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

• Published in: Biochemistry (Easton) 1990-03, Vol.29 (10), p.2488-2495
• Main Authors: Putnam-Evans, Cindy L, Harmon, Alice C, Cormier, Milton J
• Format: Article
• Language: English
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; AISLAMIENTO (TECNICA); Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Antibodies, Monoclonal; Biological and medical sciences; CALCIO; CALCIUM; CHEMICOPHYSICAL PROPERTIES; Electrophoresis, Polyacrylamide Gel; ENZYME KINETICS; Enzymes and enzyme inhibitors; ENZYMIC ACTIVITY; Fundamental and applied biological sciences. Psychology; GLYCINE MAX; Glycine max - enzymology; Immunoblotting; ISOLATION; ISOLEMENT; Kinetics; Phosphorylation; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICO-CHIMIQUE; protein kinase; Protein Kinases - isolation & purification; Substrate Specificity; TRANSFERASAS; TRANSFERASE; TRANSFERASES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00462a008

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.