Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings

• Published in: Journal of plant physiology Vol. 162; no. 2; pp. 129 - 138
• Main Authors: Kuo, Soong Yu, Chien, Lee Feng, Hsiao, Yi Yuong, Van, Ru Chuan, Yan, Kun Huang, Liu, Pei Feng, Mao, Simon J., Pan, Rong Long
• Format: Journal Article
• Language: English
• Published: Jena Elsevier GmbH 01.02.2005; Elsevier; Elsevier Science Ltd
• Subjects: Biological and medical sciences; Cations, Divalent - pharmacology; Cations, Monovalent - pharmacology; Cell physiology; Diphosphonates - pharmacology; Endoplasmic reticulum; Endoplasmic Reticulum - enzymology; enzyme activity; enzyme inhibition; enzyme inhibitors; etiolation; Fabaceae - enzymology; Fundamental and applied biological sciences. Psychology; H +-pyrophosphatase; Hydrogen-Ion Concentration; Hypocotyl - enzymology; hypocotyls; inorganic pyrophosphatase; Inorganic Pyrophosphatase - antagonists & inhibitors; Inorganic Pyrophosphatase - metabolism; mung beans; Plant physiology and development; plant proteins; Plasma membrane and permeation; proton pump; Proton translocation; seedlings; Tonoplast; Vacuole; Vigna radiata; Tonoplast; Ppase; Proton translocation; EDTA; Vacuole; ER; EGTA; H +-pyrophosphatase; FITC; TNM; CCR; NEM; PGO; Endoplasmic reticulum; DCCD; H+-pyrophosphatase; Enzyme; Vigna radiata; Inorganic pyrophosphatase; Hypocotyl; Leguminosae; Dicotyledones; Angiospermae; Hydrolases; Spermatophyta; Pyrophosphatase
• ISSN: 0176-1617; 1618-1328
• DOI: 10.1016/j.jplph.2004.07.007

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 (PP i) 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 Mg 2+/PP i (inorganic pyrophosphate) for enzymatic activity and PP i-dependent proton translocation of ER-enriched vesicles were higher than those of vacuolar membranes. The PP i-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 PP i 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.