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Eur J Biochem. 2002 Dec;269(24):6278-86.

Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

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Departments of Biology, Queen's University, Kingston, Ontario, Canada.


Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension cells. SAP1 and SAP2 were resolved during cation-exchange FPLC of culture media proteins from 8-day-old Pi-starved cells, and purified to homogeneity and final p-nitrophenylphosphate hydrolyzing specific activities of 246 and 940 micro mol Pi produced.min-1 mg.protein-1, respectively. SDS/PAGE, periodic acid-Schiff staining and analytical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and 57 kDa glycosylated monomers. SAP1 and SAP2 are purple acid phosphatases (PAPs) as they displayed an absorption maximum at 518 and 538 nm, respectively, and were not inhibited by l-tartrate. The respective sequence of a SAP1 and SAP2 tryptic peptide was very similar to a portion of the deduced sequence of several putative Arabidopsis thaliana PAPs. CNBr peptide mapping indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH 5.3 and were heat stable. Although they exhibited wide substrate specificities, the Vmax of SAP2 with various phosphate-esters was significantly greater than that of SAP1. SAP1 and SAP2 were activated by up to 80% by 5 mm Mg2+, and demonstrated potent competitive inhibition by molybdate, but mixed and competitive inhibition by Pi, respectively. Interestingly, both SAPs exhibited significant peroxidase activity, which was optimal at approximately pH 8.4 and insensitive to Mg2+ or molybdate. This suggests that SAP1 and SAP2 may be multifunctional proteins that operate: (a) PAPs that scavenge Pi from extracellular phosphate-esters during Pi deprivation, or (b) alkaline peroxidases that participate in the production of extracellular reactive oxygen species during the oxidative burst associated with the defense response of plants to pathogen infection.

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