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 Pn....P1 and P1'....Pn', 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 isoinhibitor in 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, Cys42-Cys52 and Cys54-Cys57 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alfaalfaNOEs and 3JalfaNH 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 toxinx 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 breta-trypsin and bovine alfa-chymotrypsin are 3.3 x 10+9 M-1 and 2.4 x 10+6 M-1, respectively, at pH 8.0 and 21.0 °C. The serine proteinase:inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitor do not show any similarity to other serine proteinase inhibitors exept 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.
Characterization of low -molecular-mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed
BORTOLOTTI, Fabrizio;MENEGATTI, Enea
1999
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 Pn....P1 and P1'....Pn', 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 isoinhibitor in 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, Cys42-Cys52 and Cys54-Cys57 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alfaalfaNOEs and 3JalfaNH 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 toxinx 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 breta-trypsin and bovine alfa-chymotrypsin are 3.3 x 10+9 M-1 and 2.4 x 10+6 M-1, respectively, at pH 8.0 and 21.0 °C. The serine proteinase:inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitor do not show any similarity to other serine proteinase inhibitors exept 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.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
