Salt induction and the partial purification/characterization of phosphoenolpyruvate carboxylase protein-serine kinase from an inducible crassulacean-acid-metabolism (CAM) plant, Mesembryanthemum crystallinum L.
Treatment of the common ice plant (Mesembryanthemum crystallinum) with high salinity caused the well-documented increase in phosphoenolpyruvate carboxylase (PEPC) protein and a concomitant rise in the activity of a Ca(2+)-independent PEPC-kinase (PEPC-PK). When the plants were irrigated with 0.5 M NaCl, PEPC protein level and PEPC-PK activity started to increase after 2 days of treatment and continued to rise for the next 8 days, attaining about a 14- and 8-fold total increase, respectively. This salt-induced PEPC-kinase activity was detected only in leaves harvested from the stressed plants at night. This highly regulated protein kinase was partially purified about 3500-fold from these darkened, salt-stressed plants by sequential fast-protein liquid chromatography on phenyl-Sepharose, blue dextran-agarose, and Superdex 75. The gel-filtration data indicated that the native PEPC-kinase has a molecular weight around 33,000. Complementary analysis by denaturing electrophoresis and subsequent in situ renaturation and assay of PEPC-kinase activity revealed two major PEPC-PK polypeptides with approximate molecular masses of 39 and 32 kDa. The partially purified M. crystallinum PEPC-kinase readily phosphorylated PEPCs purified from maize, M. crystallinum, and tobacco leaves and a recombinant sorghum enzyme. In contrast, this Ca(2+)-independent protein kinase phosphorylated neither a recombinant sorghum mutant PEPC in which the target residue (Ser-8) was changed by site-directed mutagenesis to Asp nor histone III-S, casein, and bovine serum albumin. The optimal pH for PEPC-PK activity was pH 8.0 and this activity was affected by both the substrate (phosphoenolpyruvate) and the negative allosteric effector (L-malate) of PEPC in a pH-dependent manner. Overall, the molecular properties of this highly regulated PEPC-kinase from M. crystallinum are strikingly similar to those reported recently by this laboratory for the reversibly light-activated C4 enzyme from maize (Arch. Biochem. Biophys., 1993, 304, 496-502, and 307, 416-419).
Li
B
B
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Chollet
R
R
D002118:Calcium / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D010944:Plants / Q000187:drug effects / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012965:Sodium Chloride; D004533:Egtazic Acid; D010730:Phosphoenolpyruvate Carboxylase; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D002118:Calcium