PURIFICATION AND MOLECULAR-PROPERTIES OF THE THYLAKOID-BOUND ASCORBATE PEROXIDASE IN SPINACH-CHLOROPLASTS

The hydrogen peroxide that is photoproduced in thylakoids is scavenged by the thylakoid-bound ascorbate peroxidase (tAPX) [Miyake and Asada (1992) Plant Cell Physiol 33: 541]. tAPX was purified from spinach thy lakoids to homogeneity as judged by SDS-polyacrylamide gel electrophor esis, and its molecular properties were studied. Spinach tAPX was a mo nomer with a molecular weight of 40,000, which is about 10,000 higher than that of the stromal ascorbate peroxidase (sAPX) from spinach chlo roplasts. tAPX cross-reacted with the antibody raised against sAPX fro m tea leaves, as determined by Western blotting, which also provided e vidence for the higher molecular weight of tAPX from spinach thylakoid s than that of tea sAPX. The amino acid sequence of the amino-terminal region of tAPX showed a low degree of homology to those of cytosolic APXs from spinach, pea and Arabidopsis thaliana, but a high degree of homology to that of stromal APX from tea. Thus, the amino-terminal reg ion of tAPX seems not to be a domain required for binding of the enzym e to the thylakoid membranes. tAPX contained protoheme IX, as identifi ed by its pyridine hemochromogen, and gave a Soret peak at 403 nm and 433 nm with an alpha band at 555 nm in its oxidized and reduced forms, respectively. Resembling sAPX but differing from cytosolic APX, tAPX showed high specificity for ascorbate as the electron donor. tAPX was inhibited by cyanide, thiol-modifying reagents, thiols and several sui cide inhibitors, such as hydroxyurea and p-aminophenol.