Comparative Studies of High MrSubunits of Rye and Wheat. III. Localisation of Cysteine Residues

Abstract

The high Mrsubunit fraction of rye glutelin was isolated from flour (cultivar Danko) by a specific extraction/precipitation procedure. The Cys content of the rye fraction (1·9 mol-%) was increased by a factor of 2–3 in comparison with wheat high Mrsubunits. To identify the positions of the Cys residues within the sequences, the high Mrsubunit fraction of rye was partially hydrolysed with chymotrypsin and the digest was separated by covalent chromatography on Thiopropylsepharose 6B. The chymotryptic digest was separated into a minor bound portion (4%) and a major non-bound portion (96%). The Cys content of the bound portion (13·2 mol-%) was strongly increased compared with the total digest (2·3%). The Cys-containing peptides of the bound portion were separated by RP-HPLC, alkylated with 4-vinylpyridine, purified by RP-HPLC and sequenced by automated Edman degradation. Altogether, 51 Cys peptides were sequenced; 24 of them could be assigned to known sequences of wheat highMr subunits. The other peptides were either derived from alpha -amylase or trypsin inhibitors or fromalpha -chymotrypsin. Four peptides could not be assigned. The peptides of the high Mrsubunits of rye represented six different Cys residues within the amino acid sequences. Five of these Cys residues corresponded to known Cys residues of wheat subunits: Ca, Cband Cdfrom domain A, Cyfrom domain B and Czfrom domain C. Three Cys residues present in wheat subunits could not be detected in rye, the adjacent Cc1Cc2from domain A of y-type subunits and Ceat the beginning of domain B of x-type subunit 5. One additional Cys residue of rye subunits (Cz*) located in domain C was found, which is not present in wheat highMr subunits. Thus, rye subunits could have a second Cys residue besides Czin domain C. This second Cys residue opens the possibility that high Mrsubunits of rye containing residue Cz*are chain terminators.