Vacuolar H + -pyrophosphatase purified from pear fruit Yasuo Suzuki a, *, Yoshinori Kanayama b , Katsuhiro Shiratake a , Shohei Yamaki a a Laboratory of Horticultural Science, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan b Laboratory of Horticulture, Faculty of Agriculture, Tohoku University, Aoba, Sendai, 981-8555, Japan Received 12 March 1998; revised 2 July 1998 Abstract A vacuolar H + -translocating inorganic pyrophosphatase was purified from pear fruit through selective detergent treatments, Superose 6 and Mono Q column chromatography. The specific activity of the purified enzyme was 850 mmol h  1 mg protein  1 . The Mr of V-PPase was 66 kDa by SDS-PAGE and the polypeptide cross-reacted with the antiserum against V-PPase of mung bean. The purified V-PPase was stimulated by potassium and inhibited by calcium and N, N 0 -dicyclohexylcarbodiimide. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Pyrus communis; Rosaceae; Pear fruit; Enzyme purification; Vacuolar H + -translocating inorganic pyrophosphatase; Tonoplast 1. Introduction The vacuole is a dynamic organelle involved in sev- eral cellular processes including storage of metabolites and ions, regulation of cytosolic homeostasis, degra- dation and recycling of cellular components, and space filling (Maeshima, Nakanishi, Matsura-Endo & Tanaka, 1996; Taiz, 1992). Many of these processes are directly or indirectly related to either the trans- membrane electrochemical gradient across the vacuolar membrane or acidic pH in the vacuole. These gradients are generated by two proton pumps in the vacuolar membrane; the vacuolar H + -ATPase (V-ATPase) and the vacuolar H + -pyrophosphatase (V-PPase). V- ATPase from the tonoplast of higher plants has been well characterized (Sze, Ward & Lai, 1992) and puri- fied from the flesh tissue of pear fruit (Hosaka, Kanayama, Shiratake & Yamaki, 1994). V-PPase, which is a universal enzyme among green plants (Maeshima, Miura & Sato, 1994), has also been iso- lated from various tissues of higher plants (Sarafian & Poole, 1989; Britten, Turner & Rea, 1989; Maeshima & Yoshida, 1989; Sato, Kasahara, Ishii, Homareda, Matsui et al., 1994; Perotti, Gavin, Widmer & Chanson, 1994; Becker, Canut, Luttge, Maeshima, Marigo & Ratajczak, 1995). However, V-PPase has not been isolated from the flesh tissue of fruit where it may play important roles in development. For approximately forty days after flowering, pear fruit development depends mainly on cell division, after which the fruit size enlarges only through cell expansion accompanied by the accumulation of sugars (Bain, 1961; Yamaki & Matsuda, 1977). V-PPase is an important enzyme for pear fruit development because it has high activity during cell division, a stage in which the relative growth rate is also high, and has essential activity in cell expansion stage (Shiratake, Kanayama, Maeshima & Yamaki, 1997). In this study, we report the purification and characterization of V- PPase from pear fruit as a first step in understanding the role that this enzyme plays in fruit development. 2. Results and discussion 2.1. Solubilization of V-PPase by lysophosphatidylcholine The following procedure was eective in solubilizing significant amount of the V-PPase from the tonoplast of pear fruit. First, peripheral proteins were removed from the tonoplast preparation by solubilization with 1% Triton X-100. Non-solubilized proteins were col- lected through ultracentrifugation, and V-PPase was solubilized from the remaining proteins with 0.5% Phytochemistry 50 (1999) 535–539 0031-9422/98 $ - see front matter # 1998 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(98)00554-8 PERGAMON * Corresponding author.