A preliminary characterization of a phenoloxidase from extracts of soluble and ionically‐bound cell wall proteins of peach (Prunus persica L. Batsch, cv. Redhaven) endocarp is described in the present study to establish differences with peroxidases from the same plant tissue. The phenoloxidase activity was detected mainly in the first stage of peach fruit growth, while peroxidase activity and lignin content increased along the second stage of growth. There were clear differences between the two enzymes. The phenoloxidase had a pI value of 5.6, different from those of peroxidases isoenzymes with various pIs ranging from 3.6 to 9.6. The oxidase molecular mass was 112 kDa, similar to other phenoloxidases described in the literature, while all peroxidase isoenzymes showed a molecular mass of around 40 kDa. The specific activities of phenoloxidase against different substrates and its inhibition by various effectors suggest that the endocarp oxidase described here is probably a metal‐dependent polyphenol oxidase, displaying attributes of both catechol oxidase (EC 1.10.3.1) and laccase (EC 1.10.3.2).
Changes of soluble and ionically bound peroxidase and indoleacetic acid (IAA) oxidase activities were followed during peach seed development. Soluble peroxidase activity was located mainly in the embryo plus endosperm tissue, whereas wall ionically bound activities were found predominantly in the integument tissue. The different peroxidase isoenzymes present in the extracts were characterized by polyacrylamide gel electrophoresis and isoelectric focusing; the main soluble isoenzyme of embryo plus endosperm tissue was an anionic isoperoxidase of R F 0.07. Basic ionically bound isoenzymes were located only in the integument tissue, but two soluble anionic isoenzymes of R F 0.23 and 0.51 were also present in this tissue. In parallel, peroxidase protein content was estimated specifically using polyclonal antibodies. The kinetic data and the changes of seed IAA oxidase activity during fruit development suggested that basic peroxidase isoenzymes from ionically bound extracts of integument might be involved in IAA degradation.The peach fruit is a useful model for studying growthrelated changes in peroxidase isoenzyme patterns and their relationships with some physiological processes because its growth occurs in three well defined development stages (Valpuesta et al. 1991). The seed apparently plays a critical role in growth and viability of the fruit during stage I (Tukey 1936). Changes in indoleacetic acid (IAA) levels and peroxidase activities have been determined previously along with the fruit growth (Valpuesta et al. 1989).The main soluble anionic peroxidase isoenzyme from peach seeds was purified, and rabbit polyclonal antibodies were obtained against this isoenzyme (Quesada et al. 1990). Changes in total IAA oxidase activity of peach complete seed peroxidases and levels of specific peroxidase proteins, detected by polyclonal antibodies, were followed during fruit growth (Alba et al. 1993).Here we report the first differential distribution of peroxidase isoenzymes and changes in the levels of specific protein in each seed tissue, integument (the outer coat of seed) and embryo plus endosperm, during fruit development. The kinetic behavior of IAA oxidase activity of the isoenzymes with the highest activity with this substrate was analyzed in order to study the possible role of peroxidases during peach seed development. Materials and Methods PlantsFruits were obtained from a commercial cultivar (cv. Redhaven) of peach (Prunus persica L. Batsch) during fruit growth. Several samples were collected during the three stages of fruit development defined previously for the cultivar Redhaven (Alba et al. 1995). Stage I extends from 0 to 55 days, stage II from 55 to 79 days, and stage III from 79 to 98 days after anthesis. Seeds were removed from frozen fruit and dissected mechanically into integument and embryo plus endosperm as a unique section. Crude Extract Isolation ProcedureSoluble protein extracts and those ionically bound to cell walls were obtained by grinding the seeds and their different sections with 50 mM sodium ...
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