Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings.
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.
Kuo
Soong Yu
SY
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.
Chien
Lee Feng
LF
Hsiao
Yi Yuong
YY
Van Ru
Chuan
C
Yan
Kun Huang
KH
Liu
Pei Feng
PF
Mao
Simon J
SJ
Pan
Rong Long
RL
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*
D002413:Cations, Divalent; D002414:Cations, Monovalent; D004164:Diphosphonates; C016514:imidodiphosphonic acid; D043564:Inorganic Pyrophosphatase