title: Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings.
abstract: Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase (V-PPase, EC 3.6.1.1) did not inhibit this novel pyrophosphatase-dependent proton translocation, excluding the possible contamination of tonoplast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPi-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PPi-dependent proton translocation of ER-enriched vesicles absolutely required the presence of monovalent cations with preference for K+, but could be inhibited by a common PPase inhibitor, F-. Furthermore, ER H+-pyrophosphatase exhibited some similarities and differences to vacuolar H+-PPases in cofactor/substrate ratios, pH profile, and concentration dependence of F-, imidodiphosphate (a PPi analogue), and various chemical modifiers. These results suggest that ER-enriched vesicles contain a novel type of proton-translocating PPase distinct from that of tonoplast from higher plants.
authors.affiliation: Department of Life Sciences, Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University, Hsin Chu 30043, Taiwan, Republic of China.
authors.forename: Soong Yu
authors.initials: SY
authors.lastname: Kuo
authors.forename: Lee Feng
authors.initials: LF
authors.lastname: Chien
authors.forename: Yi Yuong
authors.initials: YY
authors.lastname: Hsiao
authors.forename: Chuan
authors.initials: C
authors.lastname: Van Ru
authors.forename: Kun Huang
authors.initials: KH
authors.lastname: Yan
authors.forename: Pei Feng
authors.initials: PF
authors.lastname: Liu
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Mao
authors.forename: Rong Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D004164:Diphosphonates / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000201:enzymology; D043564:Inorganic Pyrophosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D002414:Cations, Monovalent; D004164:Diphosphonates; C016514:imidodiphosphonic acid; D043564:Inorganic Pyrophosphatase