The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

Lipid-protein interactions in membrane models fluorescence polarization study of cytochrome b5-phospholipids complexes

According to previous authors, cytochrome $\text{b}{}_5$, when extracted from bovine liver by a detergent method, is called cytochrome $\text{d-b}{}_5$. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome $\text{t-b}{}_5$.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, $\text{R}$, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome $\text{d-b}{}_5$, whereas $\text{R}$ does not change for cytochrome $\text{c}$ and cytochrome $\text{t-b}{}_5$. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome $\text{d-b}{}_5$ occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome $\text{d-b}{}_5$, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.     

Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

Transversal mobility in bilayer membrane vesicles: Use of pyrene lecithin as optical probe

Pyrene lecithin, a new excimer-forming lipid molecule, has been synthesized to examine the transversal mobility of probe molecules in lecithin bilayer vesicles. The rate of the lipid exchange is obtained by following the excimer yield as a function of time after mixing of fluorescence doped and undoped vesicles. A rapid exchange ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 11}\text{s}$) is followed by a slow transfer ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8}\text{h}$). Above the lipid phase transition the fast transfer can be attributed to an exchange of lipid molecules from the outer layer of one vesicle to the outer layer of another one. The slow exchange is interpreted in terms of the ‘flip-flop’ process between the two layers of a single bilayer vesicle.Using pyrene and pyrene decanoic acid as probe molecules only the fast transfer through the water phase is observed ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4}\text{s}$ for pyrene and $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7}\text{s}$ for pyrene decanoic acid). This indicates that molecules like fatty acids or apolar membrane constituents must equilibrate very rapidly in a single bilayer vesicle.The water solubility or the critical micelle concentration of the probe molecules is determined and related to the transfer rates. An exchange process through the water phase via a monomeric state can be excluded.     

The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics

(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 1 · 10}{}^{\mathrm{?8}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 2 · 10}{}^{\mathrm{?5}}\text{M}$). It is not necessary to postulate a third reaction of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 10}{}^{\mathrm{?6}}\text{M}$. (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed.     

Evidence for a protonmotive Q cycle mechanism of electron transfer through the cytochrome b-c1 complex.

A protein named oxidation factor can be reversibly removed from succinate-cytochrome $\text{c}$ reductase complex and shown to be required for electron transfer between succinate and cytochrome $\text{c}$. This protein is required for reduction of cytochrome $\text{c}{}_1$ and, in the presence of antimycin, for reduction of both cytochromes $\text{b}$ and $\text{c}{}_1$. These results are consistent with a protonmotive Q cycle mechanism in which the oxidation factor catalyzes electron transfer from reduced quinone to cytochrome $\text{c}{}_1$ and thus liberates from reduced quinone one of two protons required for energy conservation during electron transfer through the cytochrome $\text{b}\text{-}\text{c}{}_1$ complex.     

Restoration of pool function type behavior to succinate dehydrogenase having latent reconstitutive activity in ubiquinol-cytochrome c reductase complex

Mitochondrial ubiquinol-cytochrome $\text{c}$ reductase complex contains small amounts of succinate dehydrogenase. Estimates from electrophoresis indicate there is one dehydrogenase per eight complexes. This dehydrogenase transfers electrons to the $\text{b}$-$\text{c}{}_1$ complex poorly, as judged by low succinate-ubiquinone and succinate-cytochrome $\text{c}$ reductase activities. Electron transfer to the $\text{b}$-$\text{c}{}_1$ complex is restored by reconstitution of the complex with phospholipid. This phospholipid dependent restoration of electron transfer indicates that either reconstitutive activity of the dehydrogenase is preserved under conditions where electron transfer is absent, or that addition of phospholipid allows one dehydrogenase to transfer electrons to multiple $\text{b}$-$\text{c}{}_1$ complexes.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Correlation of the perceived texture of random coil polysaccharide solutions with objective parameters

A wide range of concentrated random coil polysaccharide solutions have been assessed for textural attributes by a trained sensory panel. The only textural terms invoked to describe these model systems were ‘thickness’ and ‘stickiness’, which were shown to be highly correlated, and essentially identical numerically, using a ratio scaling technique. Viscosity (η) measurements over a wide range of shear rates (γ) for all these samples gave flow curves (log η versus log γ) of the same form. Differences in flow behaviour between samples could then be characterised completely by two parameters, the maximum viscosity at low shear rates (η₀), and the shear rate ($\text{γ}\text{?}{}_{\mathrm{0·1}}$) at which $\text{η =}\text{solη}{}_0\text{10}$. A simple linear relationship was demonstrated between these two parameters and perceived thickness (T) or stickiness (S), irrespective of polysaccharide type. For Newtonian liquids, log T (or log S) varied linearly with log η. Hence the effective ‘in-mouth’ thickness of random coil polysaccharide solutions, in normal viscosity units, may be predicted directly from η₀ and $\text{γ}\text{?}{}_{\mathrm{0·1}}$ by the simple relationship: $\text{log}\text{η}{}_{\mathrm{N}}\text{= 1·13}\text{log}\text{η}{}_0\text{+ 0·45}\text{log}\text{γ}\text{?}{}_{\mathrm{0·1}}\text{? 1·72}$ where η_N is the viscosity of a Newtonian solution which would be perceived as identical in thickness (and stickiness) to the polysaccharide solution.     

A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits

Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome $\text{b}{}_5$, NADH-cytochrome $\text{b}{}_5$ reductase, and NADPH-cytochrome $\text{c}$ reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome $\text{c}$ reductase preparation (purified by a detergent method) and phosphatidyl choline.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome $\text{c}{}_1$ and cytochrome $\text{c}$ have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome $\text{c}{}_1$ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome $\text{c}$ is rather insignificant. The formation of a complex between cytochromes $\text{c}$ and $\text{c}{}_1$ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome $\text{c}$ upon mixing with cytochrome $\text{c}{}_1$ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome $\text{c}$ was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

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.     

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 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.     

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.     

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.     

Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation

Stable ubisemiquinone radical(s) in the cytochrome $\text{b}\text{?}\text{c}{}_1$-II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q₂. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (～9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved $\text{g}$-anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome $\text{b}\text{-}\text{c}{}_1$. This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome $\text{c}$ reductase from succinate dehydrogenase and the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex.     

Nanosecond fluorescence anisotropy decays of 1,6-diphenyl-1,3,5-hexatriene in membranes

Nanosecond decays of the fluorescence anisotropy, $\text{r}$, were studied for the emission of 1,6-diphenyl-l,3,5-hexatriene (DPH) embedded in a series of mixed multilamellar liposomes containing egg yolk phosphatidylcholine, phosphatidylethanolamine and cholesterol in varying molar ratios, as well as in membranes of intact cells and in virus envelopes.The relative contributions of the fast and the infinitely slow decaying component to the steady-state value, $\text{r}$, of the fluorescence anisotropy were very similar for artifical and biological membranes.Angles, θ, of the cone, by which the motion of the fluorescent molecule is limited, were calculated from the intensity of the infinitely slow decaying anisotropy component and compared with steady-state fluorescence anisotropies and with ‘microviscosities’, 〈η〉. An increase in 〈η〉 from 1.5 to 5.2 P in our systems was accompanied by a decrease in θ from 49° to 30° while the decrease in the mean motional relaxation times, $\text{φ}{}_{\mathrm{<mtext>f</mtext>}}$, of the label molecule was not more than 1 ns and due mainly to changes in the potential, by which the diffusion of DPH in the membrane is restricted. From these observations we conclude that differences in the steady-state fluorescence anisotropy and in ‘microviscosities’ of cholesterol-containing membranes ($\text{r}\text{> 0.15}$) represent changes in the degree of static orientational constraint rather than changes in diffusion rates of the label.