The effects of 5-(
N
-methyl-
N
-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na
+
/H
+
antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (
...Beta vulgaris
L.) cell suspension cultures. MIA inhibited Na
+
/H
+
exchange in a competitive manner with a
K
i
of 2.5 and 5.9 micromolar for ΔpH-dependent
22
Na
+
influx in tonoplast vesicles and Na
+
-dependent H
+
efflux in intact vacuoles, respectively. Scatchard analysis of the binding of
3
HMIA to tonoplast membranes revealed a high affinity binding component with a
K
d
of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na
+
/H
+
antiport. Photolabeling of the tonoplast with
3
HMIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.
The mechanisms of salt adaptation were studied in the cyanobacterium
Synechococcus
6311. Intracellular volumes and ion concentrations were measured before and after abrupt increases of external NaCl ...concentrations up to 0.6 molar NaCl. Equilibrium volumes, measured with a rapid and accurate electron spin resonance spin probe method, showed that at low NaCl concentrations the cells did not shrink as expected for an impermeable solute. However, when the NaCl concentration exceeded a critical value, volume losses occurred. These losses were not fully reversed by hypoosmotic treatment, suggesting membrane damage. The critical value of irreversible volume loss paralleled the increase in salinity during cell growth. Rapid mixing experiments showed that exposure of
Synechococcus
6311 to non-damaging NaCl concentrations caused water extrusion from the cells; the volume decreases were time resolved to about 200 milliseconds. Subsequently, volumes increased rapidly as NaCl moved into the cells. Controls recovered their volumes within 15 seconds, while salt-adapted cells grown at 0.6 molar NaCl required 1 minute for volume equilibration. This decrease in the rate of cell volume recovery indicates that salt adaptation is accompanied by changes in cell membrane properties. Subsequent to these initial rapid volume changes, a more gradual sequence of ion movement and sugar accumulation was observed. Under conditions for photoautotrophic growth, significant Na
+
extrusion was observed 30 min after salt shock. Sucrose accumulation reached a maximum value after 16 hours and K
+
accumulation reached equilibrium after 40 hours. The final concentrations of K
+
and Na
+
and sucrose and glucose inside the 0.6 molar NaCl-grown cells indicate that the inorganic ions and organic `compatible' solutes are the major osmotic species which account for the adaptation of
Synechococcus
6311 to salt.
The H+-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl- and NO3
-. N-Ethylmaleimide (NEM) was employed as a ...probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the Km of the enzyme for ATP but caused relatively little alteration of the Vmax. Nitrate increased Km only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl-. Other monovalent anions that specifically increased the Km of the ATPase for ATP were, in order of increasing Ki, $\text{SCN}^{-}$, $\text{ClO}_{4}{}^{-}$, and $\text{ClO}_{3}{}^{-}$. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO3
-, was a competitive inhibitor of the ATPase but ADP and NO3
- did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO3
- does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO3
- exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl- nor NO3
- are involved in substrate binding.
The H
+
-ATPase of tonoplast vesicles isolated from red beet (
Beta vulgaris
L.) storage tissue was studied with respect to the kinetic effects of Cl
−
and NO
3
−
.
N
-Ethylmaleimide (NEM) was ...employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the
K
m
of the enzyme for ATP but caused relatively little alteration of the
V
max
. Nitrate increased
K
m
only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl
−
. Other monovalent anions that specifically increased the
K
m
of the ATPase for ATP were, in order of increasing
K
i
, SCN
−
, ClO
4
−
, and ClO
3
−
. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO
3
−
, was a competitive inhibitor of the ATPase but ADP and NO
3
−
did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO
3
−
does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO
3
−
exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl
−
nor NO
3
−
are involved in substrate binding.
An improved method is described for the isolation of membrane envelope enclosing the bacteroids (peribacteroid membrane) from soybean (
Glycine max
L.) root nodules. The ATPase activity of the ...peribacteroid membrane from infected roots is compared with that of the plasma membrane from uninfected roots. The two ATPases are similar in terms of their vanadate sensitivities, pH optima, and mineral cation requirements, and show antigenic cross-reactivity. However, the ATPase of peribacteroid membrane is more sensitive to stimulation by NH
4
+
. ATP-dependent proton translocation across the peribacteroid membrane was demonstrated in broken protoplasts of infected cells, by the use of fluorescence microscopy with acridine orange. It is suggested that acidification of the peribacteroid space by the peribacteroid membrane ATPase results in the conversion of NH
3
to NH
4
+
in this space and thereby facilitates the removal of fixed-nitrogen from the bacteroid.
Na+/H+ antiporters are involved in the transport of sodium and hydrogen ions across membranes and contribute in pH regulation of actively metabolizing cells. They play a primary role in homeo-stasis ...and are found in every biological kingdom, from bacteria to humans to higher plants. In plants, vacuolar Na+/H+ antiporters use the proton electrochemical gradient generated by the vacuolar H+-translocating enzymes, H+-ATPase, and H+-PPiase to couple the downhill movement of H+ with the uphill movement of Na+. Moreover, it has been shown that they compartmentalize Na+ into the vacuoles for detoxification and improve consequently the salt tolerance in yeasts and plants. Recently, genes encoding these Na+/H+ antiporters have been identified and studied using a molecular genetic approach in the model systemsArabidopsis or Saccharomyces cerevisiae. We describe here the identification, cloning, molecular characterization and functional properties in yeast heterologous system of a vacuolar Na+/H+antiporter from grapevine. To identify a Na+/H+ antiporter from grapevine we applied a candidate gene approach. A 1.83-kb genomic sequence adjacent to the VvNHX1 gene was isolated using the thermal asymmetric interlaced-PCR. Histochemical localization of β-glucuronidase gene (GUS) activity was directed by VvNHX1 promoter-GUS fusion in transgenic Arabidopsis. To determine the subcellular localization of the VvNHX1 protein by heterologous expression in yeast and transient expression in onion epidermal cells, chimera constructions were prepared using a modified green fluorescent protein mGFP6. An RT-PCR approach was used to examine the VvNHX1 mRNA levels in different organs and tissues of grapevine plants. To assess VvNHX1 transport properties, VvNHX1 was expressed in the nhx1 mutant TY001 (that lacks the endogenous Nhx1 Na+/H+ antiporter) and the rates of H+-coupled transport was measured by fluorescence quenching. Rates of cation-dependent proton movements in vacuoles isolated from yeast expressing VvNHX1 were measured.
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