Protein kinases from spinach chloroplasts. I. Purification and identification of two distinct protein kinases

Two protein kinases (chloroplast protein kinases 1 and 2 (ChlPK1 and ChlPK2)) were isolated from spinach chloroplasts. After solubilization of the chloroplasts with octylglucoside and cholate, these kinases were purified by ammonium sulfate precipitation, sucrose gradient centrifugation, and hydroxylapatite chromatography. ChlPK1 traveled as a single band in polyacrylamide gel electrophoresis corresponding to 25,000 daltons; ChlPK2 traveled as a single band corresponding to 38,000 daltons. After exposure to 8-azido-[gamma-32P]ATP, the radioactive bands appeared in the same positions revealed by Coomassie blue staining. However, a trace of ChlPK2 was detected in the ChlPK1 preparation and a faint second lower molecular weight radioactive band was seen in the ChlPK2 preparations. Both enzymes acted on casein or histone IIIS as substrate and phosphorylated a serine residue. The proteolytic peptide maps, however, were clearly distinguishable in autoradiograms, suggesting that different serine residues were phosphorylated by ChlPK1 and ChlPK2.