PROTEIN-PHOSPHORYLATION AS A MECHANISM FOR OSMOTIC-STRESS ACTIVATION OF SUCROSE-PHOSPHATE SYNTHASE IN SPINACH LEAVES

Experiments were performed to investigate the mechanism of sucrose-pho sphate synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immu nopurification and was not the result of an increased SPS protein leve l. The previously described Ca2+-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant en zyme catalyzing phosphorylation and inactivation of dephosphoserine-15 8-SPS. A new, Ca2+-dependent SPS-protein kinase activity (peak IV kina se) was also resolved and shown to phosphorylate and activate phosphos erine-158-SPS in vitro. The peak IV kinase also phosphorylated a synth etic peptide (SP29) based on the amino acid sequence surrounding serin e-424, which also contains the motif described for the serine-158 regu latory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weig ht of approximately 150,000 as shown by gel filtration. The SP29 pepti de was not phosphorylated by the inactivating peak III kinase. Osmotic ally stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic P-32-phosphopeptide analysis of SPS from excised spinach leaves fed [P-32]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefo re, at least part of the osmotic stress activation of SPS in dark leav es results from phosphorylation of serine-424 catalyzed by a Ca2+-depe ndent, 150-kD protein kinase.