Molecular cloning of the barley seed protein CMd: a variant member of the alpha-amylase/trypsin inhibitor family of cereals.

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of alpha-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with alpha-amylases of bacterial origin.

In vitro (organ culture) studies of the toxicity of specific A-gliadin peptides in celiac disease.

Specific peptides of known amino acid sequence were prepared from alpha-gliadin (A-gliadin) by cleavage of the protein with cyanogen bromide and chymotrypsin and purification of the resulting peptides. The three peptides derived from the cyanogen bromide cleavage spanned the complete sequence of A-gliadin (266 residues). Four peptides derived from chymotryptic digestion covered the N-terminal sequence through residue 68. These peptides were tested for toxicity in celiac disease by organ culture of biopsied small intestinal tissues taken from patients with active celiac disease. Enterocyte height was used as a measure of peptide effect on cultured tissues. Five of seven peptides tested significantly inhibited increase of enterocyte height in the cultures and were considered toxic on this basis. The largest common sequences among the toxic peptides were -pro-ser-gln-gln- and -gln-gln-gln-pro-; these sequences were absent from the nontoxic peptides. The relationship of these sequences to the damaging effect of gliadins on the small intestinal mucosa in celiac disease remains to be investigated.

Nucleic acid (cDNA) and amino acid sequences of alpha-type gliadins from wheat (Triticum aestivum).

The complete amino acid sequence for an alpha-type gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar alpha-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an alpha-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature alpha-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently.

Structural homology of storage proteins coded by the Hor-1 locus of barley (Hordeum vulgare L.).

Three 'C' hordein fractions were prepared by ion-exchange chromatography of a total hordein preparation on carboxymethyl cellulose at pH 4.6 Polyacrylamide gel electrophoresis at pH 3.2 and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at pH 8.9 showed that each fraction contained a single major band. The apparent molecular weights of these were determined by SDS-PAGE as 58, 57, and 54,000. When compared by isoelectric focusing, however, the 58 and 57,000 components each separated into two major bands and the 54,000 component into four. Amino acid analysis showed that although the three fractions had similar compositions with high glutamate+glutamine (38-39%), proline (30-32%) and phenylalanine (8-9%) contents, some differences were present, notably in the relative content of lysine. The three fractions had identical amino acid sequences for the first ten residues at the N-terminal end. They also had identical sequences for the first five residues at the C-terminal end, with the exception that a mixture of two amino acids were released from position 4 of the 58,000 fraction only. Peptide mapping with three enzymes (trypsin, chymotrypsin and V8 protease) indicated that the 58 and 57,000 fractions were more closely related to each other than to the 54,000 fraction. It is suggested that the 57 and 58,000 fractions and the 54,000 fraction constitute two families of closely related polypeptides which are coded by genes derived from the duplication and divergence of a single ancestral gene.