Shu-I Tu

Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase

A tonoplast-type vesicle preparation, substantially free from other subcellular membranes, was obtained from corn roots by equilibrium sucrose density gradient centrifugation. At pH 6.5 and in the presence of chloride ions, the tonoplast-type ATPase activity as measured by Pi release, was inhibited by nitrate ions. The ATPase activity was insensitive to molybdate and vanadate, indicating a minimum nonspecific phosphatase and plasma membrane contamination. The vesicles exhibited an ATP hydrolysis-supported proton uptake which was measured by the absorption change of acridine orange. The ATP hydrolysis supported uptake and the subsequent perturbant-induced release of protons (decay) was described by a kinetic model which was previously developed to evaluate the coupling between proton pumping and the primary energy yielding process for other biomembranes. The proton pumping activity was more sensitive to nitrate ions then was ATP hydrolysis. The differential effect and the kinetic analysis of nitrate inhibition led us to suggest that (i) the coupling between Pi release and proton pumping was indirect in nature and (ii) the primary inhibitory effect of nitrate ion was originated from an interaction with a protogenic protein domain which is functionally linked to the ATPase in the tonoplast-type membrane.

Effects of Bafilomycin A1 and Metabolic Inhibitors on the Maintenance of Vacuolar Acidity in Maize Root Hair Cells.

Proton pumps of tonoplast membranes have been studied extensively in vitro, but data concerning their regulation in vivo are lacking. Effects of either anoxia, or the addition of KCN, 2-deoxy-d-glucose (deoxy-glucose), or bafilomycin-A1 (BAF) on vacuolar pH of maize (Zea mays L.) root hair cells were followed by fluorescence microscopy after loading of 2[prime]7[prime]-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein. Root hair cells were able to maintain vacuolar acidity for at least 2 h in the presence of either 10 mM KCN or 50 mM deoxy-glucose or during anoxia. Treatments with either deoxy-glucose or KCN reduced total tissue ATP more than anoxia. ADP accumulated during anoxia and treatment with KCN as detected by in vivo 31P-NMR spectroscopy, but not during deoxy-glucose treatment. With control roots and roots treated with deoxy-glucose, the presence of BAF, a specific inhibitor of the V-type ATPase, caused alkalization of the vacuolar pH. However, either in the presence of KCN or under anoxic conditions, BAF was relatively ineffective in dissipating vacuolar acidity. Therefore, under anoxia or in the presence of KCN, unlike the situation with air or deoxy-glucose, the V-type ATPase apparently is not required for maintenance of vacuolar acidity.

Selective accumulation of the fluorescent pH indicator, BCECF, in vacuoles of maize root-hair cells

Summary The vacuolar-type H+ -ATPase localized on the tonoplast membrane is believed to regulate aciditywithin the vacuolar lumen. However, the ability to monitor vacuolar pH in situ can be difficult because of the use of destructive assays based on cell sap pH or accumulation of weak bases, or elicit ion of wound responses after impaling cells with a pH-sensing microelectrode. The goal of our present study was to identify a fluorescent pH probe that is localized in vacuoles of (Zea mays L.) root cells. When excised corn root segments were exposed to the ester derivative of 2′,7′ -bis-2-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) for 30 to 60 min, the root-hair cells preferentially became fluorescent. The distribution of the fluorescence within cells followed closest to that of the vacuole when fluorescent and phase contrast images were compared. The ratio of the fluorescence intensities at 535 nm using excitation wavelengths of 440 and 490 nm varied directly with pH between 4.5 and 7.5. The ratio of fluorescent intensities of BCECF-Iabeled root-hair cells indicated that the majority of the dye was localized in a compartment with an average pH of 5.8. The above data indicated that the BCECF was localized in the vacuoles of root-hair cells from maize seedlings, and the changes in the fluorescence ratio of BCECF may provide a means of monitoring changes of vacuolar pH in situ.

In vivo 133Cs-NMR a probe for studying subcellular compartmentation and ion uptake in maize root tissue.

Three 133Cs-NMR signals were observed in the spectra of CsCl-perfused and CsCl-grown maize seedling root tips. Two relatively broad lower field resonances were assigned to the subcellular, compartmented Cs+ in the cytoplasm and vacuole, respectively. The rate of area increase of the broader cytoplasmic Cs resonance was about 9-times faster than that of the vacuolar signal during the first 300 min of tissue perfusion with CsCl. In addition, the spin lattice relaxation time of the cytoplasmic Cs resonance was approx. 3-times shorter than that of the extracellular resonance, while the Cs+ signal associated with the metabolically less active vacuolar compartment exhibited a relaxation time comparable to that of the extracellular signal. 133Cs spectra of excised, maize root tips and excised top sections of the root adjacent to the kernel, each grown in 10 mM CsCl showed a difference in the relative areas of the Cs resonance corresponding to the distinct cytoplasm/vacuole volume ratio of these well differentiated sections of the root. The high correlation of counterion concentration with 133Cs chemical shifts suggested that the larger downfield shift exhibited by the cytoplasmic confined Cs+ was due principally to the higher ionic strength and protein content in this compartment. Such observations indicate that 133Cs-NMR might be employed for studying ionic strength, and osmotic pressure associated chemical shifts and the transport properties of Cs+ (perhaps as an analogue for K+) in subcellular compartments of plant tissues.

Temperature dependence and mercury inhibition of tonoplast-type H+-ATPase

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H+-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 degrees C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was approximately 14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 degrees C. Lineweaver-Burke analysis of the activities showed that the Km of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg2+. The proton pumping process was more sensitive to the presence of Hg2+. Double-reciprocal analysis of kinetic data indicated that Hg2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H+ pumping and ATP hydrolysis.

Mechanistic investigation on the temperature dependence and inhibition of corn root plasma membrane ATPase.

The kinetics of corn root plasma membrane-catalyzed Mg-ATP hydrolysis may be satisfactorily described by a simple Michaelis-Menten scheme. It was found that the Km of the process was relatively insensitive to changes in temperature. This property allowed us to conveniently estimate the activation energy of the enzyme turnover process as approximately 14 kcal mol-1 in the temperature range of 10 to 45 degrees C. The enzyme activity was inhibited by the presence of diethystilbestrol (DES), miconazole, vanadate, and dicyclohexylcarbodiimide (DCCD). The inhibition caused by DES and miconazole was strictly uncompetitive and inhibition by vanadate was noncompetitive. The inhibition by DCCD showed a substrate concentration dependence, i.e., competitive at high and uncompetitive at low concentrations of Mg-ATP. The 1/V vs [I] plots suggested that there were different but unique binding sites for DES, vanadate, and miconazole. However, the modification of the plasma membrane by DCCD exhibited interaction with multiple sites. Unlike yeast plasma membrane ATPase, the enzyme of corn root cells was not affected by the treatment with N-ethylmaleimide. Although the enzyme activity was regulated by ADP, a product of the reaction, the presence of inorganic phosphate showed no inhibition to the hydrolysis of Mg-ATP.

Regulatory effects of adenosine diphosphate on the activity of the plasma membrane ATPase of corn roots

Plasma membrane enriched microsomal fraction was isolated from corn root cells by sucrose density centrifugation. The ATPase activity as measured by the release rate of inorganic phosphate, was decreased by the presence of modifiers which included diethylstilbestrol, vanadate, N,N-dicyclohexylcarbodiimide, and miconazole. The presence of ADP also decreased the rate of ATP hydrolysis. Furthermore, a preincubation of the membrane with ADP significantly reduced the inhibitory effects of these membrane ATPase modifiers. Since the modes of interaction of these modifiers with the enzyme are different, the results suggest that the binding of ADP may stabilize the plasma membrane ATPase in a modifier insensitive state.

The nature of proton‐translocating ATPases in maize roots

Abstract The mechanisms of the coupling between ATP hydrolysis and proton transport catalyzed by the ATPases of the tonoplast and plasma membrane of maize (Lea mays L.) roots were investigated. Proton transport by the tonoplast ATPase was found to be much more sensitive to nitrate than ATP hydrolysis, being inhibited by 80% with almost no effect on hydrolysis at 5 mM NO3. Mercury was also found to be a potent inhibitor of this enzyme, inhibiting transport and hydrolysis by 50% at 60 and 100 μM, respectively. The same type of pattern was seen with other divalent cations. Millimolar concentrations of Cd2+, Co2+, Cu2+, and Zn2+ in the presence of Mg2+ inhibited proton transport significantly more than hydrolysis whereas Ba2+ and Ca2+ had little effect. Both free and ATP‐complexed species of these inhibitory cations appeared to be effective. When the influence of temperature was investigated, both the tonoplast and plasma membrane enzymes showed a similar pattern. ATP hydrolysis by both enzymes generally obey...

Proton movement in reconstituted purple membrane of Halobacteria: Effects of pH and ionic composition of the medium☆

Abstract Bacteriorhodopsin has been incorporated into sonicated phospholipid vesicles to form the functionally active proton pump. The kinetics of the light-induced proton uptake and of the subsequent release of protons in the dark have been analyzed according to a scheme which assumes the existence in bacteriorhodopsin of a light-dependent pathway responsible for an inhibition of the proton-pumping activity. The growth stage of proton movement obeys the empirical equation ln (1 − Δ Δ s ) = −k L t , where Δ and Δ s are the extent of proton uptake at time t of illumination and at the steady state, respectively. The release of protons in the dark stage follows the decay equation ln ( Δ Δ s ) = −k D t , where k D is a light-independent rate constant. With these definitions, the rate constant for the light-dependent proton-pumping inhibition is ( k L − k D ) = k I . The initial proton-pumping rate, R 0 , is obtained either directly from the “on rate” or from the steady-state equation R 0 = k L Δ s . We find that, in vesicles made from egg yolk phosphatidylcholine, changes in pH in the range 5–7 result in changes in both k I and R 0 , but have no significant effect on k D . The two empirical kinetic parameters, k I and R 0 , do not change to the same extent with pH, indicating a pH dependence of the coupling between them. When negatively charged phospholipids, e.g., phosphatidic acid, are incorporated into the phosphatidylcholine vesicles, the inhibition k I and the initial pumping rate, R 0 , are sensitive to pH changes. However, there are differences in the pH effects in the presence and absence of acidic phospholipids, and the coupling between pumping and its inhibition is no longer pH dependent. Thus, the presence in the bilayer of phospholipids with negatively charged head groups seems to stabilize the mechanism of coupling between R 0 and k I . Vesicles made from soybean phospholipids have been employed to study the effect of external ionic composition on the kinetics of the lightinduced proton movement. The K + of the external medium is replaced by other monovalent cations, at constant ionic strength and a fixed composition of the internal medium. Replacement of K + by Na + has no effect on the rate constants k D and k D but causes a gradual increase in the initial proton-pumping rate, R 0 . Replacement of K + by Cs + causes a marked decrease in the inhibition of the proton pumping measured by k I , without any concomitant effect on the other kinetic parameters. When most of the K + has been replaced by Cs + , pronounced effects are observed in all the parameters. Replacement of K + by Li + has no significant effect on any of the kinetic parameters, except when the K + Li + ratio is very low, in which case pronounced effects are again observed in all the kinetic parameters. These results and previous work from this laboratory, including the modification of the light-dependent proton-pumping inhibition by the charge of the bilayer, support a picture in which there is an indirect link between the primary proton-pumping activity and its associated inhibitory process in bacteriorhodopsin.

Purification and immunological properties of vanadate sensitive Mg:ATPase from plasma membrane of maize roots

Abstract The vanadate-sensitive ATPase from maize ( Zea mays L.) root plasma membrane was partially purified from KI-washed microsomes by discontinuous sucrose gradient centrifugation followed by deoxycholate (DOC) treatment, l-l -lysophosphatidyl-choline (lyso-PC) solubilization and glycerol gradient centrifugation. The plasma membrane preparation had a relatively high specific activity of ATPase activity (182 μmol P i /h (mg proteins). During lyso-PC solubilization, the presence of Mg:ATP and vanadate substantially improved the recovery of active ATPase. The 100-kDa polypeptide of the plasma membrane ATPase was isolated by sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) and used to raise a specific polyclonal antiservm. The addition of antiserum to the lyso-PC solubilized plasma membrane resulted in a loss of 90% of the total ATPase activity from the supernatant fraction after centrifugation. The antiserum also inhibited the proton transport activity of reconstituted plasma membrane vesicles. Western blot experiments showed that a single band of 100 kDa reacted with the anti-ATPase antibodies. Enzyme-linked immunosorbant assay (ELISA) analysis indicated that the antiserum exhibited specificity to plasma membrane as compared to other subcellular membranes. Thus, the antiserum obtained in this study is specific for the 100-kDa polypeptide of the plasma membrane ATPase.