Isolation of a Raw Starch-Binding Fragment from Barley [alpha]-Amylase.
Wong, Dominic W. S. 1,2; Batt, Sarah B. 1; Tibbot, Brian K. 1; Robertson, George H. 1
[Article]
Journal of Protein Chemistry.
19(5):373-377, July 2000.
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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 [alpha]7-and [alpha]8-helices of the ([alpha]/[beta])8 domain.
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