tailieunhanh - Báo cáo Y học: Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

Two secreted acid phosphatases (SAP1 and SAP2) were markedly up-regulated during Pi-starvation of tomato suspension and SAP2 were resolved during cat-ion-exchange FPLC of culture media proteins from 8-day-old Pi -starved cells, and purified to homogeneity and final p-nitrophenylphosphatehydrolyzing specificactivitiesof 246 and 940lmol Pi producedÆmin )1 mgÆprotein )1 , , periodic acid-Schiff staining and analyt-ical gel filtration demonstrated that SAP1 and SAP2, respectively, exist as 84 and57 kDa glycosylatedmonomers | Eur. J. Biochem. 269 6278-6286 2002 FEBS 2002 doi Purification and characterization of two secreted purple acid phosphatase isozymes from phosphate-starved tomato Lycopersicon esculentum cell cultures Gale G. Bozzo1 Kashchandra G. Raghothama3 and William C. Plaxton1 2 Departments of1 Biology and 2Biochemistry Queen s University Kingston Ontario Canada 3Department of Horticulture and Landscape Architecture Purdue University Indiana USA 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 Ltmol Pi produced-min-1 mg-protein-1 respectively. SDS PAGE jeeii od ic acid-Schifis ttaíníng and ana ly - -ical 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 mappíng indicated that SAP1 and SAP2 are structurally distinct. Both isozymes displayed a pH optimum of approximately pH and were heat stable. Although they exhihi ted wide substrate specificities the Vmixi of SAP2 with various phosphate-esters was significantly greater than that of SAP 1. SAP 1 and S A P2 were activated by up to 80 by 5 mM Mg2 and demonstrated potent competitive inhibition by molybdate but mixed and competitive inhibition by Pr respectively. nntat-estingly both SAPs exhibited significant peroxidase activity which was optimal at approximately pH and insensitive to Mg2 or molybdate. Thís suggetss that .

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