Two ascorbate peroxidases from broccoli: identification, expression and characterization of their recombinant proteins

Abstract

Two distinct clones having high nucleotide identity to the sequences encoding ascorbate peroxidase (APX) were isolated from broccoli ( Brassica oleracea L. var. italica ). Deduced amino acid sequences of both cDNAs, BO-APX 1 (accession number AB078599) and BO-APX 2 (accession number AB078600), shared identity of 92.8% and there was more than 80% identity between BO-APX s and other plant cytosolic APXs at the protein level. Gene expression and protein levels of BO-APX 1 and BO-APX 2 were investigated in various parts of broccoli after harvest. Transcript levels of BO-APX 2 gradually increased in florets, while those of BO-APX 1 decreased in florets after harvest. BO-APX 1 and BO-APX 2 were expressed in Escherichia coli as a fusion protein with glutathione S -transferase (GST) and purified to homogeneity by glutathione sepharose 4B column chromatography. Both proteins of BO-APX 1 and BO-APX 2 appeared as a single major band on SDS-PAGE corresponding to a mass of 25 kDa and reacted with polyclonal antibodies raised against recombinant BO-APX 1 . Both enzymes showed high specificities for ascorbate and hydrogen peroxide. The km values of recombinant BO-APX 1 and BO-APX 2 for ascorbate were 395 and 526 μM and those for hydrogen peroxide were 15 and 7 μM, respectively. The role of APX was discussed in relation to ascorbate breakdown in broccoli florets during senescence.