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 con served among plant species but absent in the deduced peptide sequence of th e tomato SuSy cDNA (TOMSSF), In this study, we report the in vitro phosphor ylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogeno us Ca2+-dependent protein kinase activity. Two distinct P-32-labeled peptid es detected in the tryptic peptide map of in vitro P-32-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid anal ysis of the major P-32-labeled peptide revealed the presence of a SuSy isoz yme in young tomato fruit having the N-terminus phosphorylation site presen t in other plant species, By using Fe(III)-immobilized metal affinity chrom atography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two P-32-labeled tomato SuSy isoforms were separated from a nonradiolabele d SuSy fraction by using a pH gradient, The major P-32-SuSy isoform was pho sphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabel ed SuSy fraction was indicated by a higher retention during Fe(III)-IMAC an d by tryptic peptide snapping analysis, Kinetic analyses of SuSy isoforms p urified by Fe(III)-IMAC have revealed that phosphorylation of the major pho sphorylation site of tomato fruit SuSy was not sufficient by itself to modu late tomato SuSy activity, whereas the affinity for UDP increased about thr eefold for the multiphosphorylated SuSy isoform, (C) 1999 Academic Press.