PURIFICATION AND KINETIC PROPERTIES OF A BASIC PEROXIDASE SECRETED TO THE CULTURE MEDIUM OF TOMATO HAIRY ROOTS UNDER SALT STRESS

TALANO, MELINA; AGOSTINI, ELIZABETH; MILRAD, SILVIA; MEDINA, MARÍA I. & TIGIER, HORACIO

Villa Carlos Paz-Córdoba

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB); 2001

SAIB

Tomato hairy root cultures treated with 100 mM NaCl showed an excretion of a highly basic peroxidase to the culture medium. The aim of this work was to purify this isoperoxidase and to study the kinetic properties with coniferyl alcohol (CA) as substrate, in order to evaluate its involvement in lignification as a response to salt stress. These properties were also compared with those of pI 9.6 isoperoxidase of normal tomato roots, whose expression we have found to be increased in hydroponic cultures exposed to the same stress. Peroxidases secreted to the medium of hairy roots cultures containing 100 mM NaCl, were purified through a SP Sephadex C50 column equilibrated at pH 7.5 where some of them were retained. Then they were eluted in one step with 0.3 M NaCl. Cationic and anionic PAGE of these fractions showed a single cationic peroxidase band with Rf 0.59. Kinetic studies performed with CA (10 a 100 mM) and 4 mM H2O2 at pH 4.5 showed a Km of 55.5 mM for CA and a catalytic efficiency of 1.05 mM-1s-1. The isoenzyme affinity and its catalytic efficiency with CA were higher than those found for pI 9.6 isoperoxidase from normal tomato roots, whose expression was increased under salt stress, and that is presumably involved in lignosuberization of these tissues. So, the highly basic peroxidase from hairy root cultures may be also related to this process. Grants of: SECYT UNRC, FONCYT - Agencia Córdoba Ciencia, CONICET