Isolation of a raw starch-binding fragment from barley alpha-amylase.
Barley alpha-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with beta-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel beta-sheets in domain C and the alpha7- and alpha8-helices of the (alpha/beta)8 domain.
Wong
D W
DW
Western Regional Research Center, USDA-ARS, Albany, California 94710, USA. dwsw@pw.usda.gov
Batt
S B
SB
Tibbot
B K
BK
Robertson
G H
GH
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004798:Enzymes / Q000737:chemistry* / Q000302:isolation & purification; D001467:Hordeum / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification
D004798:Enzymes; D010446:Peptide Fragments; D013213:Starch; D000516:alpha-Amylases; D014357:Trypsin