Comparative studies of high M-r subunits of rye and wheat. II. Partial amino acid sequences

High M-r subunit fractions of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction wit h 50% (v/v) aqueous propan-1-ol under reducing conditions and 60 degrees C followed by precipitation using a 60% concentration of propan-1-ol, centrif ugation, dialysis and lyophilisation. Two single major subunits of rye Dank o were isolated by preparative RP-HPLC and characterised by N-terminal sequ encing which showed a strong homology to wheat high M-r subunits. Total hig h M-r subunit fractions of Danko, Halo and Rektor were digested with TPCK-t reated trypsin at 37 degrees C for 8 h. The partial hydrolysates were pre-s eparated into the fractions I (M-r <10 000) and II (M-r >10 000) by centrif ugation on a Centriprep 10-membrane. Both fractions were then separated by reversed-phase HPLC on C-18 silica gel. Dominating peptides were purified b y preparative rechromatography under slightly changed HPLC conditions and c haracterised by amino acid analysis and determination of amino acid sequenc es. The resulting sequences of 19 (Danko), 12 (Halo) and 15 peptides (Rekto r) were compared with known sequences from x- and y-type high M-r subunits of wheat. With the exception of three peptides, the sequences could be assi gned to the three different domains of high M-r subunits. Even when the seq uences of the rye peptides were similar to those of wheat, they were not id entical in any case; on average the variations were about 20%. Most of rye peptides corresponded to the x-type subunits of wheat. Within the most char acteristic sequence motifs belonging to the repetitive domain B, fundamenta l differences between wheat and rye were not detected. (C) 1999 Academic Pr ess.