Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramylpentapeptide translocase

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by $\text{phospho}\text{-N-}\text{acetylmuramyl}$ (MurNAc)-pentapeptide translocase (UDP-MurNAc-Ala-γ dGlu-Lys-dAla-dAla undecaprenyl phosphate phospho-MurN Acpentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30°C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}$ derivatives of 12-and 16-ketostearic acid intercalated into membranes from this organism define the lower ($\text{T}{}_1\text{= 16–22°}\text{C}$) and upper ($\text{T}{}_{\mathrm{<mtext>h</mtext>}}\text{= 30°}\text{C}$) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurN Ac-pentapeptide translocase has a significant effect on the catalytic activity of this enzyme.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Alteration of the conductance of Na+ channels in the nodal membrane of frog nerve by holding potential and tetrodotoxin

(1) Na⁺ currents and Na⁺-current fluctuations were measured in myelinated frog nerve fibres at 15°C during 7.7 ms depolarizations to $\text{V = 40, 60}\text{and}\text{80}\text{mV}$. (2) The conductance γ of a single Na⁺ channel and the number $\text{N}{}_0$ of channels per node were calculated from ensemble average values of the mean Na⁺ current and the variance of Na⁺-current fluctuations. (3) For a hyperpolarizing holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ the mean values of the channel conductance and number were $\text{γ = 9.8}\text{pS}$ and $\text{N}{}_0\text{= 74 000}$. (4) After changing the holding potential to the resting potential ($\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= 0}$) the conductance γ increased by a factor of 1.37 whereas the number $\text{N}{}_0$ decreased by a factor of 0.60. (5) Addition of 8 nM tetrodotoxin at a holding potential of $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{= ?28}\text{mV}$ increased γ by a factor of 1.55 and reduced $\text{N}{}_0$ by a factor of 0.25. (6) The increase of the channel conductance at reduced channel numbers suggests negative cooperativity between Na⁺ channels in the nodal membrane.     

The basic asymmetry of Na+-dependent glycine transport in Ehrlich cells

Influx and efflux of glycine have been examined as a function of external and internal Na⁺ concentrations, respectively, when $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$ it was found that at comparable external and cellular Na⁺ levels, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for efflux was larger by an order of magnitude than the value for influx and the $\text{V}$ for efflux was several times greater than the $\text{V}$ for influx. For both fluxes the major effect of Na⁺ was to decrease the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value. The observations are consistent with the conclusion that the Na⁺-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na⁺ concentration from 13 to 100 mM, the half-time for glycine equilibration being a function of the Na⁺ concentration in absence of an electrochemical potential difference for Na⁺. In Na⁺-free media ($\text{[}\text{Na}{}^{\mathrm{+}}\text{] < 5}\text{mM}$) equilibration of glycine between cells and medium was not achieved after 60 min at 25°C. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$, efflux (or uptake) of glycine was not affected by internal (or external) K⁺ between 20 and 120 mM suggesting that K⁺ plays no direct role in Na⁺-dependent transport of glycine in Ehrlich cells.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

Double dependence of organic acid active transport in proximal tubules of surviving frog kidney on sodium ions I. Influence of sodium ions in bath medium on the uptake and run out of fluorescein and uranin

With the aid of direct microfluorimetric determination of marker organic anions (fluorescein and uranin) accumulated in the proximal tubules the influence of Na⁺ in the bath medium on the active transport of these anions was studied. Kinetic analysis of the rate dependence of organic acid active transport into tubules on their concentration in the bath medium with a constant Na⁺ concentration permitted to define values of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}$ for uranin and fluorescein transport in the medium with different Na⁺ content. It was shown that a decrease of Na⁺ concentration in the medium increases $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and lowers the $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio with uncharged $\text{V}$. By varying the Na⁺ concentration in the medium with a constant concentration of the marker anion the and values for fluorescein and uranin transport were determined. A value for fluorescein in twice as much that for uranin. The $\text{1/K}{}_{\mathrm{<mtext>m</mtext>}}$ value for uranin transport is a linear function of Na⁺ concentration, while for fluorescein transport it is a quadratic one. Therefore it is concluded that two Na⁺ from the medium participate in active transfer of one fluorescein anion whereas only one Na⁺ from the medium is required for active transfer of one uranin anion. The run out of fluorescein from tubules preloaded with this acid is sharply reinforced by the Na⁺ omission from the medium. Thus, active transport of organic acids in proximal tubules of frog kidney is Na⁺-dependent, and Na⁺ from the medium is likely to participate directly in formation of a transport complex. When Na⁺ is absent in the medium a carrier fulfils a facilitated diffusion only.     

Temperature-dependent relationship between K+ influx,Mg2+-ATPase activity,transmembrane potential and membrane lipid composition in mycoplasma

The temperature-dependent relationship between K⁺ active influx, Mg²⁺-ATPase activity, transmembrane potential (ΔΨ) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 μg/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (?), E).Arrhenius plots of ⁴²K⁺ active influx gave a linear relationship for (chol (+), O + P) cells ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?9}\text{kcal}$). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process ($\text{t > 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?24}\text{kcal}\text{; t < 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?40}\text{kcal}$).Finally, a biphasic response with a break at approx. 23°C ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?7}\text{kcal}\text{, t > 23°}\text{C}$; $\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?44}\text{kcal}\text{, t < 23°}\text{C}$) is observed for (chol (?), E) organisms. From the lack of correspondence between these effects on the K⁺ influx and the temperature dependence of both the Mg²⁺-ATPase activity and ΔΨ, it is suggested that changes in the membrane lipid composition affect the K⁺ transport at the level of the K⁺ carrier itself.Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K⁺ carrier is associated with the most fluid lipid species present in the membrane.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Energetics of coupled Na+ and Cl− entry into epithelial cells of bullfrog small intestine

Na⁺, K⁺ and Cl^? concentrations ($\text{c}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$) and activities ($\text{a}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$), and mucosal membrane potentials ($\text{E}{}_{\mathrm{<mtext>m</mtext>}}$) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na⁺ and Cl^? and 5.4 mM K⁺. Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry and Cl^? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO₃. ¹⁴C-labelled inulin was used to determine extracellular volume. $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ was measured with conventional open tip microelectrodes, $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with solid-state Cl^?-selective silver microelectrodes and $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with Na⁺- and K⁺-selective liquid ion-exchanger microelectrodes. The average $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ recorded was ?34 mV. $\text{c}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{c}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{c}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 51, 105 and 52 mM. The corresponding values for $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na⁺ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K⁺ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl^?. $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na⁺ and Cl^? is implicated in intracellular Cl^? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na⁺ and Cl^? electrochemical potential differences (Δμ&#x0304;Na and Δμ&#x0304;Cl). Δμ&#x0304;Na (?7000 J · mol^?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ&#x0304;Cl (1000–2000 J · mol^?1).     

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans

The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na⁺. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na⁺. The extent of transport inhibition by Na⁺ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na⁺ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values for Na⁺ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order $\text{Li}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Rb}{}^{\mathrm{+}}\text{> Cs}{}^{\mathrm{+}}$. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na⁺, in addition to the competitive inhibition of transport Na⁺ induces an increase in membrane permeability for amino acids.     

Lipid dynamics and protein-lipid interactions in rat colonic epithelial cell basolateral membranes

Lipid dynamics and lipid-protein interactions were examined in basolateral membranes prepared from rat proximal and distal colonic epithelial cells. The results demonstrate that: (1) these membranes have a high lipid fluidity, as assessed by steady-state fluorescence polarization studies using seven fluorescent probes; (2) lipid compositional differences exist between these membranes but their fluidity is similar; (3) fluorescence polarizations studies, using diphenylhexatriene (DPH), detect a thermotropic transition at 22–23°C in each membrane; (4) several membrane protein activities, including adenylate cyclase and sodium-potassium dependent adenosine triphosphatase ($\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$) appear to be functionally dependent on the physical state of the proximal basolateral membrane's lipid.     

Phlorizin as a probe of the small-intestinal Na+,d-glucose cotransporter. A model

(1)‘Uptake’ of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of d-glucose, by the simultaneous presence of Na_out⁺ and $\text{Δψ?0}$. However, phlorizin contrary to d-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na⁺,d-glucose cotransporter. (2) The stoicheiometry of Na⁺/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Na_out⁺-dependence of phlorizin binding. (3) The preferred order of binding at $\text{Δψ?0}$ is Na⁺ first, phlorizin second (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to Δψ. These observations while agreeing with the effect of $\text{Δψ?0}$ on the $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na⁺,Δψ-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na⁺,d-glucose cotransport. (6) The $\text{k}{}_{\mathrm{<mtext>on</mtext>}}$, $\text{k}{}_{\mathrm{<mtext>off</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ constants of phlorizin interaction with the Na⁺,d-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

The uptake of l-glutamic acid into brush-border membrane vesicles isolated from rat renal proximal tubules is Na⁺-dependent. In contrast to Na⁺-dependent uptake of d-glucose, pre-equilibration of the vesicles with K⁺ stimulates l-glutamic acid uptake. Imposition of a K⁺ gradient ($\text{[}\text{K}{}_{\mathrm{i}}{}^{\mathrm{+}}\text{] > [}\text{K}{}_{\mathrm{o}}{}^{\mathrm{+}}\text{]}$) further enhances Na⁺-dependent l-glutamic acid uptake, but leaves K⁺-dependent glucose transport unchanged. If K⁺ is present only at the outside of the vesicles, transport is inhibited. Intravesicular Rb⁺ and, to a lesser extent, Cs⁺ can replace intravesicular K⁺ to stimulate l-glutamic acid uptake. Changes in membrane potential incurred by the imposition of an H⁺-diffusion potential or anion replacement markedly affect Na⁺-dependent glutamic acid uptake only in the presence of K⁺. Experiments with a potential-sensitive cyanine dye also indicate that, in the presence of intravesicular K⁺ a charge movement is involved in Na⁺-dependent transport of l-glutamic acid.The data indicate that Na⁺-dependent l-glutamic acid transport can be additionally energized by a K⁺ gradient. Furthermore, intravesicular K⁺ renders Na⁺-dependent l-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference.