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Isolation and Amino Acid Sequence of a Serine Proteinase Inhibitor from Common Flax ( Linum usitatissimum ) Seeds
Author(s) -
LorencKubis Irena,
Kowalska Jolanta,
Pochroń Bogusława,
Żużło Aneta,
Wilusz Tadeusz
Publication year - 2001
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20010105)2:1<45::aid-cbic45>3.0.co;2-#
Subject(s) - trypsin , chymotrypsin , cyanogen bromide , linum , chemistry , serine , biochemistry , peptide sequence , biology , enzyme , botany , gene
LUTI ( Linum usitatissimum trypsin inhibitor), a member of the potato inhibitor I family, has been isolated from seeds of flax by ethanol fractionation, ion exchange chromatography on CM‐Sephadex C‐25, affinity purification on immobilized methylchymotrypsin ( α ‐chymotrypsin in which His 57 has been converted to 3‐methylhistidine) in the presence of 5 M NaCl, and finally by reversed‐phase HPLC. The 7655 Da inhibitor consists of a single polypeptide chain of 69 residues with one disulfide bridge. The molecule is acetylated at the N terminus. Its primary structure has been determined after limited proteolysis of the native molecule with trypsin at the reactive site, cleavage with cyanogen bromide or arginyl endopeptidase (Arg‐gingipain), and alcoholytic deacetylation of the N‐terminally blocked serine. The association constants ( K a ) of LUTI with bovine β ‐trypsin and α ‐chymotrypsin are 3.58×10 10 M −1 and 5.02×10 5 M −1 , respectively. High NaCl concentration (3 M ) increased the association constant of LUTI with α ‐chymotrypsin to 6.64×10 7 M −1 . To our knowledge, LUTI is the first serine‐proteinase‐type inhibitor isolated from a plant of the Linaceae family.