Influence of sterols on ion transport through lipid bilayer membranes

Charge-pulse relaxation experiments with the negatively charged lipophilic ions, dipicrylamine and tetraphenylborate, (as well as with the positively charged carrier system Rb⁺-valinomycin) have been carried out in order to study the influence of sterols on the ion transport through the lipid bilayer membrane. The mol fraction of the sterols (cholesterol, epicholesterol, ergosterol, stigmasterol, dihydrocholsterol, epicoprostanol and cholesterololeate) as referred to total lipid was varied in a wide range (mol fractions 0–0.8).The monoolein/sterol or dioleoylphosphatidylcholine/sterol mixtures were dissolved in $\text{n}$-hexadecane in order to minimize effects of the sterol on the membrane thickness.Cholesterol had a strong influence on the transport of the lipophilic ions. Its incorporation into monoolein membranes increased the rate constant ${}_{\mathrm{<mtext>i</mtext>}}$ of translocation up to 8-fold, but incorporation into phosphatidylcholine membranes had virtually no influence on $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$. The other sterols with one hydroxy group and cholesterololeate had no influence on the rate constant or the partition coefficient β. The results are discussed on the basis of a possible change of dipole potential of the membrane caused by cholesterol and its derivatives.In the case of valinomycin-mediated Rb⁺ transport only cholesterol had a strong influence on transport properties. The rate constants of association ($\text{k}{}_{\mathrm{<mtext>R</mtext>}}$) as well as the rate constants of translocation of the complex ($\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$) and of the free carrier ($\text{k}{}_{\mathrm{<mtext>S</mtext>}}$) were reduced by incorporation of cholesterol up to eight-fold. The decrease of $\text{k}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$ are possibly caused by a decrease of membrane fluidity, whereas the decrease of $\text{k}{}_{\mathrm{<mtext>R</mtext>}}$ may be due to an increase of surface potential. The different action of cholesterol on the two transport systems is discussed under the assumption that the adsorption plane of the lipophilic ion is located more towards the aqueous side and that of the ion-carrier complexes more towards the hydrocarbon side of the dipole layer.     

Electrical capacity of black lipid films and of lipid bilayers made from monolayers

Planar bilayer membranes were formed from monolayers of a series of monounsaturated monoglycerides and lecithins. The hydrocarbon thickness of these membranes, as calculated from the electrical capacity, increases with the length of the fatty acid chain. The specific capacity of monoolein bilayers was found to be 0.745 μF/cm² which is nearly twice that of a monoolein black film made in the presence of decane, but is close to that obtained after freezing out the solvent from the black film. The hydrocarbon thickness of the bilayer, as calculated with a dielectric constant of 2.1, is considerably less than twice the length of the extended hydrocarbon chain of the monoglyceride.The specific capacity ($\text{C}{}_{\mathrm{<mtext>m</mtext>}}$) of bilayers made from monoolein monolayers showed a negligible voltage dependence, whereas the $\text{C}{}_{\mathrm{<mtext>m</mtext>}}$ increased significantly at a voltage of 150 mV in the case of Mueller-Rudin-type monoolein films with $\text{n-}\text{decane}$ as a solvent.     

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes $\text{? 4 · 10}{}^{\mathrm{?5}}$. A minimal concentration of about 6 · 10¹¹ dipicrylamine ions per cm² of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ is compared with the concentrations $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ obtained from simultaneous electrical relaxation measurements. $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ and $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ agreed at low dipicrylamine concentrations (10^?8–10^?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10^?6 M). In the saturation range $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ was maximally four times higher than $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$. The significance of this difference is discussed together with general aspects of the saturation phenomenon.     

Flow analysis in a biological system adopting the buffer capacitor concept.

The flow measurement of each component in each compartment is important in works on transport phenomena in a biological system. The method of flow measurement was studied adopting the capacitor concept derived from network thermodynamics.A biologically active component i in a compartment is defined as follows,

$\text{n}{}_{\mathrm{i}}\text{=n}{}_1\text{+n}{}_2\text{=c}{}_1\text{V+c}{}_2\text{V}$

where the total quantity ni consists of a measurable form n_i (free form, conc. c₁) and concealed form n₂ (conc, c₂). Capacitor for the species i of a compartment is defined as follows,

$\text{C=}\text{d}\text{n}{}_{\mathrm{i}}\text{d}\text{μ}{}_{\mathrm{i}}\text{=}\text{1+}\text{c}{}_2\text{c}{}_1\text{c}{}_1\text{dV}\text{dμ}{}_{\mathrm{i}}\text{+}\text{1+}\text{dc}{}_2\text{dc}{}_1\text{v}\text{dc}{}_1\text{dμ}{}_{\mathrm{i}}$

,

$\text{=Ac}{}_1\text{dV}\text{dμ}{}_{\mathrm{i}}\text{+BV}\text{d}\text{c}{}_1\text{d}\text{t}$

Thus flow of each component is expressed as,

$\text{J}{}_{\mathrm{i}}\text{=}\text{d}\text{n}{}_{\mathrm{i}}\text{d}{}_{\mathrm{i}}\text{=}\text{d}\text{n}{}_{\mathrm{i}}\text{dμ}{}_{\mathrm{i}}\text{dμ}\text{n}{}_{\mathrm{i}}\text{dt}\text{=C}\text{d}\text{μ}{}_{\mathrm{i}}\text{dt}$

,

$\text{=Ac}{}_1\text{d}\text{V}\text{dt}\text{+BV}\text{d}\text{c}{}_1\text{dt}$

Method of determination of capacitor coefficients A and B by titration experiment was also considered. For an experimental case, the capacitance for H⁺ of blood compartment was determined. The relationship between the capacitor concept and the buffer value of Van Slyke was discussed.     

Inhibition of chymotrypsin by alkyl phosphonates: a quantitative structure-activity analysis.

A quantitative structure-activity relationship has been formulated for 53 alkyl phosphonates [R₂OPO(CH₃)SR₃] inhibiting chymotrypsin: . In this so-called bilinear model, k_i is the bimolecular rate constant (m^?1 s^?1), β is a disposable parameter evaluated by a computerized iterative procedure, MR is the molar refractivity of a substituent, $\text{σ}{}_3{}^1$ is Taft's polar parameter, and I is an indicator variable for substituents containing a sulfonium group. The correlation coefficient for this equation is 0.985. This quantitative structure-activity relationship is compared with those previously formulated for the action of chymotrypsin on acylamino acid ester substrates.     

Characterization of the β-adrenergic receptors of hamster white fat cells: Evidence against an important role for the α2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the β-adrenergic antagonist, [³H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±1.3}\text{nM}$) but low capacity ($\text{32±17}\text{fmol/mg}$ membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±0.5}\text{nM}$) binding sites with a binding capacity of $\text{53 ± 13}\text{fmol/mg}$ membrane protein (corresponding to $\text{4000 ± 980}$ sites per cell), and independent kinetic analysis provided a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a $\text{β}{}_1\text{-}\text{receptor}$ subtype, yielding $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values in good agreement with the $\text{K}{}_{\mathrm{<mtext>act</mtext>}}$ and the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [³H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the $\text{α}{}_2\text{-}\text{component}$ of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the $\text{α}{}_2\text{-}\text{receptors}$ in the adrenergic control of lipolysis in hamster white adipocytes.     

Imipramine binding site Temperature dependence of the binding of 3H-labeled imipramine and 3H-labeled paroxetine to human platelet membrane

The characteristics of ³H-labeled imipramine and ³H-labeled paroxetine binding to human platelet membranes were determined at various temperatures between 0 and 37°C. Both paroxetine and imipramine probably bind to the same molecular complex in the platelet membrane, but the binding characteristics are different for the two molecules. The dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) for imipramine increases from 0.3 nM to 7.0 nM with increasing incubation temperature in a continuous way, whereas $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for paroxetine is almost constant, about 0.05 nM, between 0 and 19°C, and first begins to increase from 0.06 nM to 0.16 nM between 20 and 37°C. This suggests that the binding of paroxetine to the binding site induces a conformational change in the molecular complex of the binding site, whereas the binding of imipramine takes place without conformational changes in the binding site.     

Dielectric dispersion of a single spherical bilayer membrane in suspension

Single spherical bilayer membranes of the Pagano-Thompson type (Pagano, R. and Thompson, T.E. (1967) Biochim. Biophys. Acta 144, 666–669), formed from monooleyl phosphate and cholesterol dissolved in CHCl₃/CH₃OH/n-decane, were subjected to a fast impedance analysis of high precision. Dielectric behavior of the whole system, as monitored from outside the spherical membrane, was sensitive to changes in the membrane state from the thick colored to the thin black state. With a spherical membrane 2–3 mm in diameter formed in the sample cavity containing 0.12 ml 10 mM NaCl, the former state was characterized by a dielectric dispersion having dielectric increment (Δ?) of some 10² and characteristic frequency ($\text{?}{}_{\mathrm{<mtext>c</mtext>}}$) around 10⁶ Hz, while the latter had $\text{Δ? ? 10}{}^5\text{and}\text{?}{}_{\mathrm{<mtext>c</mtext>}}\text{? 10}{}^3\text{Hz}$. Complex plane plots for both dispersions traced semicircles, indicating that the present system may be unequivocally analyzed to yield spherical radius and membrane capacity (C_m) on the basis of a well-established dielectric theory. C_m for the thin membranes has thus been determined to be 0.54 μF · cm^?2, in excellent agreement with a separate determination on planar membranes. The applicability of the present type of spherical membranes under dielectric monitoring to the study of membrane fusion or of exocytosis is suggested.     

Ligand-dependent tryptic inactivation of the ouabain sensitivity of ADP-ATP exchange catalyzed by canine renal Na+, K+-ATPase.

Phosphate transporter of bovine heart mitochondria was purified by solubilization of submitochondrial particles with octylglucoside and fractionation of the extract with ammonium sulfate. After reconstitution into liposomes the purified protein catalyzed phosphate transport which was sensitive to mersalyl and other SH reagents. Transport measured either as $\text{P}{}_{\mathrm{i}}\text{OH}$ or $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange was proportional to protein concentration and time. The $\text{P}{}_{\mathrm{i}}\text{OH}$ but not the $\text{P}{}_{\mathrm{i}}\text{P}{}_{\mathrm{i}}$ exchange was stimulated several fold by valinomycin plus nigericin in the presence of K⁺. The reconstituted system provides a suitable assay during purification of the mitochondrial phosphate transporter.     

Model of singlet oxygen scavenging by α-tocopherol in biomembranes

Quenching of singlet molecular oxygen (${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$) by α-tocopherol (I) involves the hydroxy function of the chromanol ring of I. In phosphatidylcholine (PC) uni- and multilamellar vesicles this structural element of I is localized at the interface polar headgroup/hydrophobic core. A dielectric constant of ? ～ 25 was determined for this special region of the PC bilayer. The ratio k_Q/k_R of rate constants of quenching processes (k_Q) and irreversible reactions (k_R) of I with ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ increases with decreasing polarity of the solvent. In ethanolic solutions where ? = 25.5, k_Q/k_R is about 40. Extrapolation of these results to phospholipid bilayers suggests that at the nearness of the ester carbonyl oxygen of the PC fatty acid moieties, α-tocopherol can deactivate approximately 40 ${}^1\text{Δ}{}_{\mathrm{g}}\text{O}{}_2$ molecules before being destroyed. It is concluded that in vivo, one may expect to find a higher k_Q/k_R ratio if the chromanol ring of I hides within the more hydrophobic interiors of the membrane surface peptides.     

Stoichiometry versus coupling ration in the cotransport of Na and different neutral amino acids

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions.The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2.These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acid (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na.The variation in coupling ratio values can be used to calculate K_A, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio $\text{k}{}_1\text{k}{}_2$, where k₁ and k₂ are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of K_A for methionine is 3.6 ± 1.1 mM and the $\text{k}{}_1\text{k}{}_2$ ratio is 1.01 ± 0.07. The constant coupling ratio value of 1 for alanine indicates that the value for K_A is extremely high or that the k₁ value is extremely low.     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

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).     

On equivalent forms of Whittaker's similarity index

For two N-species assemblages A, B with specific proportionate abundances of the ith species a_i, b_l respectively, we consider the equality

$\text{∑}\text{t=1}\text{N}\text{c}{}_{\mathrm{i}}\text{= 1?}\text{1}\text{2}\text{∑}\text{t=1}\text{N}\text{|a}{}_{\mathrm{i}}\text{?b}{}_{\mathrm{i}}\text{|, c}{}_{\mathrm{i}}\text{=}\text{a}{}_{\mathrm{i}}\text{a}{}_{\mathrm{i}}\text{? b}{}_{\mathrm{i}}\text{b}{}_{\mathrm{i}}\text{a}{}_{\mathrm{i}}\text{> b}{}_{\mathrm{i}}\text{, 0?a,b,c?1}$

. The left-hand term is known as Sanders' minimum faunal abundance value, while the right side is referred to as Whittaker's similarity index. Both measures are commonly used in community studies. Equality between these two measures obtains only when proportionate abundances are utilized. We develop equivalent formulation which is valid for absolute abundance data, reduces to the Sanders-Whittaker equality when proportionate abundance data are employed, and is more sensitive to differences in species abundance distributions. Namely, we show that

$\text{2}\text{α+β}\text{∑}\text{t=1}\text{N}\text{c}{}_{\mathrm{i}}\text{= 1 ?}\text{1}\text{α+β}\text{∑}\text{t=1}\text{N}\text{|a}{}_{\mathrm{i}}\text{?b}{}_{\mathrm{i}}\text{|}$

, where

$\text{α =}\text{∑}\text{t=1}\text{N}\text{a}{}_{\mathrm{i}}\text{, β =}\text{∑}\text{t=1}\text{N}\text{b}{}_{\mathrm{i}}$

, and the a's, b's c's are as defined above.     

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.     

The binding of cytochrome b5 to plasma membranes of rat liver. Its implication for membrane specificity and biogenesis

The in vitro incorporation of cytochrome $\text{b}{}_5$ into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome $\text{b}{}_5$ than did microsomal preparations; 60% of this cytochrome $\text{b}{}_5$ could not be reduced by the NADH-cytochrome $\text{b}{}_5$ reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome $\text{b}{}_5$ clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome $\text{b}{}_5$. These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell.     

A unified approach to ion transport through membranes. I. Membrane-soluble ions.

Theoretical membrane potential transient produced by applying a current step to nerve cells has been derived based on the compartment neuron model and also on the equivalent cylinder model developed by W. Rall. It is expressed as a sum of exponential functions as

$\text{∑}\text{i=0}\text{n?1}\text{E}{}_{\mathrm{i}}\text{[1?}\text{exp}\text{(}\text{t}\text{τ}{}_{\mathrm{i}}\text{)]}$

where n is the number of compartments. The ratio of the amplitudes of the first and the second largest exponential functions, ($\text{E}{}_1\text{E}{}_0$), was found to be proportional to that of their respective time constants, ($\text{τ}{}_1\text{τ}{}_0$), in these neuron models. The constant of proportionality is given in a form that depends on the number of compartments as $\text{E}{}_1\text{E}{}_0\text{= (1 +}\text{cos}\text{π}\text{n}\text{)}\text{τ}{}_1\text{τ}{}_0$. This theoretical result is discussed in the light of recent experimental results in cat red nucleus neurons.     

Heterozygote frequencies in small subpopulations.

The mean fixation index within subpopulations (F_IS) has been defined as $\text{F}\text{̄}{}_{\mathrm{IS}}\text{= ∑w}{}_{\mathrm{i}}\text{F}{}_{\mathrm{ISi}}\text{or as}\text{F}\text{̂}{}_{\mathrm{IS}}\text{=}\text{∑w}{}_{\mathrm{i}}\text{p}{}_{\mathrm{i}}\text{q}{}_{\mathrm{i}}\text{F}{}_{\mathrm{ISi}}\text{∑w}{}_{\mathrm{i}}\text{p}{}_{\mathrm{i}}\text{q}{}_{\mathrm{i}}$. The latter definition is preferred because it can be obtained from the two other fixation indices, F_ST and F_IT and because it is unaffected by the mean gene frequency. The expected frequency of heterozygotes in small subpopulations of dioecious organisms will exceed Hardy-Weinberg expectations and this can be measured by $\text{F}\text{̂}{}_{\mathrm{IS}}$. In an isolated subpopulation of constant variance effective size $\text{N,}\text{F}\text{̂}{}_{\mathrm{IS}}$ rapidly tends to $\text{1 − 4N}{}^2\text{(N − 1 + [N}{}^2\text{+ 1]}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{)}{}^2$. In the Island model of population structure, $\text{F}\text{̂}{}_{\mathrm{IS}}$ is approximately $\text{−(1 − m)}\text{N}\text{where}\text{m}$ is the immigration rate.When a sample is drawn from a natural population, the observed F_IS will depend upon the genetic structure of the population. The values of F_IS expected in three different types of population structure are discussed.