictly conserved histidines. The pore entry is constricted by two highly conserved phenylalanines, 'twin-Phe.' In this study, RhCG function was investigated by stopped-flow spectrofluorometry measuring kinetic pH variations in HEK293E cells in the presence of an ammonium gradient. The apparent unitary NH3 permeability of RhCG was determined and was found to be close to that of AmtB. With a site-directed mutagenesis approach, critical residues involved in Rh N[H.sub.3] channel activity were highlighted. In the external vestibule, the importance of both the charge and the conformation of the conserved aspartic acid was shown. In contrast to AmtB, individual mutations of each phenylalanine of the twin-Phe impaired the function while the removal of both resulted in recovery of the transport activity. The impact of the mutations suggests that, although having a common function and a similar channel structure, bacterial AmtB and human Rh vary in several aspects of the N[H.sub.3] transport mechanisms. ammonium transport; HEK cells; Rhesus family, pH variations; ammonia channel

This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein.

Byline: Tilman J. Schober (1), Manfred Kuhn (2) Keywords: Capillary electrophoresis; Spelt; Triticum aestivum ssp. spelta; Wheat; Gliadin; Cultivars; Breeding programs Abstract: Gliadins of 27 European spelt [Triticum aestivum ssp. spelta (L.) Thell.] cultivars (cvs) were separated by capillary zone electrophoresis (CZE). A great amount of diversity was found within the CZE gliadin patterns of these cvs however, only 15 had unique patterns, whereas 2 triples and 3 pairs were identical, i.e., differences were within the uncertainty of the method and environmental effects. Two of these identical pairs were attributed to mislabeling: affected were one of the two Swiss spelt cvs 'Ostar' and 'Sertel', and the German spelt cv 'Steiners Roter Tiroler'. The German cv 'Franckenkorn' showed considerable similarity with the Belgian cv 'Rouquin'. CZE gliadin patterns of 12 modern German bread wheat cvs were distinctly different from those of spelt cvs selected from old land varieties like the Swiss cv 'Oberkulmer Rotkorn'. In the gliadin patterns of 11 spelt cvs, bread wheat elements were found. 7 of the 24 cvs not mislabeled are known to be progeny of wheat/spelt crosses, 4 cvs are described as pure spelt, and the pedigrees of 13 are unknown. Wheat elements were spotted in only 4 of the 7 wheat/spelt crosses and in none of the 4 pure spelt cvs. The results suggest that crossing spelt with modern wheat may be, but is not necessarily, reflected in the gliadin pattern. Author Affiliation: (1) Department of Food and Nutritional Sciences (Food Technology), University College Cork, National University of Ireland, College Road, Cork, Ireland (2) Institute 150 (Food Technology), Section Cereal Technology, University of Hohenheim, Garbenstrasse, 25, 70599, Stuttgart-Hohenheim, Germany Article History: Received Date: 13/02/2003 Online Date: 22/07/2003

Byline: Werner Seilmeier (1), Herbert Wieser (1) Keywords: Wheat species; Gliadin analysis; Immunoassay; Coeliac disease Abstract: The only commercially available immunoassay for gliadin determination in gluten-free food which has been ring-tested and in use for many years, is a test kit based on monoclonal antibodies against [omega]-gliadins. Various studies of the literature have shown that different gliadin standards resulted in different calibration curves, and it has been proposed that the affinity of [omega]-gliadins to the monoclonal antibodies varied among wheat varieties. To clarify this fundamental problem, total gliadins and the [omega]-gliadins from a winter wheat ('Rektor'), a spring wheat ('CWRS'), a wheat rye hybrid ('Herzog') and varieties of spelt, durum wheat, emmer and einkorn, were isolated and analyzed by means of an enzyme-linked immunoabsorbent assay (ELISA) kit based on antibodies against [omega]-gliadins. Additionally, single [alpha]- and I3-gliadins of Rektor wheat were studied. The results demonstrated that the calibration curves derived for total gliadins differed, in parts, strongly from that of the kit gliadin standard only the curves for the durum wheat and spelt gliadins were in congruence with the kit gliadin. Single [omega]-gliadins revealed strong differences between and within wheat species and ELISA equivalents had a range from 10 to 220% according to kit gliadin. The affinity of [omega]-gliadins was not correlated with the calibration curves of total gliadins. Some of the [alpha]-gliadins of Rektor wheat showed ELISA equivalents similar to those of [omega]-gliadins. Because the proportions of [alpha]-gliadins in total gliadins were significantly higher than those of [omega]-gliadins, the unspecific binding of [alpha]-gliadins contributed much more to the total affinity of gliadins than the specific binding of [omega]-gliadins. I3-Gliadins, however, did not show any detectable affinity. Author Affiliation: (1) Deutsche Forschungsanstalt fur Lebensmittelchemie and Kurt-Hess-Institut fur Mehl- und Eiweissforschung, Lichtenbergstrasse 4, 85748 Garching, Germany Article History: Received Date: 03/03/2003 Online Date: 15/07/2003