Selectivity of interaction of univalent cations with mammalian ribosomes studied by equilibrium dialysis in the presence of the K+ analogue, 204Tl+

The interaction of K⁺ with mammalian ribosomes was studied by equilibrium dialysis and compared with that of other univalent cations. The heavy K⁺ analogue, Tl⁺, binds more firmly than K⁺ to ribosomes and, unlike K⁺, has a practically useful isotope. With ²⁰⁴Tl⁺ as a marker of K⁺-selective binding the ribosome-cation interaction could be followed down to levels below 0.1 average Tl⁺-occupied site per ribosome. The Tl⁺/ribosome ratio varied with the free Tl⁺ concentration in a multiple way. At high Tl⁺ saturation Tl⁺ was easily displaced by Mg²⁺. With decreasing Tl⁺ saturation the competitive activity of Mg⁺⁺ was strikingly reduced, indicating that Tl⁺ and Mg⁺⁺ compete with different efficiency for different classes of sites.The experiments on univalent cations were performed at 1.5 mM Mg²⁺ under two complementary conditions: (1) Ribosomes were pretreated with 5 × 10^?2, 5 × 10^?3, and 5 × 10^?4 M LiNO₃, NaNO₃, KNO₃, and CsNO₃, and then equilibrated with different concentrations of ²⁰⁴TlNO₃ in the same buffers. (2) Ribosomes were pretreated with 10^?2, 10^?4, and 10^?6 M ²⁰⁴TlNO₃, and then equilibrated with different concentrations of LiNO₃, NaNO₃, KNO₃, and CsNO₃ (displacement experiments). At high Tl⁺ saturation Na⁺ and Li⁺ were about as active as K⁺ and Cs⁺ in competing with ²⁰⁴Tl⁺. With decreasing Tl⁺ saturation a differentiation occurred in favor of K⁺ and Cs⁺, with some preference for K⁺. It is concluded that ribosomes contain a limited number of sites with pronounced ion specificity. Of physiological cations K⁺ is most firmly bound to these sites.     

Inhibition of human red cell Nak-ATPase by magnesium and potassium.

The inhibition of NaK-ATPase (EC 3.6.1.3) from human red cells by Mg²⁺ is markedly dependent on the relative concentrations of Na⁺ and K⁺. Inhibition increases with increasing K⁺ and decreases with increasing Na⁺. The inhibition appears to be a combined effect of Mg²⁺ and K⁺ at sites distinct from the sites at which these cations activate the enzyme. The kinetics of activation of the enzyme by Na⁺ with inhibitory levels of Mg²⁺ and K⁺ are biphasic, indicating both low and high affinity Na⁺ sites. At noninhibitory levels of Mg²⁺ and K⁺ only high affinity Na⁺ sites are seen. The results are inconsistent with any model in which Mg²⁺ and K⁺ compete with Na⁺ at a single site. A kinetic model is proposed to explain the mechanism of inhibition by Mg²⁺ and K⁺.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Clustered Distribution of Calcium Sensitivities: An Indication of Hetero-tetrameric Gating Components in Ca2+-activated K+ Channels Reconstituted from Avian Nasal Gland Cells

High-conductance, Ca²⁺-activated K⁺ channels from the basolateral membrane of rabbit distal colon epithelial cells were reconstituted into planar phospholipid bilayers to examine the effect of Mg²⁺ on the single-channel properties. Mg²⁺ decreases channel current and conductance in a concentration-dependent manner from both the cytoplasmic and the extracellular side of the channel. In contrast to other K⁺ channels, Mg²⁺ does not cause rectification of current through colonic Ca²⁺-activated K⁺ channels. In addition, cytoplasmic Mg²⁺ decreases the reversal potential of the channel. The Mg²⁺-induced decrease in channel conductance is relieved by high K⁺ concentrations, indicating competitive interaction between K⁺ and Mg²⁺. The monovalent organic cation choline also decreases channel conductance and reversal potential, suggesting that the effect is unspecific. The inhibition of channel current by Mg²⁺ and choline most likely is a result of electrostatic screening of negative charges located superficially in the channel entrance. But in addition to charge, other properties appear to be necessary for channel inhibition, as Na⁺ and Ba²⁺ are no (or only weak) inhibitors. Mg²⁺ and possibly other cations may play a role in the regulation of current through these channels. Received: 25 August 1995/Revised: 16 November 1995     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.     

Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase

1. An adenosine triphosphatase membrane system, dependent on Mg²⁺ and activated further by Na⁺+K⁺, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na⁺ in the presence of K⁺+Mg²⁺, for K⁺ in the presence of Na⁺+Mg²⁺ and for Mg²⁺ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na⁺+K⁺ were added to preparations from 8°-acclimatized fish the affinity for Mg²⁺ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg²⁺-activated adenosine triphosphatase was inhibited by Na⁺ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na⁺-inhibited adenosine triphosphatase was separated from the (Na⁺+K⁺)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg²⁺-activated and more (Na⁺+K⁺)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.     

Role of the choline exchanger in Na-independent Mg efflux from rat erythrocytes

Two types of Na⁺-independent Mg²⁺ efflux exist in erythrocytes: (1) Mg²⁺ efflux in sucrose medium and (2) Mg²⁺ efflux in high Cl⁻ media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na⁺-independent Mg²⁺ efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K⁺,Cl⁻- and Na⁺,K⁺,Cl⁻-symport, Na⁺/H⁺-, Na⁺/Mg²⁺-, Na⁺/Ca²⁺- and K⁺(Na⁺)/H⁺ antiport, Ca²⁺-activated K⁺ channel and Mg²⁺ leak flux. We suggest that, in choline Cl medium, Na⁺-independent Mg²⁺ efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg²⁺ efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg²⁺ to the same degree. The K_d value for inhibition of [¹⁴C]choline efflux and for inhibition of Mg²⁺ efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg²⁺ efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg²⁺ efflux was reduced to the same degree by these inhibitors as was the [¹⁴C]choline efflux.     

Effects of Cation Levels of the Nutrient Medium on the Biochemistry of Chlorella: II. Factorial Experiment

A factorial experiment was designed to study the effects of Mg²⁺, K⁺, and Na⁺ on the growth and biochemistry of Chlorella sorokiniana. Raising Mg²⁺ or K⁺ concentration in the nutrient medium increased growth rates as well as total N levels and Mg²⁺ and K⁺ accumulation by the cells. The total N effect was Mg²⁺-dependent—if Mg²⁺ was below a certain level in the medium—increasing the K⁺ concentration did not raise the total N level of cells. Low nutrient levels of K⁺ decreased the levels of unsaturated fatty acids (especially 18:1 and 18:3), while increasing the levels of palmitic acid (16:0), total fatty acids, and total lipid. Increasing nutrient K⁺ concentrations were accompanied by increases in levels of some unsaturated fatty acids, with a concomitant reduction in 16:0, total fatty acids and total lipid. Low Mg²⁺ levels in the nutrient medium reduced the cellular levels of palmitic acid, total fatty acids, total lipid, and certain unsaturated fatty acids (though this last effect also depended on the nutrient level of K⁺). These relationships indicate that Mg²⁺ may be important in the initial steps of fatty acid synthesis, whereas K⁺ may be necessary for the formation of certain unsaturated fatty acids. Variations in Na⁺ concentration did not have any significant effect on the growth and biochemistry of C. sorokiniana.     

Variable ATPase composition of human tumor plasma membranes

Purified plasma membranes from several transplantable human tumors exhibit very high Mg²⁺-dependent ATPase activities. Three types of Mg²⁺-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na⁺, K⁺-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg²⁺-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na⁺ (or K⁺) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg²⁺-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Ion permeation and rectification in ATP-sensitive channels from insulin-secreting cells (RINm5F): Effects of K+, Na+ and Mg2+

Summary Patch-clamp techniques were used to study the permeability to ions of an ATP-sensitive channel in membranes from the pancreatic B-cell line (RINm5F). With patches in the outside-out configuration, theI-V curves for different Na⁺–K⁺ mixtures in the bath and 140 mM K⁺ in the pipette were almost linear, and crossed the zero-current axis at voltages that indicated a variable permeability ratio. When K⁺ was added symmetrically, the plot of the conductancevs. K⁺ activity exhibited saturation, with aG _max of about 160 pS and a half-maximal activity of 216 mM. TheI-V behavior for different K⁺–Na⁺ mixtures in the bath could be accurately described with a model based on Eyring theory, assuming two sites and one-ion occupancy. For K⁺, the dissociation constants (K_K) of the two sites were 290 and 850 mM, the lower value pertaining to the site close to the intracellular medium. In experiments with inside-out patches, both Na⁺ and Mg²⁺, when present in the bath, induced a voltagedependent block of the outward current. Fitting the data with the model suggested that for these ions only one of the two sites binds significantly, the corresponding dissociation constants being (mM): 46 for Na⁺ and 34 for Mg²⁺. Blocking by Na⁺ and Mg²⁺ may account for the low outward current seen in intact cells. This hypothesis is consistent with the observation that such current is further reduced by addition of 2,4-DNP, since metabolism inhibitors are expected to lower the ATP level, thereby liberating Mg²⁺ from the Mg²⁺-ATP complex, as well as inducing accumulation of Na⁺ by decreasing the rate of the Na⁺–K⁺ pump.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

The isolation and properties of cardiac ribosomes and polysomes

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg²⁺ ions. The degradation in the absence of Mg²⁺ ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2·4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na⁺ and K⁺ ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.     

The role of membrane-bound magnesium in the permeability of ghosts to K+

Mg²⁺ is required for restoring a low cation permeability of erythrocyte membranes after osmotic lysis. These ions promote binding of haemoglobin to the ghost membrane. Because the optimal binding occurs at a pH where the K⁺ retention by ghosts is maximal, the possibility exists that Mg²⁺ exerts an indirect effect by facilitating the adsorption of haemoglobin, which may be essential for the maintenance of a low permeability. In order to investigate this possibility, a systematic study was done of the effects of pH and hypotonic washing on the retention of haemoglobin, Mg²⁺ and K⁺ by human erythrocyte ghosts.Between pH 5.5 and 7.5, the retention of haemoglobin paralleled that of K⁺ and Mg²⁺ on resealing immediately after lysis. Such a parallelism was not observed if ghosts were both washed with fresh haemolytic media and resuspended in a medium of lower osmolarity before reversal.No correspondence was found between ghost K⁺ content and membrane-bound haemoglobin, indicating that the latter is not involved in the maintenance of a low permeability. By contrast, Mg²⁺ binding was altered by pH in the same way as ghost K⁺ and a strict relationship between membrane-bound Mg²⁺ and K⁺ retention was obtained.The results suggest that K⁺ permeability is mainly determined by the amount of Mg²⁺ associated with the ghost membrane.     

Surface Charge-Mediated Effects of Mg on K Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg²⁺ external to the isolated intact chloroplast interacts with stromal K⁺, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed hypothesis that millimolar levels of external, unchelated Mg²⁺ result in lower stromal K⁺, which somehow is linked to stromal acidification. Stromal acidification directly results in photosynthetic inhibition. These effects were attributed to Mg²⁺ interaction (binding) to negative surface charges on the chloroplast envelope. Chloroplast envelope-bound Mg²⁺ was found to decrease the envelope membrane potential (inside negative) of the illuminated chloroplast by 10 millivolts. It was concluded that Mg²⁺ effects on photosynthesis were likely not mediated by this effect on membrane potential. Further experiments indicated that envelope-bound Mg²⁺ caused lower stromal K⁺ by restricting the rate of K⁺ influx; Mg²⁺ did not affect K⁺ efflux from the stroma. Mg²⁺ restriction of K⁺ influx appeared consistent with the typical effects imposed on monovalent cation channels by polyvalent cations that bind to negatively charged sites on a membrane surface near the outer pore of the channel. It was hypothesized that this interaction of Mg²⁺ with the chloroplast envelope likely mediated external Mg²⁺ effects on chloroplast metabolism.     

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg²⁺ diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg²⁺. These results suggest the presence of a Mg²⁺/H⁺ antiporter. The maximum Mg²⁺/H⁺ exchange rate was observed at pH 8.5. The K_m values for Mg²⁺ (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K⁺ and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg²⁺/H⁺ exchange was electroneutral. Mg²⁺/H⁺ exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K_m for Mg²⁺ are similar to those reported for the Mg²⁺/2Na⁺ antiporter in animals cell. These data are consistent with the existence of a Mg²⁺/2H⁺ antiporter in a plant tonoplast.