In vitro phosphorylation of purified tobacco‐leaf phosphoenolpyruvate carboxylase

• Published in: FEBS letters Vol. 328; no. 1-2; pp. 215 - 218
• Main Authors: Wang, Yue-Hao, Chollet, Raymond
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 09.08.1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; C3 plant; CAM, Crassulacean acid metabolism; Chromatography, Gel; Chromatography, High Pressure Liquid; DTT, dithiothreitol; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; FPLC, fast-protein liquid chromatography; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; Lyases; Molecular Sequence Data; MOPS, 3-(N-morpholino)propanesulfonic acid; Nicotiana - enzymology; PEG, polyethylene glycol; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; Phosphoenolpyruvate Carboxykinase (GTP) - metabolism; Phosphoenolpyruvate carboxylase; Phosphoenolpyruvate Carboxylase - isolation & purification; Phosphoenolpyruvate Carboxylase - metabolism; Phosphorylation; PK, protein kinase; Plants, Toxic; PMSF, phenylmethylsulfonyl fluoride; Protein kinase; Protein phosphorylation; Tobacco (Nicotiana tabacum L.); Zea mays - enzymology; Phosphorylation; Enzyme; Protein kinase; Dicotyledones; Angiospermae; Spermatophyta; Plant leaf; Solanaceae; Nicotiana tabacum; C3-Type; Phosphoenolpyruvate carboxylase
• DOI: 10.1016/0014-5793(93)80995-7
• ISSN: 0014-5793

C3‐leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was purified about 1,000‐fold from tobacco and displayed a final specific activity of 35 μmol/min/mg protein, an apparent K m (total PEP) of 95 mM (both at (pH 8.0, 30°C), and an I50(l‐malate) value of 0.14 mM at pH 7.3, 0.2 mM PEP. The rapid, 5‐step protocol involved polyethylene glycol fractionation and sequential FPLC on hydroxylapatite, phenyl‐Sepharose, Mono Q and Superose 12. The electrophoretically pure protein and purified C4‐leaf PEPC were phosphorylated in vitro in a reconstituted system with PEPC‐kinase isolated from illuminated tobacco and maize leaves. These reciprocal phosphorylation experiments (i) indicate that Ser11 of tobacco PEPC is the likely target residue, situated in the plant‐invariant Glu/Asp‐Lys/Arg‐X‐X‐Ser phosphorylation motif near the N‐terminus, and (ii) lend support to the recent hypothesis that C3‐leaf PEPC is subject to regulatory phosphorylation in vivo.