Density Gradient Localization of Plasma Membrane and Tonoplast from Storage Tissue of Growing and Dormant Red Beet

ABSTRACITThe effects of NO3-and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H+-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KN03 when assayed at 24°C or above and was tentatively identified as the tonoplast H+-ATPase. A smaller peak of H+-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KN03 and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H'-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO3 inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO3. The time course for fluorescence quench indicated that addition of ATP in the presence of KN03 caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KN03 was explained by the temperature-dependent delay of the inhibition by KNO3. The plasma membrane H'-ATPase maintained a pH gradient in the presence of KN03 for up to 30 minutes at 24°C.The characteristic that is used most frequently to identify the tonoplast ATPase is that the enzyme is inhibited by NO3-but not by vanadate or azide (3, 5, 9, 19-21, 24, 26, 31). Other inhibitors ofthe tonoplast ATPase, such as DCCD, are less useful, because they affect all of the proton-translocating ATPases (31).However, the effects of NO3-on the tonoplast H+-ATPase are complex (4, 18) and NO3-inhibits other ATPases as well. For example, a putative Golgi membrane H+-ATPase was partially inhibited by N03 (7) and the mitochondrial F,F0-ATPase was inhibited by NO3- (27,31). It is important to clarify the effects of nitrate on the tonoplast ATPase for several reasons. The criteria for using NO3-to identify the tonoplast ATPase need to be defined, particularly since there is some confusion in the literature between the effects of NO3-as an inhibitor of the ATPase and as a substrate for a NO3-/H' symport (4). The effect of NO3 on the tonoplast ATPase in vivo also needs to be elucidated. The physiological significance of the N03 -inhibition is unclear and puzzling, because the vacuole stores nitrate (23,29). Nitrate inhibits the mitochondrial F,F0-ATPase and the tonoplast H+-ATPase with a Ki of only 5 to 10 mm (27,31) although it was estimated that the cytoplasmic concentration of nitrate was approximately 4 mM and the vacuolar concentration was greater than 40 mm for barley leaf protoplasts (23).Barley roots have been used extensively for studies of ion transport and nitrogen metabolism, but until recently it has been difficult to obtain membrane preparations from barley that are suitable for studies of ion...

Section: Resultsmentioning

confidence: 73%

“…2A). The activity of the peak at 1.11 g-cm-3 was almost entirely inhibited by KNO3, and this is a density commonly reported for tonoplast vesicles (3,19,26,31). For ease of discussion, this will be referred to as the tonoplast peak.…”

Section: Resultsmentioning

confidence: 99%

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

DuPont

1987

“…Vanadate-sensitive ATPase activity, representative of the plasma membrane ATPase (and possibly nonspecific phosphatase) (10,19) and Triton X-100 stimulated UDPase, representative of Golgi membranes were first enriched to the KI-extracted microsomal pellet and then substantially decreased when the membranes were centrifuged on a 6% dextran cushion. Comparisons between the properties of ATPase activity associated with isolated vacuoles (14 and references therein, 15) and the properties of ATPase andH+-transport activity in sealed vesicle fractions isolated from microsomes (2,3,17,20,22) Values in parentheses represent the percent of the control activity. b Assayed in the presence of 5 mM ATP (bis-tris-propane salt, pH 7.2), 5 mM MgSO4, 50 mM KCI, 2 Mm Quinacrine, 250 mm sorbitol, and 25 mM bistris-propane/Mes (pH 7.2).…”

Section: Resultsmentioning

confidence: 99%

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot

Dp¹,

Wr²,

Re³

1985

Self Cite

Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta valgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H'-transport, and this is consistent with the observation that Hf-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N'-dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I-> Br-> Cl1 while P was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl1, alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H'/ alkali cation exchange. Based upon the properties of the HW-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary.Energy dependent, primary proton transport appears to be a ubiquitous property of higher plant cells (21 and references therein). The energy conserved in the electrochemical potential gradient of protons, the proton motive force, can then provide the driving force for the transport of other solutes such as sugars according to the chemiosmotic hypothesis (18). The latter process has been termed 'secondary' transport since the coupling to metabolic energy is indirect via an ion gradient (11 and references therein). Studies mary proton transport can occur both at the plasma membrane (cytoplasm to cell exterior) and at the tonoplast (cytoplasm to vacuole lumen) (27 and references therein). When membrane fractions enriched with plasma membrane (16) or tonoplast (15) are prepared, they are found to contain ATP hydrolytic activity which has been postulated to reflect the presence of ATP-fueled proton pumps responsible for carrying out these primary transport events (16,21,27).A significant advance to the study of membrane transport in higher plants came with the development of the methodology to isolate sealed membrane vesicles by Sze (25). The sealed vesicle system allowed the demonstration that these membrane-associated ATPases isolated from higher plant cells could directly transport protons (27). Several laboratories have since characterized ATPase-mediated proton transport in sealed vesicles thought to be d...

“…ATPase activity was measured in a 0. rated from the bulk of membrane protein as well as from vanadate-sensitive ATPase (marker for plasma membrane), Cyt c oxidase (mitochondria), latent UDPase (Golgi), and the bulk of the antimycin-insensitive NADH-Cyt c reductase (ER). A small fraction of the latter enzyme is thought to belong to the tonoplast (12). In the present work, a 0 to 16% sucrose step was used to isolate tonoplast vesicles, which eliminated essentially every trace of contaminating markers.…”

Section: Methodsmentioning

confidence: 99%

Salt Tolerance in Suspension Cultures of Sugar Beet

Blumwald

Poole²

1987

Self Cite

157

ABSTRACICell suspension cultures of sugar beet were grown at various salinities (0-200 millimolar NaCI). Their tolerance to Na' was comparable to that of the intact plant. Tonoplast vesicles were prepared by sucrose density gradient centrifugation of microsomal membranes and shown to be highly purified. The vesicles were subjected to a pH jump in the presence of acridine orange and the rate of recovery of fluorescence after addition of Na' was used as a measure of Na'-dependent H' efflux. In the presence of K+ and valinomycin, the Naf/H' antiport showed saturation kinetics.Increasing Na' in the growth medium did not change the apparent K.for Nat, but increased V,,, to about twice the control value, suggesting a specific induction of antiport synthesis by salt.In a previous study (3) we characterized a Na+/H+ antiport in tonoplast vesicles isolated from storage tissue of red beet and sugar beet. Since the vacuolar accumulation of sodium is characteristic of salt-tolerant species (7,8) this tonoplast Na+/H+ antiport may be one of the principal physiological factors conferring salt tolerance on the plant. Further work on the expression and regulation of this transport system is therefore needed. A recent report ( 14) shows that the marked increase in capacity for Na+ accumulation which develops during washing of sliced storage tissue of red beet (11) is accompanied by a progressive decrease in apparent Km of the tonoplast Na+/H+ antiport.In the present work, we use sugar beet cell suspensions to investigate the regulation of antiport activity in response to extracellular Na+, and present evidence for a specific induction of antiport activity by salt. Bartlesville, OK). The homogenization medium consisted of 5% PVP, 0.5% BSA, 1 mm PMSF,3 30 mM Tris, 5 mM DDT, 5 mM EGTA, 5 mM MgSO4, 0.5 mm butylated hydroxytoluene, 0.5 mM dibucaine, and 0.25 M mannitol, adjusted to pH 8.0 with H2SO4. The isolation was performed at 4°C throughout. The homogenate was filtered through four layers of cheesecloth and centrifuged for 20 min at 8,000g to remove debris and mitochondria. Pellets were discarded and the supernatants were centrifuged for 35 min at 80,000g in a Beckman type 35 rbtor. The supernatant was aspirated and the microsomal pellet was resuspended with a Teflon pestle homogenizer in a medium containing 1.1 M glycerol, 1 mM Tris-EDTA, 0.5 mM dibucaine, 0.5 mM butylated hydroxytoluene, 3 mM DDT, 10 mM Tris-Mes (pH 7.5), and 0.15 M KI. The membranes were again sedimented at 80,000g for 35 min, resuspended in suspension medium without KI, and layered on sucrose gradients. For linear sucrose gradients, the KI-treated membranes were resuspended in 4 ml suspension medium and layered on a 33 ml linear gradient of 10 to 45% (w/w) sucrose in suspension medium. After centrifugation at 80,000g for 2 h in a Beckman SW 27.1 rotor, the gradient was divided into 22 to 24 fractions for subsequent assay. MATERIALS AND METHODSFor routine preparation of tonoplast vesicles, the KI-treated microsomal pellet was resuspended in 20 ml suspension ...