Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L.; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed ≈ 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit (≈ 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine β-trypsin (trypsin) and bovine α-chymotrypsin were 6.3 × 109m−1 and 2.0 × 106 m−1, respectively, at pH 8.0 and 21.0 °C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 × 105 m−1·s−1 and 8.9 × 10−5 m−1·s−1, respectively, at pH 8.0 and 21.0 °C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.
Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.)seed
MENEGATTI, Enea;
2000
Abstract
Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 family were identified from white mustard (Sinapis alba L.; MTI-2) seed. Purified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at position 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutamic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of the serine proteinase inhibitor. MTI-2 showed ≈ 70% amino-acid identity with low-molecular-mass trypsin inhibitors isolated from oil rape (Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhibitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MTI-2 was homologous to brazzein, the sweet-tasting protein from Pentadiplandra brazzeana Baillon fruit (≈ 30% amino-acid identity). Although snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, and Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, being different from that of brazzein. Therefore, protein similarity might be attributable to the three-dimensional arrangement rather than to the amino-acid sequence. Values of Ka for MTI-2 binding to bovine β-trypsin (trypsin) and bovine α-chymotrypsin were 6.3 × 109m−1 and 2.0 × 106 m−1, respectively, at pH 8.0 and 21.0 °C. Moreover, values of kon for MTI-2 binding to trypsin and of koff for the dissociation of the serine proteinase:inhibitor complex were 5.6 × 105 m−1·s−1 and 8.9 × 10−5 m−1·s−1, respectively, at pH 8.0 and 21.0 °C. Despite the heterogeneity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
