Protein kinases of the thylakoid membrane.

Abstract The claim of Racker and co-workers (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156 and Lucero, H. A., Lin, Z. F., and Racker, E. (1982) J. Biol. Chem. 257, 12157-12160) that two protein kinases, designated CPK1 (25 kDa) and CPK2 (38 kDa), are present in spinach thylakoid membranes was investigated in light of results from this laboratory (Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385) showing that 75-80% of the measurable protein kinase activity of isolated thylakoids is attributable to a protein kinase of 64 kDa apparent molecular mass. Extraction of thylakoid membranes with octyl glucoside/cholate according to the procedure of Lin et al. (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156) released proteins assignable to CPK1 and CPK2 on the basis of photoaffinity labeling with 8-azido-[32P]ATP. The 64-kDa protein kinase was present in this extract and accounted for greater than 80% of the total phosphotransferase activity toward lysine-rich histone as substrate; it was not labeled by the photoaffinity reagent. The three presumptive kinases were purified by ammonium sulfate precipitation, sucrose density gradient centrifugation, hydroxylapatite chromatography, and affinity chromatography. CPK1 was specifically eluted from Cibacron blue-Sepharose by 10 mM ATP; it electrophoresed on denaturing polyacrylamide gels as a single band with apparent molecular mass of 25 kDa. Its specific activity toward lysine-rich histone as substrate was approximately 250 pmol of phosphate transferred (mg protein)-1 min-1. The 64-kDa protein kinase was eluted from the affinity column by 1% (w/v) lithium dodecyl sulfate or from a histone IIIs-Sepharose affinity column by 0.25 M NaCl. Its specific activity towards lysine-rich histone was 100-200 times greater than that of CPK1. CPK2 eluted from the Cibacron blue affinity column in 10 mM NADP+; it had an apparent molecular mass of 38 kDa, possessed NADPH-dependent diaphorase activity (specific activity: 225 nmol of ferricyanide reduced (mg protein)-1 min-1), and cross-reacted with immunoglobulin raised against purified ferredoxin:NADP+ oxidoreductase, with which it was thus identified. Kinase activity was not detectable in CPK2 or in reductase isolated by conventional procedures.