Phosphorylation of thylakoid proteins by a purified kinase.
A simplified method is given for the purification of a 64-kilodalton protein kinase from spinach or pea thylakoid membranes (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385). In a heterogeneous reconstitution system comprised of purified kinase and washed thylakoids (having their intrinsic kinase inactivated or removed), endogenous light-harvesting pigment protein of photosystem II could serve as a substrate. Its phosphorylation did not require rebinding of kinase to the thylakoid membrane and, like the phosphorylation of solubilized pigment protein, was not under redox control. No reconstitution was observed upon replacing 64-kilodalton protein kinase with 25-kilodalton protein kinase (Coughlan, S., and Hind, G. (1986) J. Biol. Chem. 261, 14062-14068). Tryptic digestion of phosphorylated membranes removed the site of phosphorylation; the phosphorylated amino acid present in light-harvesting pigment protein and its tryptic peptide was threonine. Immunoglobulin from a polyclonal antiserum, raised against the purified enzyme, fully inhibited kinase activity toward solubilized and endogenous pigment protein. At higher titers, the antibody was effective in totally inhibiting the redox-sensitive phosphorylation of thylakoid proteins by endogenous kinase; inhibition profiles for phosphorylation of pigment protein and thylakoid proteins of 32, 16, and 9 kilodaltons were essentially identical. The 64-kilodalton protein kinase would thus appear to be responsible for all of the observed phosphorylation of thylakoid phosphoproteins.
Coughlan
S
S
Hind
G
G
D000936:Antigen-Antibody Complex; D002734:Chlorophyll / Q000378:metabolism*; D007887:Fabaceae; D007074:Immunoglobulin G; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D020930:MAP Kinase Kinase Kinases; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D010946:Plants, Medicinal; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
D000936:Antigen-Antibody Complex; D002734:Chlorophyll; D007074:Immunoglobulin G; D045342:Light-Harvesting Protein Complexes; D020930:MAP Kinase Kinase Kinases; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011494:Protein Kinases; D000099151:MAP Kinase Kinase Kinase 7