Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative crassulacean‐acid‐metabolism plant

Abstract

Phosphoenolpyruvate PyrP carboxylase (PyrPC) and PyrPC kinase were copurified from darkadapted leaves of the common ice plant Mesembryanthemum crystallinum L . with crassulacean-acid metabolism (CAM). Purification by (NH&S04 fractionation, chromatography on Fractogel-DEAE and hydroxylapatite resulted in a PyrPC preparation with a specific activity of 23 -25 U/mg protein and a protein kinase activity of255 pmol P,. mol-' PyrPC . s-'. After in vitro phosphorylation, the most prominently phosphorylated polypeptide was identified as PyrPC by immunoblotting and sequencing. Phosphorylation of PyrPC in vitro by incubation with 400 pM MgATP decreased its sensitivity towards malate. When purified in the absence of the protease inhibitor chymostatin, PyrPC lost an N-terminal sequence of 128 amino acids. Although the carboxylation reaction was unaffected, the truncated PyrPC could neither be phosphorylated in vitro nor inhibited by malate. This result and data obtained by limited proteolysis concur with the hypothesis [Jiao, J.-A. & Chollet, R. (1989) Arch. Biochem. Biophys. 283, 300 -3051 that Serll is the phosphorylation site of the CAM PyrPC of M . crystallinum. At pH 7.0, the K,,, for ATP of the protein kinase was 25 pM; phosphorylation of PyrPC was maximal after 30 min at pH 7.0. The kinase showed also activity with histone 111-S but not with dephosphorylated casein. It was inhibited by malate. The results show, that reversible protein phosphorylation is an important factor in the regulation of PyrPC in the facultative CAM plant M . crystallinum, similar to C, and constitutive CAM plants.

Partial Purification and Characterization of a Calcium-Dependent Protein Kinase and an Inhibitor Protein Required for Inactivation of Spinach Leaf Nitrate Reductase

“…4). This value is similar to those reported for spinach SPS-protein kinase (5 p~; Huber and Huber, 1990) and PEP carboxylase-protein kinase isolated from maize (40 PM; Wang and Chollet, 1993) or Mesembryanthemum (25 p~; Baur et al, 1992).…”

Evolution of the Enzymatic Characteristics of C4Phosphoenol Pyruvate Carboxylase

“…This truncation may not change the overall activity of the enzyme but, as the lost part harbours the phosphorylation site, the regulatory properties are most certainly affected (Bauer et al, 1992). The intactness of the purified P-pyruvate carboxylases was therefore investigated by N-terminal sequencing through Edman degradation.…”

Posttranscriptional and Posttranslational Control of Enolase Expression in the Facultative Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L

“…In M . crystallinum, phosphorylation of PEPC by a PEPC protein kinase is one of the key mechanisms of modulating diurnal enzyme activity (Baur et al, 1992). In the case of enolase, phosphorylation has been invoked as a possible explanation for the disparity between protein levels and activities found in castor bean (Miernyk and Dennis, 1992).…”