Purification and characterization of a novel calcium-dependent protein kinase from soybean.
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., & Cormier, M.J. (1987) Plant Physiol. 83, 830-837], was purified to greater than 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 microM 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 microM and a Vmax of 1.7 mumol 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.
Putnam-Evans
C L
CL
Department of Biochemistry, University of Georgia, Athens 30602.
Harmon
A C
AC
Cormier
M J
MJ
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D017871:Calcium-Calmodulin-Dependent Protein Kinases
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases