Protein inhibitors are molecules secreted by many plants. In a functional genomics approach, an invertase inhibitor (SolyCIF) of Solanum lycopersicum was identified at the Solanaceae Cornell University data bank (www.sgn.cornell.edu). It was established that this inhibitor is expressed mainly in the leaves, flowers and green fruit of the plant and localized in the cell wall compartment. The SolyCIF cDNA was cloned by performing RT-PCR, fully sequenced and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by performing ion-exchange chromatography and gel filtration was further biochemically characterized and used to perform affinity chromatography. The latter step made it possible to purify natural vacuolar invertase (TIV-1), which showed high rates of catalytic activity (438.3 U mg(-1)) and efficiently degraded saccharose (K-m = 6.4 mM, V-max = 2.9 mu mol saccharose min(-1) and k(cat) = 7.25 x 10(3) s(-1) at pH 4.9 and 37 degrees C). The invertase activity was strongly inhibited in a dose-dependent manner by SolyCIF produced in P. pastoris. In addition, Gel-SDS-PAGE analysis strongly suggests that TIV-1 was proteolyzed in planta and it was established that the fragments produced have to be tightly associated for its enzymatic activity to occur. We further investigated the location of the proteolytic sites by performing NH2-tenninal Edman degradation on the fragments. The molecular model for TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two halves, which confirms that the enzymatic activity is possible only when the fragments are tightly associated. (c) 2008 Elsevier Masson SAS. All rights reserved.
Molecular characterization of a novel apoplastic invertase inhibitor from tomato / Reca, Ida Barbara; Brutus, ALEXANDRE RENE MAURICE; D'Avino, R; Villard, C; Bellincampi, Daniela; Giardina, T.. - STAMPA. - 90:44176(2008), pp. 50-50. (Intervento presentato al convegno XLVII congresso SIFV tenutosi a Pisa; Italy nel 30 Giugno-2 Luglio 2008).
Molecular characterization of a novel apoplastic invertase inhibitor from tomato
RECA, Ida Barbara;BRUTUS, ALEXANDRE RENE MAURICE;BELLINCAMPI, Daniela;
2008
Abstract
Protein inhibitors are molecules secreted by many plants. In a functional genomics approach, an invertase inhibitor (SolyCIF) of Solanum lycopersicum was identified at the Solanaceae Cornell University data bank (www.sgn.cornell.edu). It was established that this inhibitor is expressed mainly in the leaves, flowers and green fruit of the plant and localized in the cell wall compartment. The SolyCIF cDNA was cloned by performing RT-PCR, fully sequenced and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by performing ion-exchange chromatography and gel filtration was further biochemically characterized and used to perform affinity chromatography. The latter step made it possible to purify natural vacuolar invertase (TIV-1), which showed high rates of catalytic activity (438.3 U mg(-1)) and efficiently degraded saccharose (K-m = 6.4 mM, V-max = 2.9 mu mol saccharose min(-1) and k(cat) = 7.25 x 10(3) s(-1) at pH 4.9 and 37 degrees C). The invertase activity was strongly inhibited in a dose-dependent manner by SolyCIF produced in P. pastoris. In addition, Gel-SDS-PAGE analysis strongly suggests that TIV-1 was proteolyzed in planta and it was established that the fragments produced have to be tightly associated for its enzymatic activity to occur. We further investigated the location of the proteolytic sites by performing NH2-tenninal Edman degradation on the fragments. The molecular model for TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two halves, which confirms that the enzymatic activity is possible only when the fragments are tightly associated. (c) 2008 Elsevier Masson SAS. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
