Plant Science 161 (2001) 861–869 Characteristics of phosphoenolpyruvate phosphatase purified from Allium cepa Takuro Shinano *, Ryoma Yonetani, Nahoko Ushihara, Hirofumi Adachi, Jun Wasaki, Hirokazu Matsui, Mitsuru Osaki Laboratory of Plant Nutrition, Graduate School of Agriculture, Hokkaido Uniersity, Sapporo, Hokkaido 060 -8589, Japan Received 30 March 2001; received in revised form 15 June 2001; accepted 15 June 2001 Abstract A phosphatase has been purified 1767-fold, to a final specific activity of 53.0 (mol pyruvate produced per min per mg protein), from the bulb of Allium cepa L. The enzyme has high specificity for a phosphoenolpyruvate (PEP) substrate. The molecular mass of the enzyme is approximately 240 kDa, as determined by gel column chromatography. Although 52 and 42 kDa polypeptides were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), their N-terminal amino acid sequences were the same. We deduce, therefore, that the enzyme consists of four polypeptide subunits of 52 kDa. The purified enzyme hydrolyzes a wide variety of phosphate esters, and the lowest K m was obtained with PEP (0.44 mM). The enzyme consumed 1 mol of PEP, with the release of 1 mol of phosphate and 1 mol of pyruvate, with no ATP formation during the enzymatic reaction in the presence of ADP and Mg 2 + . Therefore, the enzyme is thought to be a PEP phosphatase. A cDNA that encodes PEP phosphatase, the deduced amino acid sequence of which resembles that of the plant acid phosphatases, was also isolated from onion bulb RNA. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Allium cepa ; Phosphoenolpyruvate phosphatase; Protein purification; cDNA sequence; Biochemical properties www.elsevier.com/locate/plantsci 1. Introduction Phosphate is one of the major elements found in plants. The available phosphate concentration in many soils is quite low, however, because most phosphorus exists as phytic acid (inositol hexaphosphate), as alu- minum- or ferrous-bound forms, or as organic forms in the soil. Therefore, a large amount of phosphorus fertilizer is required for intensive agriculture. As the sources of phosphorus become limited, improvements in the efficiency of phosphorus absorption and use in plants are becoming necessary. Plants have developed several different mechanisms for overcoming phospho- rus deficiency, such as expansion of the root surface [1], coexistence with mycorrhiza [2,3], the release of protons [4,5] or organic acids [5–7], and the secretion of acid phosphatase (EC 3.1.3.2) [8–10] or phytase (EC 3.1.3.26) [11 – 13] from the roots. Under phosphate-limiting conditions, plant cells re- lease phosphate into the cytosol from vacuoles [8,14]. In addition, ribonuclease (EC 3.1.27.1) releases phos- phorus by catabolizing ribosomal RNA [15,16]. The induction of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) by phosphate deficiency has been reported in Brassica nigra [10] and Catharanthus roseus [17,18]. The first report of phosphoenolpyruvate (PEP) phos- phatase was made by Duff et al. [19], in B. nigra. This PEP phosphatase activity is more pronounced under conditions of phosphorus deficiency [17]. PEP phos- phatase protein is synthesized de novo in response to phosphorus deficiency in B. nigra [20]. Duff et al. [20] have proposed that the role of PEP phosphatase is to allow the ADP-dependent pyruvate kinase (PK, EC 2.7.1.40) reaction to be bypassed under phosphate-de- pleted conditions (when ADP and phosphate [Pi] levels decrease [10,19]), by converting PEP (and other es- Abbreiations: Fru-6-P, fructose-6-phosphate; Glc-6-P, glucose-6- phosphate; LDH, lactate dehydrogenase; PEG, polyethylene glycol; PEP, phosphoenolpyruvate; PK, pyruvate kinase; p NPP, p -nitro- phenyl phosphate. * Corresponding author. Tel./fax: +81-11-706-3845. E-mail address: takuro@chem.agr.hokudai.ac.jp (T. Shinano). 0168-9452/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(01)00480-0