Comparative studies of high M-r subunits of rye and wheat. III. Localisation of cysteine residues

The high M-r subunit fraction of rye glutelin was isolated from flour (cult ivar Danko) by a specific extraction/precipitation procedure. The Cys conte nt of the rye fraction (1.9 mol-%) was increased by a factor of 2-3 in comp arison with wheat high M-r subunits. To identify the positions of the Cys r esidues within the sequences, the high M-r subunit fraction of rye was part ially hydrolysed with chymotrypsin and the digest was separated by covalent chromatography on Thiopropylsepharose 6B. The chymotroptic digest was sepa rated 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 co mpared with the total digest (2.3%). The Cys-containing peptides of the bou nd portion were separated by RP-HPLC, alkylated with 4-vinylpyridine, purif ied by RP-HPLC and sequenced by automated Edman degradation. Altogether, 51 Cys peptides were sequenced, 24 of them could be assigned to known sequenc es of wheat high M-r subunits. The other peptides were either derived from alpha-amylase or trypsin inhibitors or from alpha-chymotrypsin. Four peptid es could not be assigned. The peptides of the high M-r subunits of rye repr esented six different Cys residues within the amino acid sequences. Five of these Cys residues corresponded to known Cys residues of wheat subunits: C -a, C-b and C-d from domain A, C-y from domain B and C-z from domain C. Thr ee Cys residues present in wheat subunits could not be detected in rye, the adjacent (CCc2)-C-c1 from domain A of y-type subunits and C-c at the begin ning of domain B of x-type subunit 5. One additional Cys residue of rye sub units (C-2*) located in domain C was found, which is not present in wheat h igh M-r subunits. Thus, rye subunits could have a second Cys residue beside s C-z in domain C. This second Cys residue opens the possibility that high M-r subunits of rye containing residue C-z* are chain terminators. (C) 2000 Academic Press.