Stromal protein phosphorylation in spinach (Spinacia oleracea) chloroplasts.
When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.
D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D003624:Darkness; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008565:Membrane Proteins / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010788:Photosynthesis; D014674:Plant Proteins, Dietary / Q000378:metabolism*
D008565:Membrane Proteins; D010446:Peptide Fragments; D014674:Plant Proteins, Dietary; C021261:thylakoid polypeptides; D015636:Magnesium Chloride; D008274:Magnesium; D010100:Oxygen