A 64-kDa protein is a substrate for phosphorylation by a distinct thylakoid protein kinase

Abstract

Solubilization of spinach thylakoids with the nonionic detergent n-octyl-β-D-glucopyranoside (OG) releases active protein kinase from the membrane. Further purification was reported to demonstrate that a 64-kDa protein is the origin of this kinase activity (Coughlan S J and Hind G (1986) J Biol Chem 261: 11378-11385). The N-terminal sequence of this protein was subsequently determined (Gal A, Herrmann R, Lottspiech F and Ohad I (1992) FEBS Lett 298: 33-35). Liquid phase isoelectric focusing of the OG extract and an hydroxylapatite-purified fraction, derived from the OG preparation, reveals that the 64-kDa protein with this documented N-terminal sequence can be separated from the protein kinase activity. Experimental conditions were optimised by manipulation of ampholyte and detergent concentrations to maximise protein solubility and enzyme activity. The kinase-containing fraction was able to catalyze the phosphorylation of several proteins including the 64-kDa which was identified using antibodies raised against a synthetic peptide corresponding to the N-terminal sequence. The results described indicate that this 64-kDa protein is not the protein kinase responsible for the phosphorylation of the light-harvesting complex associated with Photosystem II.