Purification and characterization of a novel calcium-dependent protein kinase from soybean

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Abstract
A novel calcium-dependent protein kinase (CDPK) previously reported to be activated by the direct binding of Ca2+, and requiring neither calmodulin nor phospholipids for activity [Harmon, A. C., Putnam-Evans, C. L., and Cormier, M. J. (1987) Plant Physiol. 83, 830-837], was purified to > 95% homogeneity from suspension-cultured soybean cells (Glycine max, L. Wayne). Purification was achieved by chromatography on DEAE-cellulose, phenyl-Sepharose, Sephadex G-100, and blue Sepharose. The purified enzyme (native molecular mass = 52,200 Da) resolved into two immunologically related protein bands of 52 and 55 kDa on 10% SDS gels. Enzyme activity was stimulated 40-100-fold by micromolar amounts of free calcium (K0.5 = 1.5 .mu.M free calcium) and was dependent upon millimolar Mg2+. CDPK phosphorylated lysine-rich histone III-S and chicken gizzard myosin light chains but did not phosphorylate arginine-rich histone, phosvitin, casein, protamine, or Kemptide. Phosphorylation of histone III-S, but not autophosphorylation, was inhibited by KCl. CDPK displayed a broad pH optimum (pH 7-9), and kinetic studies revealed a Km for Mg2+-ATP of 8 .mu.M and a Vmax of 1.7 .mu.mol min-1 mg-1 with histone III-S (Km = 0.13 mg/mL) as substrate. Unlike many other protein kinases, CDPK was able to utilize Mg2+-GTP, in addition to Mg2+-ATP, as phosphate donor. The enzyme phosphorylated histone III-S exclusively on serine; however, CDPK autophosphorylated on both serine and threonine residues. These properties demonstrate that CDPK belongs to a new class of protein kinase.