Similarities of omega gliadins from Triticum urartu to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing.

The omega-gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6 x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2 x=14, AA ). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified omega-gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded omega-gliadins were smaller than 1B- or 1D-encoded omega-gliadins. The N-terminal amino acid sequences for 1A omega-gliadin mature peptides were nearly identical to those for the T. urartu omega-gliadins and were more similar to 1D omega-gliadin sequences than to sequences for T. monococum omega-gliadins, barley C-hordeins, or rye omega-secalins. They diverged greatly from the N-terminal sequences for the 1B omega-gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those omega-gliadins with N-terminal sequences beginning with KEL.

Characterisation and chromosomal localisation of C-type low-molecular-weight glutenin subunits in the bread wheat cultivar Chinese Spring.

Low-molecular-weight glutenin subunits are classically divided into the B, C and D groups. Most attention has been paid to the characterisation of the B and D groups, whereas C subunits, although represented by a large number of protein components, have not been thoroughly characterised, mainly because they tend to separate with the gliadins in many fractionation procedures. Here we describe a procedure for obtaining a fraction strongly enriched in C subunits that has allowed us to determine the chromosomal location of these subunits in the bread wheat cultivar Chinese Spring. This analysis has shown that these subunits are coded on chromosome groups 1 and 6. Comparison between N-terminal amino acid sequencing of B and C subunits has shown that, whereas the former group includes mainly subunits with typical LMW-GS type sequences (76%), the C subunit group is made up almost completely of subunits with gliadin-like sequences (95%), including the alpha-type. These results indicate that the LMW-GSs are likely to be coded not only by the typical Glu-3 loci, but also by loci tightly linked to, and possibly included within, the Gli-1 and Gli-2 loci.

Purification and characterization of wheat alpha-gliadin synthesized in the yeast, Saccharomyces cerevisiae.

The development of efficient methods for production and purification of plant seed storage proteins in heterologous microbial hosts would facilitate structure-function studies of these proteins. This report describes such methods applied to the production and isolation of wheat alpha-gliadin, a prolamine-type seed storage protein, from Saccharomyces cerevisiae. Beginning with the vector, growth conditions, and extraction methods of Neill et al. [Gene 55 (1987) 303-317], we implemented several improvements to increase the yields of alpha-gliadin per volume of yeast cell culture. The CYCl::Gli-A2-Y transcriptional fusion vector, pAY31 (Neill et al., 1987), was modified by replacing the ARS1 region of replication with that of the 2 mu plasmid of yeast. We formulated a new medium, a derivative of synthetic defined (SD) medium supplemented with several nitrogen sources, that allows both selection for maintenance of plasmids and growth to high cell densities. Stationary phase cultures of cells bearing the modified expression vector, and grown in this medium with glycerol and lactate as carbon sources, contain significantly higher levels of alpha-gliadin than log-phase cultures grown in SD glucose. Sonication in 80% ethanol selectively and efficiently extracts the alpha-gliadin from cell pellets of small- or large-scale cultures, allowing the purification of several hundred micrograms of the wheat protein per liter in just a few high-yield steps. The alpha-gliadin isolated from yeast elutes at the same position in HPLC as the A-gliadin fraction purified from wheat flour. N-terminal amino acid (aa) sequencing reveals that the signal peptide is removed from the gliadin precursor in yeast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

An active pseudopeptide analog of the leucokinin insect neuropeptide family.

A pseudopeptide analog of the active core of the leucokinin insect neuropeptide family was synthesized and found to retain myotropic activity. No reports of active pseudopeptide analogs of an insect or other invertebrate neuropeptide have previously appeared in the literature. The pseudopeptide (Phe psi [CH2-NH] Phe-Ser-Trp-Gly-NH2) contains a reduced-amide linkage between the two N-terminal Phe residues. Unlike its amide-bond containing counterpart, the activity of the pseudopeptide was not destroyed upon exposure to aminopeptidase M.