Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K⁺ concentration ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{: 200–300}\text{mM}$) which greatly exceeds that of the growth medium, and a low Na⁺ concentration ($\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{: 20}\text{mM}$). Unlike $\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{, K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ varies with cell aging.The K⁺ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K⁺ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ decreases and is partially compensated by a gain in Na⁺. This substitution completely reverses when metabolic substrate is added (K⁺ reaccumulation process). Kinetic analysis of K⁺ movement in cells with steady K⁺ level shows that most of K⁺ influx is mediated by an autologous K⁺-K⁺ exchange mechanism. On the other hand, during K⁺ reaccumulation by K⁺-depleted cells, a different mechanism (a K⁺ uptake mechanism) with higher transport capacity and affinity drives the net K⁺ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K⁺ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent ATPase activity.     

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.     

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1)

Studies of the localization of the Na⁺-dependent sugar transport in monolayers of LLC PK₁ cells show that the uptake of a methyl α-d-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [³H]phlorizin binding to monolayers of LLC PK₁ cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) of 1.2 μM and a concentration of high affinity receptors of 0.30 μmol binding sites per g DNA. The other class involving low affinity sites had a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of 240 μM with the number of binding sites equal to 12 μmol/g DNA. Phlorizin binding at high affinity binding sites is a Na⁺-dependent process. Binding at the low affinity sites on the contrary is Na⁺-independent. The mode of action of Na⁺ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na⁺ dependence and the matching of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for high affinity binding sites with the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of phlorizin for the inhibition of methyl α-d-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl α-d-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na⁺ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na⁺-dependent sugar transport system in the apical membrane of LLC PK₁ cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.     

Fluctuation analysis of Na+ channels modified by batrachotoxin in myelinated nerve

(1) Single myelinated nerve fibres of Rana esculenta were treated with the steroidal alkaloid batrachotoxin, and Na⁺ currents and Na⁺-current fluctuations were measured near the resting potential under voltage-clamp conditions. Between test pulses fibres were held at hyperpolarizing membrane potentials. (2) The spectral density of Na⁺-current fluctuations was fitted by the sum of a $\text{1}\text{f}$ component and a Lorentzian function. The time constant $\text{τ}{}_{\mathrm{<mtext>c</mtext>}}\text{= 1/(2π?}{}_{\mathrm{<mtext>c</mtext>}}\text{)}$ obtained from the corner frequency ?_c of the Lorentzian function approximately agreed with the activation time constant τ_m of the macroscopic currents. (3) The conductance γ of a single Na⁺ channel modified by batrachotoxin was calculated from the integral of the Lorentzian function and the steady-state Na⁺ current. At the resting potential V = O we obtained γ = 1.6 pS, higher γ-values of 3.2 and 3.45 pS were found at V = ?8 and ?16 mV, respectively. (4) The conductance of a modified Na⁺ channel is significantly lower than the values 6.4 to 8.85 pS reported in the literature for normal Na⁺ channels. Hence, our experiments are in agreement with the view that batrachotoxin acts in an ‘all-or-none’ manner on Na⁺ channels and creates a distinct population of modified channels.     

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.     

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.     

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).     

Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

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.     

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.     

Rat enterocyte Na+ transport in vitro action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na⁺ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na⁺ efflux rate constant (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na⁺ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca²⁺-free medium, bovine PTH lost its capacity to inhibit (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$). Thus, the presence of extracellular Ca²⁺ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$, bovine PTH induced no change in net Na⁺ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na⁺ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca²⁺ in the incubation medium to be operative.     

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.     

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 .     

Interaction of substituted guanidines with the tetrodotoxin-binding component in Electrophorus electricus.

In efforts to understand the molecular properties of ion channels in biomembranes, we have investigated the interaction of substituted guanidines with the Na⁺ channel site in membranes isolated from Electrophorus electricus. This interaction was measured by equilibrium competitive binding studies with [${}^3\text{H}$]tetrodotoxin ([${}^3\text{H}$]TTX); TTX has been shown to bind specifically to the Na⁺ channel in electrically excitable membranes. Although guanidine and small substituted guanidines such as methylguanidine or aminoguanidine competed with [${}^3\text{H}$]TTX for the membrane binding site, the apparent K_I values for these derivatives were nearly seven orders of magnitude higher than the K_d for TTX. On the other hand, the binding of the guanidines was considerably enhanced by introducing a substituent aromatic ring or aliphatic chain. Detailed analysis of the binding of aliphatic guanidines of varying chain length clearly demonstrated the contribution made by hydrophobic interactions. These results suggest that the channel site may include a hydrophobic region in close proximity to the carboxylate previously postulated to be involved in TTX binding.     

Effect of N-ethylmaleimide on leucine transport in the chang liver cell. II. Effect on the kinetics of Na+-independent transport

The Na⁺-independent leucine transport system is resolved into two components by their different affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ about 44 μM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with $\text{N-}\text{ethylmaleimide}$ (1 mM) specifically stimulates the high-affinity component of the Na⁺-independent system by greatly increasing its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value, whereas the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of the low-affinity component is markedly lowered. The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na⁺-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na⁺-independent leucine uptake itself. We conclude that the stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.     

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana)

In the presence of the Na⁺-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value ($\text{S}{}_0$) decreased while the corner frequency ($\text{f}{}_{\mathrm{<mtext>c</mtext>}}$) increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ values and the amiloride concentrations it was possible to determine the binding ($\text{k′}{}_{01}$) and unbinding ($\text{k}{}_{10}$) constants for amiloride to its receptor on the Na⁺ channel. With these parameters as well as short-circuit current and $\text{S}{}_0$ values, the current through the individual Na⁺ channels ($\text{i}$) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na⁺ channels ($\text{M}$) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na⁺ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ with the amiloride concentration did not occur in these animals.