Purification of tomato sucrose synthase phosphorylated isoforms by Fe(III)-immobilized metal affinity chromatography.
The major phosphorylation site of maize sucrose synthase (SuSy) is well conserved among plant species but absent in the deduced peptide sequence of the tomato SuSy cDNA (TOMSSF). In this study, we report the in vitro phosphorylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogenous Ca2+-dependent protein kinase activity. Two distinct 32P-labeled peptides detected in the tryptic peptide map of in vitro 32P-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid analysis of the major 32P-labeled peptide revealed the presence of a SuSy isozyme in young tomato fruit having the N-terminus phosphorylation site present in other plant species. By using Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two 32P-labeled tomato SuSy isoforms were separated from a nonradiolabeled SuSy fraction by using a pH gradient. The major 32P-SuSy isoform was phosphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabeled SuSy fraction was indicated by a higher retention during Fe(III)-IMAC and by tryptic peptide mapping analysis. Kinetic analyses of SuSy isoforms purified by Fe(III)-IMAC have revealed that phosphorylation of the major phosphorylation site of tomato fruit SuSy was not sufficient by itself to modulate tomato SuSy activity, whereas the affinity for UDP increased about threefold for the multiphosphorylated SuSy isoform.
Anguenot
R
R
Centre de Recherche en Horticulture, Département de phytologie, Université Laval, Sainte-Foy, Québec, G1K 7P4, Canada.
Yelle
S
S
Nguyen-Quoc
B
B
D002846:Chromatography, Affinity / Q000379:methods; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D018551:Solanum lycopersicum / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D020033:Protein Isoforms; D005964:Glucosyltransferases; C022699:sucrose synthase; D011494:Protein Kinases