Isolation and amino acid sequence of a serine proteinase inhibitor from common flax (Linum usitatissimum) seeds.
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 (alpha-chymotrypsin in which His57 has been converted to 3-methylhistidine) in the presence of 5M 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 beta-trypsin and alpha-chymotrypsin are 3.58x10(10) M(-1) and 5.02x10(5) M(-1), respectively. High NaCl concentration (3M) increased the association constant of LUTI with alpha-chymotrypsin to 6.64x10(7) M(-1). To our knowledge, LUTI is the first serine-proteinase-type inhibitor isolated from a plant of the Linaceae family.
Lorenc-Kubis
I
I
Institute of Biochemistry and Molecular Biology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland.
Kowalska
J
J
Pochroń
B
B
Zuzło
A
A
Wilusz
T
T
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019597:Flax / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification
D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin