Browsing by Author "Van, RC"

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Differential response of vacuolar proton pumps to osmotica
(2006)
Chiu, FS
;
Hsu, SH
;
Chen, JH
;
Hsiao, YY
;
Pan, YJ
The vacuole is a fundamental and dominant organelle and occupies a large part of the total cell volume in most mature plant cells. The higher-plant vacuole contains two types of proton-translocating pumps, H+-ATPase (EC 3.6.1.3) and H+-pyrophosphatase (EC 3.6.1.1), residing on the same membrane. These two enzymes generate roughly equal proton gradients across the vacuolar membrane for the secondary transport of ions and metabolites. However, the pumps respond differentially to stress in order to maintain critical functions of the vacuole. In this work, tonoplasts from etiolated mung bean seedlings ( Vigna radiata L.) were used to investigate the function of these two enzymes under high osmotic pressure. At high concentrations of sucrose or sorbitol, the light scattering and volume of isolated vesicles were progressively changed. Concomitantly, enzymatic activities, proton translocation, and coupling efficiencies of these two proton-pumping enzymes were inhibited to various extents under high osmotic pressure. No significant change in enzymatic activities of purified vacuolar H+-PPase and H+-ATPase under similar conditions was observed. We thus believe that the membrane structure is an important determinant for proper function of proton pumping systems of plant vacuoles. Furthermore, kinetic analysis shows different variation in apparent V-max but not in K-M values of vacuolar H+-PPase and H+-ATPase at high osmolarity of sucrose and sorbitol, respectively, suggesting probable alterations in substrate hydrolysis reactions but not substrate-binding affinity of the enzymes. A working model is proposed to interpret supplemental roles of vacuolar H+-PPase and H+-ATPase to maintain appropriate functions of plant tonoplasts.
Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings
(2005-02-01)
Kuo, SY
;
Chien, LF
;
Hsiao, YY
;
Van, RC
;
Yan, KH
Endoplasmic reticulurn (ER)-enriched vesicles from etiolated hypocotyts 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 tonoptast vesicles in the ER-enriched membrane preparation. The optimal ratios of Mg2+/PPi (inorganic pyrophosphate) for enzymatic activity and PPj-dependent proton translocation of ER-enriched vesicles were, higher than those - of vacuolar membranes. The PPj-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 PPj anatogue), and various chemical modifiers.