Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

Abstract

ABSTRACrThe substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by C-and inhibited by N03-. NaCI and KCI support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K2S04 supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by KV. KCI and K2SO4 support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.The primary active transport process across the plasma and vacuolar membranes of plant cells is considered to be the ATPdependent electrogenic translocation of protons (19,24). By extension of the chemiosmotic hypothesis (17), the proton electrochemical potential difference generated by proton translocation is thought to provide the requisite motive force for the secondary transport of a wide range of chemical species.While the primacy of ATP for the energization of transport across the membranes of plant cells is not in dispute, the involvement of other phosphoesters, PPi in particular, has yet to be thoroughly evaluated. Walker and Leigh (27) (22). The homogenization medium consisted of 53% (v/v) sterilized coconut milk or 10 mm glycerophosphate, 0.65 M ethanolamine (adjusted to pH 8.0 with concentrated H2SO4), 0.28 M choline chloride, 26 mm K-metabisulfite, 2 mM salicylhydroxamic acid, 0.2% (w/v) BSA (fraction, V, essentially fatty acid-free), 10% (w/v) insoluble PVP, 5 mM DTT, 0.5 mM butylated hydroxytoluene, and 1 mM nupercaine buffered to pH 8.0 with 70 mM Tris-Mes. The suspension medium consisted of 1.1 M glycerol, 1 mm Tris-EDTA, 1 mM nupercaine, 0.5 mM butylated hydroxytoluene, and 5 mM DTT buffered to pH 8.0 with 5 mM Tris-Mes. Choline and ethanolamine were included in the homogenization medium to minimize membrane degradation by phospholipase D while nupercaine and coconut milk, glycerophosphate, or glycerol were added to inhibit phospholipase A and phosphatidic acid phosphatase activity, respectively (22).The KI solution, employed to diminish contamination of the tonoplast vesicles with acid phosphatase (20), and the sucrose density gradient solutions were prepared in suspension medium.Sucrose Density Centrifugation. For the routine preparation of tonoplast vesicles, the 0.25 M KI-treated pellet from 300 to 350 g beet was resuspended in 12 ml suspension medium and 3 ml volumes were layered onto each of four step grad...

Common Identity of Substrate Binding Subunit of Vacuolar H+-Translocating Inorganic Pyrophosphatase of Higher Plant Cells

“…PPi-and ATP-Dependent HW-Translocation PPi-and ATP-dependent intravesicular acidification were assayed fluorimetrically at 250C with the monoamine dye acridine orange at excitation and emission wavelengths of 495 and 540 nm, respectively, and a slitwidth of 5 nm for both excitation and emission (19,22 …”

“…A striking feature of the vacuo-lysosomal compartment of higher plant cells is the presence of two primary H+ pumps on the bounding (vacuolar) membrane: an H+-ATPase (EC 3.6.1.3) (13) and a PPi-energized H+-PPase2 (EC 3.6.1.1) (5,19). Both enzymes catalyze inward electrogenic H+-translocation (from cytosol to vacuole lumen) to establish an insideacid, inside-positive ATH+ for the energization of secondary H+-coupled solute transport (26), but the H+-PPase has the unusual characteristic of exclusively utilizing PPi as energy source (20,21).…”

Pharmacology of Ca2+ release from red beet microsomes suggests the presence of ryanodine receptor homologs in higher plants

“…Microsomes were isolated from red beet (Beta vulgaris), based on the protocol given in [12]. Storage root (330 g) of fresh, greenhouse Changes in cytosolic free Ca 2+ concentration are now recgrown red beet was homogenised at pH 8.0 in a Kenwood blender for ognised as being crucial in the transduction of a wide variety approx.…”

“…Storage root (330 g) of fresh, greenhouse Changes in cytosolic free Ca 2+ concentration are now recgrown red beet was homogenised at pH 8.0 in a Kenwood blender for ognised as being crucial in the transduction of a wide variety approx. 30 s. 1 ml of homogenisation medium [12] was used per g of stimuli in plant cells [1]. It has been established that inositol tissue.…”

“…The supernatant was re-centriCa 2+ concentration in plant cells [2]. Furthermore, we have fuged at 80000 ×g for 30 min to give a crude microsomal pellet which was resuspended in a suspension medium at pH 8.0, as detailed in shown previously that red beet microsomes and whole va- [12]. A further centrifugation step followed at 80000xg for 30 min.…”