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

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

doi:10.5586/asbp.2003.042

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.

Highlights

Plants and seeds contain considerable amounts of proteins and peptides inhibiting serine proteinase activity
In a previous paper (Lorenc-Kubis et al 2001) we reported on the purification of a trypsin inhibitor (LUTI) from the resting seeds of common flax (Linum usitatissimum)
LUTI shows a high level of sequence similarity to the potato I inhibitor family, and it is the first serine proteinase inhibitor isolated from Linaceae

Summary

INTRODUCTION

Plants and seeds contain considerable amounts of proteins and peptides inhibiting serine proteinase activity. The role of these inhibitors in the physiology of the plant is not fully understood. In a previous paper (Lorenc-Kubis et al 2001) we reported on the purification of a trypsin inhibitor (LUTI) from the resting seeds of common flax (Linum usitatissimum). Sepharose 4B and CM-Sephadex C-25 were from Pharmacia LKB Biotechnology (Uppsala, Sweden); acetonitrile (HPLC grade), trifluoroacetic acid (TFA), reagents for PAGE and edestin were from ICN Biomedicals (Costa Mesa, CA, USA); YM 3 membrane filters were purchased from Amicon (Danvers, MA, USA). A reverse phase Delta PAK C18 column was from Waters Division of Millipore (USA)

ANALYTICAL METHODS

METHODS

RESULTS AND DISCUSSION