Purification and subunit determination of H + -pyrophosphatase from endoplasmic reticulum-enriched vesicles of mung bean seedlings Soong Yu Kuo a,b , Lee Feng Chien c , Ru Chun Van a , Kun Huang Yan a , Pei Feng Liu a , Wen Chi Chang a , Jung Kai Wang a , Rong Long Pan a, * a Department of Life Sciences, Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC b Basic Medical Science Education Center, Fooyin University, Kaohsiung 83101, Taiwan, ROC c Department of Life Sciences, National Chung Hsing University, Tai Chung 40227, Taiwan, ROC Received 11 October 2004; received in revised form 27 May 2005; accepted 1 June 2005 Available online 22 June 2005 Abstract Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.) were isolated by Ficoll gradient and two-polymer phase partition. These ER-enriched vesicles contain a new type of H + -pyrophosphatase (H + -PPase) distinct from that of tonoplasts in higher plants. H + -PPase was then solubilized differentially by deoxycholic acid and lyso-phosphatidylcholine. The solubilized fraction was then subjected to Sephacryl S-200 gel filtration and Mono-Q anion exchange chromatography. The final purified protein complex of ER H + -PPase (ER-PPase) was successfully obtained to high homogeneity. An approximate molecular mass of 170 kDa was determined for the purified ER-PPase by size-exclusion gel filtration chromatography. However, only a single polypeptide of 74 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, radiation inactivation analysis of ER-enriched vesicles and purified ER-PPase yielded functional masses of 178.6  9.2 and 143.4  4.7 kDa for inorganic pyrophosphate hydrolysis activity, respectively, indicating that ER-PPase was functionally homodimeric. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Endoplasmic reticulum; H + -pyrophosphatase; Mung bean; Functional mass; Radiation inactivation 1. Introduction Proton pumping enzymes are essential for generating transmembrane H + -electrochemical gradients to provide the energy for diverse physiological functions including the cytoplasmic homeostasis, osmoregulation, protein sorting, and transport of metabolites. Membrane-associated H + - pyrophosphatases (H + -PPase; EC 3.6.1.1) are unique proton pumping enzymes utilizing a simple and low-cost substrate inorganic pyrophosphate (PP i ) instead of ATP to generate an H + -electrochemical gradient for secondary transport of solutes [1–5].H + -PPases were originally isolated from the purple bacterium Rhodospirillum rubum in 1960s [6], and later found abundant among in most plants, algae, protozoa, and bacteria [7–9]. In plants, H + -PPases were discovered widely distributed in various organelles; therefore, many laboratories have long been interested in the question of whether plant cells contained different types of H + -PPases [10,11]. It has been reported that chloroplasts possessed an H + -PPase requiring Mg 2+ as an essential cofactor to display PP i -dependent phosphorylation [12,13]. Afterwards a thylakoid PPase of 55 kDa was successfully purified from spinach but failed to pump protons [14].H + -PPases were also known to be www.elsevier.com/locate/plantsci Plant Science 169 (2005) 847–853 Abbreviations: BHT, butylhydroxytoluene; BSA, bovine serum albu- min; DCCD, N,N 0 -dicyclohexylcarbodiimide; DOC, deoxycholic acid; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; EGTA, ethy- leneglycol-bis-(aminoethyl ether) N,N,N 0 ,N 0 -tetraacetic acid; ER, endoplas- mic reticulum; LPC, lyso-phosphatidylcholine; NEM, N-ethylmaleimide; PEG, polyethylene glycol; PMSF, phenylmethylsulfonyl fluoride; PPase, pyrophosphatase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis * Corresponding author. Tel.: +886 3 5742688; fax: +886 3 5742688. E-mail address: rlpan@life.nthu.edu.tw (R.L. Pan). 0168-9452/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.06.001