Purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil-rape (itBrassica napus) seed

• Published in: FEBS letters 1994-04, Vol.342 (2), p.221-224
• Main Authors: Ceciliani, Fabrizio, Bortolotti, Fabrizio, Menegatti, Enea, Ronchi, Severino, Ascenzi, Paolo, Palmieri, Sandro
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Binding Sites - genetics; Biological and medical sciences; Brassica - chemistry; Brassica - genetics; Brassica napus var. oleifera; Bz-l-Arg-PNA, N-α-benzoyl-l-arginine-p-nitroanilide; chymotrypsin, bovine α-chymotrypsin; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Molecular Sequence Data; MTI-2, low molecular weight mustard trypsin inhibitor; Mustard Plant - chemistry; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plants, Medicinal; Rapeseed; RTI, rapeseed trypsin inhibitor III; Seeds - chemistry; Sequence Homology, Amino Acid; Serine proteinase inhibitor; TFA, trifluoroacetic acid; TLCK-chymotrypsin, Bovine chymotrypsin trated with N-α-tosyl-l-lysine chloromethyl ketone; TPCK-trypsin, bovine trypsin treated with tosyl-l-phenylalanine chloromethyl ketone; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - genetics; Trypsin Inhibitors - isolation & purification; trypsin, bovine β-trypsin; Z-l-Tyr-ONP, N-α-carbobenzoxy-l-tyrosine-para-nitrophenyl ester
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(94)80505-9

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed ( Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine β-trypsin and bovine α-chymotrypsin with apparent dissociation constants of 3.0 × 10 −10 M and 4.1 × 10 −7 M, at pH 8.0 and 21°C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M r, of about 6.7 kDa. The p 1-p i, reactive site bond has been tentatively identified at position Arg 20-Ile 21. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.