ATP-dependent proton uptake by proteoliposomes reconstituted with purified Na+,K+-ATPase

Proton transport catalyzed by the sodium pump was demonstrated using proteoliposomes reconstituted with purified pig kidney Na+,K+-ATPase. Intravesicular pH was monitored with fluorescence from fluorescein isothiocyanate dextran introduced into the vesicles. An ATP-induced ouabain-sensitive acidification of the intravesicular medium was observed, when the vesicles were incubated with ATP and without Na+. The ATP-induced acidification was blocked by either extravesicular Na+ or pretreatment of the enzyme with ouabain before reconstitution. Protonophores, X-537A or carbonyl cyanide m-chlorophenylhydrazone, abolished the intravesicular acidification. The acidification was not inhibited by 3 mM tetra-n-butylammonium. The initial rate of the H+ uptake was increased with a decrease in pH of the extravesicular medium, and the maximum rate was obtained at pH 5.5-5.6. It is concluded that H+ can be transported in place of Na+ by the sodium pump.     

Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline

We examined the role of pH gradient and membrane potential in dipeptide transport in purified intestinal and renal brush-border membrane vesicles which were predominantly oriented right-side out. With an intravesicular pH of 7.5, changes in extravesicular pH significantly affected the transport of glycyl-L-proline and L-carnosine, and optimal dipeptide transport occurred at an extravesicular pH of 5.5-6.0 in both intestine and kidney. When the extravesicular pH was 5.5, glycyl-L-proline transport was accelerated 2-fold by the presence of an inward proton gradient. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated glycyl-L-proline transport, and the stimulation was observed in the presence and absence of Na+. A carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced H+ diffusion potential (interior-positive) reduced dipeptide transport. It is suggested that glycyl-L-proline and proton(s) are cotransported in intestinal and renal brush-border membrane vesicles, and that the process results in a net transfer of positive charge.     

Active alanine transport in isolated brush border membranes.

Uptake of L-alanine against a concentration gradient has been shown to occur with isolated brush border membranes from rat small intestine. An alanine transport system, displaying the following characteristics, was shown: (a) L-alanine was taken up and released faster than D-alanine; (b) Na+ as well as Li+ stimulated the uptake of both stereoisomers; (c) the uptake of L- and D-alanine showed saturation kinetics; (d) countertransport of L-alanine was shown; (e) other neutral amino acids inhibited L-alanine but not D-alanine entry when an electrochemical Na+ gradient across the membrane was present initially during incubation. No inhibition occurred in the absence of a Na+ gradient. The electrogenicity of L-alanine transport was established by three types of experiments: (a) Gradients of Na+ salts across the vesicle membrane (medium concentration greater than intravesicular concentration) supported a transient uptake of L-alanine above equilibrium level, and the lipophilic anion SCN- was the most effective counterion. (b) A gradient of K= across the membrane (vesicle greater than medium) likewise supported active transport of L-alanine into the vesicles provided the K= conductance of the membrane was increased with valinomycin. (c) Similarly, a proton gradient (vesicle greater than medium) in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an agent known to increase the proton conductance of membranes, produced an overshooting L-alanine uptake. A consideration of the possible forces, existing under the experimental conditions, suggests that the gradients of SCN-, K+ in the presence of valinomycin, and H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone contribute to the driving force for L-alanine transport by creating a diffusion potential. Since the presence of Na+ was required in all experiments with active L-alanine transport these results support the existence of a transport system in the brush border membrane which catalyzes the co-transport of Na+ and L-alanine across this membrane.     

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

Trans-potassium effects on the chloride/proton symporter activity of guinea-pig ileal brush-border membrane vesicles.

To investigate the inhibitory effect of trans potassium on the Cl-/H+ symporter activity of brush-border membrane vesicles from guinea pig ileum, we measured both 36Cl uptake and, by the pyranine fluorescence method, proton fluxes, in the presence of appropriate H+ and K+ gradients. In the absence of valinomycin, a time-dependent inhibitory effect of chloride uptake by trans K+ was demonstrated. This inhibition was independent of the presence or absence of any K+ gradient. Electrical effects cannot be involved to explain these inhibitions because the intrinsic permeability of these vesicles to Cl- and K+ is negligibly small. Rather, our results show that, in the absence of valinomycin, the inhibitory effect of intravesicular K+ involves an acceleration of the rate of dissipation of the proton gradient through an electroneutral exchange of trans K+ for cis H+, catalyzed by the K+/H+ antiporter also present in these membranes. Valinomycin can further accelerate the rate of pH gradient dissipation by facilitating an electrically-coupled exchange between K+ and H+. To evaluate the apparent rate of pH-dissipating, downhill proton influx, we measured chloride uptake by vesicles preincubated in the presence of alkaline-inside pH gradients (pHout/pHin = 5.0/7.5), charged or not with K+. In the absence of intravesicular K+, proton influx exhibited monoexponential kinetics with a time constant k = 11 s-1. Presence of 100 mM K+ within the vesicles significantly increased the rate of pH gradient dissipation which, furthermore, became bi-exponential and revealed the appearance of an additional, faster proton influx component with k = 71 s-1. This new component we interpret as representing the sum of the electroneutral and the electrically-coupled exchange of trans K+ for cis H+, mentioned above. Finally, by using the pH-sensitive fluorophore, pyranine, we demonstrate that, independent of the absence or presence of a pH gradient, either vesicle acidification or alkalinisation can be generated by adding, respectively, Cl- or K+ to the extravesicular medium. Such results confirm the independent existence of both Cl-/H+ symporter and K+/H+ antiporter activities in our vesicle preparations, the relative activity of the former being larger under the conditions of the present experiments. The possible interplay of these two proton-transfer mechanisms in the regulation of the intracellular pH is discussed.     

Proton gradient-dependent transport of glycine in rabbit renal brush-border membrane vesicles

This study describes evidence for the existence of a H+/glycine symport system in rabbit renal brush-border membrane vesicles. An inward proton gradient stimulates glycine transport across the brush-border membrane, and this H+-driven glycine uptake is attenuated by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is a positive rheogenic process, i.e. the H+-dependent glycine uptake is further enhanced by an intravesicular negative potential. Glycine uptake is stimulated to a lesser degree by an inward Na+ gradient. H+-dependent glycine uptake is inhibited by sarcosine (69%), an analog amino acid, imino acids (proline 81%, hydroxy proline 67%), and beta-alanine (31%), but not by neutral (L-leucine) or basic (L-lysine) amino acids. The results demonstrate that H+ glycine co-transport system in rabbit renal brush-border membrane vesicles is a carrier-mediated electrogenic process and that transport is shared by imino acids and partially by beta-alanine.     

Leucine-proton cotransport system in Chang liver cell

The stimulatory effect of an inward H+ gradient on the Na+-independent L-leucine uptake by the plasma membrane vesicles from Chang liver cells (Mohri, T., Mitsumoto, Y., and Ohyashiki, T. (1983) Biochem. Int. 7, 159-167) has been shown to be due to the increase of the Km value without changing the Vmax value in the transport kinetics. The uptake of leucine by the vesicles is accompanied by intravesicular acidification, and a stimulated uptake of leucine by the countertransport with a high concentration of leucine in the vesicles enhances the acidification. All of these uptakes of leucine and proton and their stimulations are amplified by imposing an inward proton gradient. These results suggest appreciably different affinities of proton for the leucine transport carrier in the inner and outer sides of the plasma membrane. A rapid decrease in the cytoplasmic pH was observed only in the first minute of incubation of intact cells with leucine in Na+-containing medium. But the leucine-dependent decrease of the cytoplasmic pH persisted longer when either Na+ in the medium was replaced by choline or amiloride was present along with Na+. Addition of amiloride to Na+-containing medium was inhibitory on the leucine uptake of cells, without effect on the early phase of glycine uptake. We conclude that Chang liver cells are provided in their plasma membrane with an amino acid-H+ cotransport system, and this is coupled to the amiloride-sensitive Na+/H+ exchange system.     

Leucine transport coupled to proton movement in membrane vesicles from Chang liver cells

Leucine transport into membrane vesicles obtained from Chang liver cells was stimulated by an inward H+ gradient. The stimulatory effect of the proton gradient on the rate of leucine uptake (1 min) was inhibited by the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. When the vesicles had been preloaded with a high concentration of KCl, addition of valinomycin stimulated leucine uptake by the vesicles, showing that the leucine transport is dependent on potential gradient. Leucine-coupled H+ accumulation inside the vesicles was confirmed by measuring leucine dependent quenching of the fluorescence of 9-aminoacridine added to medium. These results imply that electrochemical gradient of proton can serve as a driving force for leucine transport across the cell membrane and proton movement is coupled to leucine transport.     

Studies on calcium binding to brush-border membranes from rabbit small intestine

A study was made of the uptake of Ca2+ by brush-border membrane vesicles prepared from rabbit small intestine. The process was found to be time, temperature and substrate concentration dependent, displayed saturability, did not depend on added energy sources and occurred optimally in a pH range of 7.5-8.0. Although the transport of D-glucose by these membrane vesicles responded to changes in osmotic pressure as modified by adding cellobiose to the medium, the uptake of Ca2+ was found not to be osmotically-sensitive. Moreover, the equilibrium uptake value obtained when vesicles were exposed to 0.36 mM Ca2+ was some 60-fold higher than the amount that could have been accommodated by the intravesicular space, calculated from the equilibrium uptake of D-glucose. It was concluded from these results that the uptake involved complete binding of the Ca2+ to the membrane. The ionophore A23187 enhanced the rates of uptake and efflux of Ca2+ without affecting equilibrium values, which suggests that the binding of Ca2+ measured under our conditions was to interior sites of the membrane. The binding capacity was decreased in the presence of 10 mM lidocaine as indicated by a diminution of the equilibrium binding values. Generating an electrochemical potential (negative inside) by addition of valinomycin to vesicles pre-equilibrated with K2SO4, enhanced the rate of uptake of Ca2+. Addition of metal ions, on the other hand, inhibited the uptake, La3+ and Tb3+ being most effective followed by Mn2+, Ba2+ and Mg2+. Na+ and K+ were the least inhibitory. The properties of the Ca2+ uptake process found in rabbit brush-border membranes were compared to those of similar processes occurring in other species.     

ATP-dependent Cl- uptake by plasma membrane vesicles from the rat brain

Uptake of Cl- by plasma membrane vesicles from the rat brain was stimulated by ATP at 37 degrees C, but not by beta, gamma-methylene ATP or at 0 degrees C. The addition of Triton X-100 or sucrose to the incubation medium diminished the ATP-stimulated Cl- uptake, suggesting that Cl- was transported across the membranes into the intravesicular space. This ATP-stimulated Cl- uptake was not affected by 1 mM ouabain. 1 microM oligomycin, 0.1 mM gamma-aminobutyric acid or 0.1 mM picrotoxin. Thus, non-mitochondrial ATP-driven Cl- transport through a system other than Na, K-ATPase or Cl- channels occurs in neuronal plasma membrane vesicles.     

Effect of natural or synthetic detergents on the transport of D-glucose in the membranes of vesicles of the brush border of the intestine of the rabbit

We describe here the effects of natural and synthetic detergents on the D-glucose transport into brush-border membranes of vesicles of rabbit's intestine. Two synthetic detergents: Triton X-100 and dodecyltrimethylammonium bromide have been found very strong inhibitors (more than 50 p. 100 of inhibition of maximal D-glucose uptake). Kinetic studies showed that these detergents behaved as mixed type inhibitors. The Na+-dependent transport of amino acids (aspartic acid, lysine, phenylalanine) is only poorly affected by dodecyltrimethylammonium bromide, while Triton X-100 inhibits unspecifically all the transport studied.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.     

Biotin uptake mechanisms in brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex

Biotin transport was studied using brush-border and basolateral membrane vesicles isolated from rabbit kidney cortex. An inwardly directed Na+ gradient stimulated biotin uptake into brush-border membrane vesicles and a transient accumulation of the anion against its concentration gradient was observed. In contrast, uptake of biotin by basolateral membrane vesicles was found to be Na+-gradient insensitive. Generation of a negative intravesicular potential by valinomycin-induced K+ diffusion potentials or by the presence of Na+ salts of anions of different permeabilities enhanced biotin uptake by brush-border membrane vesicles, suggesting an electrogenic mechanism. The Na+ gradient-dependent uptake of biotin into brush-border membrane vesicles was saturable with an apparent Km of 28 microM. The Na+-dependent uptake of tracer biotin was significantly inhibited by 50 microM biotin, and thioctic acid but not by 50 microM L-lactate, D-glucose, or succinate. Finally, the existence in both types of membrane vesicles of a H+/biotin- cotransport system could not be demonstrated. These results are consistent with a model for biotin reabsorption in which the Na+/biotin- cotransporter in luminal membranes provides the driving force for uphill transport of this vitamin.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

A systematic study was made of the action of 4-acet-amido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer 45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS, Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton.     

Specific interaction of 5-(N-methyl-N-isobutyl)amiloride with the organic cation-proton antiporter in human placental brush-border membrane vesicles. Transport and binding.

The interaction of 5-(N-methyl-N-isobutyl)amiloride (MIBA) with brush-border membrane vesicles isolated from normal human term placentas was investigated using two parameters: binding and transport. The binding of MIBA to placental membranes was specific and temperature- and pH-dependent, and the apparent dissociation constant (Kd) for the process was 58 +/- 2 microM. The binding was inhibited by other amiloride analogs and also by clonidine and cimetidine with a rank order potency: MIBA > benzamil > dimethylamiloride > amiloride > clonidine > cimetidine. These compounds also inhibited Na(+)-H+ exchanger activity in these membrane vesicles, but with a different order of potency: dimethylamiloride > MIBA > amiloride > benzamil > cimetidine > clonidine. The membrane vesicles were also able to transport MIBA into the intravesicular space, and the transport was stimulated many-fold by the presence of an outwardly directed H+ gradient across the membrane. The H+ gradient was the driving force for uphill accumulation of MIBA inside the vesicles. The transport process was electrically silent. The transport of MIBA was inhibited by other amiloride analogs and by clonidine and cimetidine, and the order of potency was the same as the order with which these compounds inhibited the binding of MIBA. The Michaelis-Menten constant (Kt) for the transport process was 46 +/- 2 microM. The binding as well as the transport were also inhibited by Na+ and Li+. Interestingly, tetraethylammonium and N1-methylnicotinamide, two of the commonly used substrates in organic cation transport studies, failed to inhibit the binding and transport of MIBA. Furthermore, although the outwardly directed H+ gradient-dependent uphill transport of tetraethylammonium could be demonstrated in renal brush-border membrane vesicles, there was no evidence for the presence of a transport system for this prototypical organic cation in placental brush-border membrane vesicles. It is concluded that the human placental brush-border membranes possess an organic cation-proton antiporter which accepts MIBA as a substrate, the low affinity binding site for MIBA observed in these membranes represents this antiporter, and that the placental organic cation-proton antiporter is distinct from the widely studied renal organic cation-proton antiporter.     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na+/L-glutamate (L-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K+ and Cl-. The presence of 100 mM K+ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K+ concentration and that in the absence of any sodium gradient, an outward K+ gradient was unable to influence the Na+/acidic amino acid transport system. It was also found that Cl- could specifically activate the Na+-dependent L-glutamate (L-aspartate) uptake either in the presence or in the absence of K+. Also the effect of Cl- was observed only in the presence of an inward Na+ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na+ gradient and in the presence of chloride gradient. L-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K+ or Cl- did not show any translocation of net charge.     

Mutual inactivation of valinomycin and protonophores by complex formation in liposomal membranes

The stimulation presence of a protonophore [3,5-di(ter-butyl)-4-hydroxybenzylidenemalononitrile or carbonyl cyanide m-chlorophenylhydrazone] and valinomycin in a liposome suspension results in time-dependent inactivation of ion transport by both the protonophore and valinomycin. Correlation of the inactivation with spectrophotometric observations on the formation of a complex between the protonophore and valinomycin strongly suggests that the complex observed has no (or very low) activity for the transport of either H+ or K+. The stoichiometry of valinomycin and the protonophore in the inactive complex is shown to be 1:1.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Pyridine nucleotide oxidation by a plasma membrane fraction from red beet (Beta vulgaris L.) storage tissue

The potential role of pyridine nucleotide oxidation in the energization and/or regulation of membrane transport was examined using sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue. In this system, pyridine nucleotide oxidation, which was enhanced in the presence of ferricyanide, occurred. In the presence or absence of ferricyanide, the oxidation of NADH was several-fold greater than the oxidation of NADPH, indicating that it was the preferred substrate for oxidation in this system. Ferricyanide reduction coupled to NADH oxidation did not require the transmembrane movement of reducing equivalents since ferricyanide incorporated inside the vesicles could not be reduced by NADH added externally to the vesicles, unless the vesicles were made leaky by the addition of 0.05% (v/v) Triton X-100. Using fluorescent probes for the measurement of transmembrane pH gradients and membrane potentials, it was determined that NADH oxidation did not result in the production of a proton electrochemical gradient or have any effect upon the proton electrochemical gradient produced by the plasma membrane H+-ATPase. The oxidation of NADH in the presence of ferricyanide did result in the acidification of the reaction medium. This acidification was unaffected by the addition of Gramicidin D and stimulated by the addition of 0.05% (v/v) Triton X-100, suggesting a scalar (nonvectorial) production of protons in the oxidation/reduction reaction. The results of this study suggest that the oxidation of pyridine nucleotides by plasma membrane vesicles is not related to energization of transport at the plasma membrane or modulation of the activity of the plasma membrane H+-ATPase.