Purification and characterization of a membrane-bound protein kinase from spinach thylakoids.
A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.
D001345:Autoradiography; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D006863:Hydrogen-Ion Concentration; D010750:Phosphoproteins / Q000032:analysis; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D014675:Vegetables / Q000201:enzymology*
D005960:Glucosides; D010750:Phosphoproteins; C018619:octyl-beta-D-glucoside; D011494:Protein Kinases