title: Characterization of low-molecular-mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed.
abstract: A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.
authors: Dipartmento di Biologia, Universitá di Roma Tre, Italy. ascenzi@bio.uniroma3.it
authors: P
authors: P
authors: Ascenzi
authors: M
authors: M
authors: Ruoppolo
authors: A
authors: A
authors: Amoresano
authors: P
authors: P
authors: Pucci
authors: R
authors: R
authors: Consonni
authors: L
authors: L
authors: Zetta
authors: S
authors: S
authors: Pascarella
authors: F
authors: F
authors: Bortolotti
authors: E
authors: E
authors: Menegatti
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001937:Brassica / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004220:Disulfides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D014357:Trypsin