Does the oxidation of methionine residue precede the inactivation of the trypsin inhibitor (LUTI) in germinating seeds of common flax (Linum usitatissimum)?

Irena Lorenc-Kubis, Agnieszka Czerwińska-Goleń, Jolanta Kowalska, Tadeusz Wilusz

Abstract


Antitrypsin activity in germinating common seeds of flax (Linum usitatissimum) was investigated. At the early stage of germination an increase in antitrypsin activity was observed, followed by its decrease during the development of the seedlings. From 6-day-old seedlings a trypsin inhibitor (gerLUTI) was purified. The purification procedure involved fractionation of proteins from seedling homogenate with alcohol and successive chromatography on CM-Sephadex C-25 on immobilised methylchymotrypsin in the presence of 5 M NaCl, and finally on a C18 column in RP-HPLC.

The gerLUTI migrated in SDS PAGE as a single band, but in mass spectroscopy analysis it exhibited the presence of at least three forms with molecular masses of 7654 ± 3 Da, 7668/7670 ± 3 Da, and 7687 ± 3 Da. The preparation of LUTI isolated from resting seeds contained only one form, with a molecular mass of 7655 ± 3 Da. LUTI and gerLUTI differed also in methionine contents. LUTI contained two methionine residues, whereas in gerLUTI only a trace of methionine was detected. The obtained results might suggest that during flax seeds germination the inhibitor molecules undergo selective modification, e.g. oxidation at methionine residues, before being degraded by proteolytic enzymes.


Keywords


Linum usitatissimum seedlings; trypsin inhibitor; protein; germination; immobilised methylchymotrypsin; mass spectra

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