Frances M. DuPont

Characterization of the 1B-type ω-gliadins from Triticum aestivum cultivar Butte.

ABSTRACT ω-Gliadins were purified from wheat (Triticum aestivum L. ‘Butte’) flour and characterized. Although reversed-phase HPLC (RP-HPLC) separated the 1B-encoded ω-gliadins into two fractions, 1B1 and 1B2, these fractions had nearly identical amino acid compositions, three similar protein bands in SDS-PAGE, 10 similar spots in two-dimensional PAGE, and two similar N-terminal amino acid sequences. The main components had a range in mass of 48,900–51,500 when estimated by mass spectrometry, significantly less than the mass estimated by SDS-PAGE. The 1B fractions were digested with thermolysin, the peptides were separated by RP-HPLC, the peptide mass was determined, and nine peptides from each fraction were sequenced with nearly identical results for the 1B1 and 1B2 digests. A possible consensus sequence of the 1B-encoded ω-gliadin internal repeat was QQQXP, where X was F, I, or L in descending order of occurrence. The 1D-encoded ω-gliadins were purified by RP-HPLC as a single fraction that had one band i...

Two cDNA Clones Encoding Isoforms of the B Subunit of the Vacuolar ATPase from Barley Roots

The vacuolar H+-ATPase of higher plants is a member of the V-ATPase family, which comprises complex, multisubunit ATPases found in a11 eukaryotes. The electrochemical gradient created by the V-ATPase is thought to provide the driving force for the iecondary transport of other ions and metabolites (Taiz, 1992). In barley (Hordeum vulgare L.) roots this enzyme may be involved in the sequestration of Na+ and Ca2+ ions in the vacuole, because the proton gradient produced by the ATPase is used by Na+/H+ and Ca2+/H+ antiports to drive the uptake of Na+ and of Ca2+ (Garbarino and DuPont, 1989; DuPont et ai., 1990). The quatemary structure of the ATPase from barley roots is very similar to that from other organisms, with approximately 10 different subunits (DuPont and Momssey, 1992). Two of the best-characterized subunits of the V-ATPases are the A and B subunits of the large, knob-like head group on the cytoplasmic side of the vacuolar membrane. The A subunit is between 67 and 70 kD, and the B subunit is between 53 and 60 kD in various organisms. Both A and B subunits are present in a stoichiometry of three per enzyme complex and contain ATP binding sites, although the B subunit is not directly involved in ATP hydrolysis (Puopolo et al., 1992). The primary structures of the A and B subunits are highly conserved among widely divergent organisms, making them useful for evolutionary studies. Isoforms of the B subunit have been described for mammals, and the distribution of the isofonns seems to be tissue specific (Puopolo et al., 1992). It was of some interest to leam whether there are also isoforms of the B subunit in plants. Two different cDNA clones for the B subunit of the barley V-ATPase, designated HTBl and HTB2, were selected from a barley root cDNA library. The two clones were very similar to each other, having higher sequence identity to each other than to any other B subunit sequence in the GenBank data base (see Table I for details). Since barley is an inbred diploid organism, it is likely that HTBl and HTB2 are encoded by different genes, rather than alleles of the same gene. The next most similar sequence was that for the Arubidopsis

Subunit composition and Ca2+-ATPase activity of the vacuolar ATPase from barley roots

The vacuolar ATPase was purified from a tonoplast-enriched membrane fraction from barley (Hordeum vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KNO3 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purified holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca(2+)-ATPase were obtained from the Sephacryl S-400 column. A peak of Ca(2+)-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO3- and DCCI. It is proposed that this Ca(2+)-ATPase is a partial reaction of the plant vacuolar ATPase. The second Ca(2+)-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO3- or DCCI. The second Ca(2+)-ATPase is a major component of ATP hydrolysis by the native membranes.

Surface-Associated Proteins of Wheat Starch Granules : Suitability of Wheat Starch for Celiac Patients

Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems. In this study, starch surface-associated proteins were extracted from four commercial wheat starches, fractionated by high-performance liquid chromatography and gel electrophoresis, and identified by tandem mass spectrometry analysis. More than 150 proteins were identified, many of which (for example, histones, purothionins, and glutenins) had not been recognized previously as starch-associated. The commercial starches were analyzed by the R-5 enzyme-linked immunosorbent assay method to estimate the amount of harmful gluten protein present. One of these starches had a low gluten content of 7 ppm and actually fell within the range proposed as a new Codex Alimentarius Standard for naturally gluten-free foods (maximum 20 ppm). This low level of gluten indicates that the starch should be especially suitable for use by celiac patients, although wheat starches with levels up to 100 ppm are deemed safe in the proposed Codex standards.

Extraction of up to 95% of wheat (Triticum aestivum) flour protein using warm sodium dodecyl sulfate (SDS) without reduction or sonication.

Extraction of glutenin polymers without sonication is an essential prerequisite for accurate determination of their composition and molecular size distribution. Sequential fractionation of wheat flour with 0.1 M KCl and 0.25% sodium dodecyl sulfate (SDS) at 21 degrees C and 2% SDS at 60 degrees C extracted up to 95% of total protein. We propose that 2% SDS at 60 degrees C disrupts hydrogen bonds in glutenin and gliadin aggregates, reduces hydrophobic interactions, and facilitates solubilization. Analysis by size-exclusion high-performance liquid chromatography (SE-HPLC), reverse-phase (RP)-HPLC, and SDS-polyacrylamide gel electrophoresis (PAGE) revealed that partitioning of gliadins and glutenins among the extracts differed for two flours with good baking quality (Butte 86 and Jagger) and one with poor baking quality (Chinese Spring). More gliadin was associated with the 0.25% SDS extract for Chinese Spring, whereas more gliadin was associated with the 2% SDS extract for Butte 86 and Jagger. Unextractable glutenin polymer was only 4-5% of total protein for Butte 86 and Chinese Spring and 14% for Jagger.