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.     

Transport kinetics of dipicrylamine through lipid bilayer membranes. Effects of membrane structure

Charge-pulse current-relaxation studies have been performed with lipid bilayer membranes in the presence of the hydrophobic ion dipicrylamine. From the analysis of the relaxation times and amplitudes the translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ of dipicrylamine as well as the partition coefficient β between membrane surface and water could be evaluated. In a first series of experiments membranes made from monoolein or dioleoylphosphatidylcholine in a number of different $\text{n-}\text{alkane}$ solvents were studied, as well as virtually solvent-free bilayer membranes made from monolayers. The thickness $\text{d}$ of the hydrocarbon layer of these membranes varied between 5.0 and 2.5 nm. While β was almost insensitive to variations in $\text{d}$, a strong decrease of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ with increasing membrane thickness was found; the observed dependence of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ on $\text{d}$ approximately agreed with the theoretically expected influence of membrane thickness on the height of the dielectric barrier. No specific differences between Mueller-Rudin films and solvent-free (Montal-Mueller) membranes other than differences in thickness were found. In a further series of experiments the chemical structure of the lipid was systematically varied (number and position of double bonds in the hydrocarbon chain, nature of the polar head group). The translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ was much larger in phosphatidylethanolamine membranes than in phosphatidylcholine membranes. A strong increase of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ was found when the number of double bonds in the hydrocarbon chain was increased from one to three. These changes were discussed in terms of membrane fluidity and dielectric barrier height. Much higher values of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ were observed in lipids with ester linkage between hydrocarbon chain and glycerol backbone, as compared with the corresponding ether analogs. This finding is qualitatively consistent with determinations of dipolar potentials in monolayers of ester and ether lipids. When cholesterol is added to phosphatidylcholine membranes, the translocation rate constant $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ increases up to five-fold, while the partition coefficient β remains virtually constant. The variation of $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ in this case can be largely accounted for by a decrease in membrane thickness and a concomitant reduction in dielectric barrier height. In membranes made from the negatively charged lipid phosphatidylserine the partition coefficient of dipicrylamine strongly increased with ionic strength, as expected from the Gouy-Chapman theory of the surface potential.     

Chloroplast membrane damage during freezing: the lipid phase

In order to study the effect of freeze damage to chloroplast membranes microviscosity of spinach thylakoids was probed by stearic acid spin labels. Changes in ESR parameters have been determined either as a function of temperature or during freezing at ?15 °C as a function of time. An empirical parameter $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ (ratio of height of a low field line component h₊ over height of the central line h₀) proved to be very sensitive to minute changes in membrane structure.In cryoprotected chloroplast membranes Arrhenius plot breaks indicative of phase changes are observed at +15 and ?10 °C. Breaks in the Arrhenius plots were not observed in vesicles prepared from chloroplast lipids by sonication. Instead, a melting zone was indicated below ?30 °C.Freeze damage of thylakoids during storage at ?15 °C is reflected in an increase of $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ and a decrease in central line width W₀. At +20 °C, differences between the ESR parameters of active as compared to freeze-damaged membranes could be detected, if the osmolarity of the suspending medium exceeded 200 mosm. The observed changes in line shapes are interpreted as an increase in mobility and/or orientation of the lipids following the swelling of thylakoids. They do not indicate a disorganization of the lipid phase. Sedimentation experiments indicated that the freeze-damaged swollen membranes still exhibited osmotic responses. It is suggested that freezing which is known to dissociate proteins from the membranes altered the charge distribution of the membranes leading first to membrane swelling and finally, by the opening of hydrophilic channels, to membrane collapse.     

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.     

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 .     

A NMR study of the ionization of fatty acids,fatty amines and N-acylamino acids incorporated in phosphatidylcholine vesicles

The ionization of fatty acids, fatty amines and $\text{N-}\text{acylamino}$ acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with ¹³C and titrated by using NMR spectroscopy. The apparent p$\text{K}{}_{\mathrm{a}}$ of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p$\text{K}{}_{\mathrm{a}}$ values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p$\text{K}{}_{\mathrm{a}}$ value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p$\text{K}{}_{\mathrm{a}}$ is essentially determined by the low polarity of the lipidic matrix.     

Effect of vitamin B-12 deprivation on the rates of synthesis and degradation of rat liver fatty acid synthetase

To characterize the basis for the increased hepatic fatty acid synthetase activity in vitamin B-12 deprivation, the content and rates of synthesis and degradation for the enzyme were determined. Animals were in a dietary steady state on normal chow or a B-12-deprived diet; animals on the latter diet were further divided into a “supplemented” group given B-12 and those “B-12-deprived.” The B-12-deprived animals had tissue B-12 depletion. Both total and specific activity of fatty acid synthetase were increased in the B-12-deprived animals, and this was due to increased enzyme protein. Rates of synthesis and degradation were studied in each group after a pulse of 20 μCi of l-[U-¹⁴C]leucine. Radioactivity was determined in the immunoprecipitate of the purified enzyme. Relative rates of synthesis in the B-12-deprived group were increased 8.8-fold over the normal and 3.6-fold over the B-12-supplemented group. Degradation of hepatic fatty acid synthetase was 63 hr ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) in the normal and 65 hr in the B-12-supplemented group. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ in the B-12-deprived group was 35 hr. Degradation rates for the soluble protein pool were not affected by B-12 deprivation. The rate constant for synthesis of hepatic fatty acid synthetase in the B-12-deprived group was 14-fold that of the normal and 6-fold that of the B-12-supplemented animals. Thus, although vitamin B-12 deprivation results in increased rate of degradation of fatty acid synthetase, enzyme synthesis is markedly increased yielding a severalfold net increase in synthetase content and activity.     

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.     

The effect of ammonium ions upon isolated reactions of the glycolytic scheme

In the glycolytic system derived from rat brain acetone powder, ammonium ion has been found to stimulate three different reactions: (a) the transphosphorylase reaction from phosphoenolpyruvate, (b) the phosphohexokinase reaction, and (c) the hexokinase reaction. The transphosphorylases are affected differently depending upon whether adenosine diphosphate or adenylic acid is the phosphate acceptor; in the case of the latter, the dependency upon $\text{NH}{}_4{}^{\mathrm{+}}$ is particularly marked. A highly active myokinase is present in these extracts and its activity influences the transphosphorylase reaction to a considerable extent. The phosphohexokinase reaction is stimulated to a greater extent by $\text{NH}{}_4{}^{\mathrm{+}}$ than is the hexokinase reaction. In contrast to these reactions which require the participation of the adenylic system, triose phosphate oxidase activity is uninfluenced by the presence of $\text{NH}{}_4{}^{\mathrm{+}}$.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

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.     

Involvement of the proton electrochemical gradient in genetic transformation in Escherichia coli

Plasmid pIY2 DNA which encodes for ampicillin-resistance was used to study the energetics of Ca⁺⁺-induced transformation in Escherichia coli. When cells are exposed to DNA in the presence of carbonylcyanide-m-chlorophenylhydrazone or 2,4-dinitrophenol, two protonophores that collapse the proton electrochemical gradient across the cell membrane ($\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$), transformation to ampicillin-resistance is drastically reduced with little or no effect on viability. Furthermore, when the components of $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$ are altered by varying ambient pH or by performing transformation in the presence of valinomycin or nigericin, the efficiency of transformation is directly correlated with the magnitude of the membrane potential and changes in the pH gradient have no significant effect. It is concluded that $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$, more specifically the membrane potential, plays a critical role in Ca⁺⁺-induced transformation.     

A restatement of melittin-induced effects on the thermotropism of zwitterionic phospholipids

Perturbations induced by melittin on the thermotropism of dimyristoyl-, dipalmitoyl-, distearoylphosphatidylcholine and natural sphingomyelin are investigated and rationalized from data obtained by fluorescence polarization, differential scanning calorimetry and Raman spectroscopy. Depending on the technique and / or experimental conditions used, the observed effects differ at the same lipid to protein molar ratio, due to partial binding of melittin. The binding is more efficient for tetrameric than for monomeric melittin, but in both cases its affinity is weaker for phosphatidylcholine dispersions in the gel phase than for sonicated vesicles. For temperatures $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$ efficient binding occurs whatever the initial state of the lipids is. One can summarize the effects induced by melittin on the transition temperature as follows: (i) No upward shift is observed on synthetic phosphatidylcholines when lipid degradation is avoided. This is achieved by using highly purified melittin, phospholipase inhibitors, and / or non-hydrolysable lipids. (ii) Melittin monomer does not change $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$. (iii) When melittin tetramer is stabilized, it decreases $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ by 10–15 deg. C. The transition broadens, and is finally abolished for $\text{R}{}_{\mathrm{<mtext>i</mtext>}}\text{? 2}$. Very similar results are found for natural sphingomyelin. Fluorescence polarization indicates similar changes in order and dynamics of the acyl chains for all lipid studied. For $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, fluorescence and Raman show that melittin decreases the amount of CH₂ groups in ‘trans’ conformation and the intermolecular order of the chains. According to fluorescence data, there is an increase of the rigid-body orientational order at $\text{T ? T}{}_{\mathrm{<mtext>m</mtext>}}$, while from Raman the positional intermolecular order decreases without significant change in the CH₂ groups ‘trans’/‘gauche’ ratio.     

A mechanism for ethanol-induced damage to liver mitochondrial structure and function

Mitochondria isolated from rats chronically fed ethanol demonstrated a marked inability to produce energy. The respiratory control ratio, the ADP/O ratio and state 3 respiration rates were all decreased. Coupled with other data, a progression of ethanol-induced changes is proposed with site I being altered prior to site II. Quantitation of mitochondrial cytochromes revealed decreases in cytochromes $\text{b}$ and $\text{aa}{}_3$ and an increase in $\text{c}{}_1$. Evaluation of respiration activity in relation to temperature showed ethanol-induced changes in the transition temperature ($\text{T}{}_{\mathrm{f}}$) which may have been related to changes in the lipid composition of the inner membrane. Mitochondrial membranes were separated, and analysis of fatty acids and phospholipids was performed. Various fatty acids were altered in both membranes; however, the outer membrane was altered more severely. A decrease in the arachidonate : linoleate ratio was observed only in the outer membrane; however, there was no ethanol-induced change in degree of unsaturation in either membrane. Phospholipid quantitation showed a reduction of total lipid phosphorous/mg protein in both membrane fractions; however, the inner membrane was most affected. Cardiolipin was the only phospholipid in this membrane which remained unaltered. The evidence indicates that the mechanism for ethanol-induced damage to the liver mitochondrion involves lipid compositional changes as well as changes in cytochromes and possibly other proteins.     

The influence of charge on phosphatidic acid bilayer membranes

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, 1,3-dimyristoylglycerol-2-phosphoric acid and $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid.The phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 ($\text{p}\text{K}{}_1$). The regions between the first and the second $\text{p}\text{K}$ of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H⁺ concentration results in an abrupt change of the transition temperature. The slope of the $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagram beyond $\text{p}\text{K}{}_2$ becomes very steep. This is the