Phosphorylation of a stromal enzyme protein in maize (Zea mays) mesophyll chloroplasts

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Abstract
When intact maize (Zea mays) mesophyll chloroplasts were illuminated in the presence of [32P]orthophosphate and subsequently subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, a major polypeptide species of Mr 100000 was found to be heavily labelled. This polypeptide was not found in maize mesophyll thylakoid or cytoplasmic fractions, but was localized solely in the chloroplast stroma. No phosphorylation of polypeptides in the 100000-Mr region was observed in the mesophyll chloroplasts from C3 species (where the primary product of CO2 fixation is a 3-carbon compound), suggesting that this polypeptide arises from a protein associated with C4 metabolism (where the first product of CO2 fixation is a 4-carbon compound). The 100kDa polypeptide was major component of the maize mesophyll chloroplast, comprising 10-15% of the total protein, which banded in an identical position to the apoprotein of the enzyme pyruvate, orthophosphate dikinase, which catalyses a reaction of the C4 cycle [Edwards & Walker (1983) C3, C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis, Blackwell Scientific Publications, Oxford and London]. Phosphorylation in the 100kDa species was prohibited by treatment of lysed chloroplasts with antibody to pyruvate, orthophosphate dikinase (EC 2.7.9.1). These data suggest that the phosphorylated polypeptide observed after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis is the monomeric form of this enzyme. The 100kDa polypeptide was partially phosphorylated in darkness, but a significant increase in the degree of phosphorylation was found on illumination. This polypeptide was found to be dephosphorylated only slowly when the chloroplasts were returned to darkness. Maximum phosphorylation was observed in the presence of pyruvate or dihydroxyacetone phosphate, which also caused maximum activation of pyruvate, orthophosphate dikinase. Phosphorylation of the 100kDa polypeptide did not coincide with deactivation of pyruvate, orthophosphate dikinase, but maximum phosphorylation occurred under conditions that promoted maximum activity of the enzyme, at which time one phosphate group was associated with each enzyme molecule. Protein phosphorylation did not appear to arise from the reaction mechanism of the enzyme.