The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

The effects of n-alcohols on sarcoplasmic reticulum vesicles

Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous $\text{n-}\text{alcohols}$ from methanol to $\text{n-}\text{heptanol}$ caused an inhibition of calcium uptake and an enhancement of ATPase activity. The $\text{n-}\text{alcohol}$ treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of $\text{n-}\text{alcohol-treated}$ vesicles was about twice that of control vesicles. With increasing number ($\text{n}$) of carbon atoms of the $\text{n-}\text{alcohols}$, the maximum increment of ATPase activity increased, and both the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$) required to inhibit calcium uptake by 50% and the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.

$\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}\text{=23.5·10}{}^{\mathrm{?0.593n}}\text{M}$

$\text{N}{}_{\mathrm{ATPase}}\text{=35.5·10}{}^{\mathrm{?0.593n}}\text{M}$

The ratio, $\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$ to $\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$, was constant for all $\text{n}$ values. The apparent free energy of binding of the methylene groups of $\text{n-}\text{alcohols}$ to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of $\text{n-}\text{alcohols}$ in octanol and water (?670 cal/mole). The effects of $\text{n-}\text{alcohols}$ on membrane vesicles are discussed on the basis of these data.     

The quenching of an intramembrane fluorescent probe. A method to study the binding and permeation of phloretin through bilayers

Phloretin and phloretin-like dipolar non-electrolytes strongly quench the fluorescence of several membrane-bound probes, including 1,6-diphenylhexa-1,3,5-triene and anthroyl derivatives of long-chain fatty acids. Fluorescence intensity measurements therefore provide a simple and sensitive method to study the equilibrium binding properties and permeability of phloretin-like molecules in biological and artificial membrane systems. The dissociation constants for the binding of phloretin and naringenin to phosphatidylcholine vesicle membranes are determined, assuming the Stern-Volmer relation, from the fluorescence intensity of intramembrane probes as a function of phloretin and naringenin concentrations. Results (phloretin, $\text{9 ± 1}\text{μM}$; naringenin, $\text{21 ± 4}\text{μM}$) agree with the dissociation constants obtained using absorption titration performed in the absence of fluorescent probes. Fluorescence nanosecond lifetime measurements show that the mechanism of quenching of diphenylhexatriene and 16-anthroylpalmitic acid by phloretin and naringenin is largely diffusional in nature. The transmembrane movement of phloretin through phosphatidylcholine vesicles was observed by the stopped-flow technique, in which phloretin is mixed rapidly with a vesicle solution containing a membrane-bound fluorescent probe. The time course obtained by fluorescence measurements was identical to that obtained in a parallel measurement of the time course of optical absorption of phloretin. Stopped-flow data for the permeability of phosphatidylcholine liposomes and red blood cell membranes are also presented. The use of a membrane-bound indicator greatly extends the range of concentrations and pH values as well as the types of systems which can be characterized by optical means.     

Effects of n-Alkanols on the membrane fluidity of chick embryo heart microsomes

The $\text{n-}\text{alkanols}$ from butanol through octanol are membrane perturbing agents that fluidize the microsomal membranes of 20-day-old chick embryo hearts as measured by the fluorescence depolarization of 1,6-diphenylhexatriene. In terms of the aqueous concentrations of $\text{n-}\text{alkanols}$ the fluidizing effect increases with increasing number of carbons per $\text{n-}\text{alkanol}$. In terms of the membrane concentrations of $\text{n-}\text{alkanols}$ the fluidizing effect is roughly equivalent for all the $\text{n-}\text{alkanols}$ studied.     

Comparison of the promoting activity of pristane and n-alkanes in skin carcinogenesis with their physical effects on micellar models of biological membranes

In 1976 (Horton, A.W., Butts, C.K. and Schuff, A.R. (1976) Colloid Interface Sci. 5, 159–168) we assayed pristance (2,6,10,14-tetramethylpentadecane) in a model interfacial system that has given excellent correlation with cocarcinogenic activity among $\text{n-}\text{alkanes}$, as tested in cycloalkane diluents. It was predicted that this branched-chain derivative of the diterpenes would have higher activity than $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$, one of the most cocarcinogenic of the $\text{n-}\text{alkanes}$ in such diluents. Pristane was compared with $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$ and two other $\text{n-}\text{alkanes}$ for their promoting activities in cyclohexane for C3H male mice after a single application of 7,12-dimethylbenz$\text{[a]}\text{anthracene}$. The branched-chain alkane proved to be more active. 20% $\text{n-}\text{tetracosane}$ in cyclohexane was inactive, which correlated with its effects in this diluent in the physical assay system. The promoting activity of 75% $\text{n-}\text{octane}$ in cyclohexane, predicted by the physical assay, was confirmed by tests on mice. The combined by-products of the synthesis of tetracosane, including C₁₂ alkanes and alkenes, C₁₉ and C₂₀ alkylbenzenes, and C₂₄ alkenes, proved to be a very active promoter. However, a mixture in cyclohexane of purified tetracosane with this composite of potential impurities was inactive. From the alkanes behavior in physical systems involving vatious membrane phospholipids and steroids, it is hypothesized that the primary step in their biological activity is a chain-chain interaction with membrane lipids that alters the properties of liquid-crystalline phases at aqueous interfaces. Resulting changes in the microfluidity of the lipid phase and the lateral mobility of critical hormone receptors and enzyme systems, such as the nucleotidyl cyclases, would perturb control systems that maintain the normal behavior of the initiated cell. Thus, its progression to a proliferating neoplasm may be favored.     

Initial membrane reaction in peptidoglycan synthesis. Perturbation of lipid-phospho-N-acetylmuramyl-pentapeptide translocase interactions by n-Butanol

$\text{Phospho}\text{-N-}\text{acetylmuramyl-pentapeptide translocase}$, the initial membrane enzyme in the biosynthesis of peptidoglycan, requires a lipid microenvironment for function. $\text{n-}\text{Butanol}$ was reversibly intercalated into membranes to perturb the hydrophobic interactions in this microenvironment in order to define further the role of lipid. In the concentration range for maximal stimulation of enzymic activity (0.12–0.18 M), $\text{n-}\text{butanol}$ causes a 40% decrease in the fluorescence emission of the dansylated product, undecaprenyl $\text{diphosphate}\text{-(N}{}^{\mathrm{?}}\text{-}\text{dansyl)pentapeptide}$. Since no change in emission maximum occurs below 22°C in the presence of 0.12 M $\text{n-}\text{butanol}$, it is concluded that intercalation of this alkanol causes an increase in fluidity. Above 22°C this concentration of $\text{n-}\text{butanol}$ causes both a decrease in the fluorescence emission and a red shift in the emission maximum. It is concluded that a polarity change as well as fluidity change occurs above 22°C. $\text{n-}\text{Butanol}$ also causes a significant change in the phase transition experienced by the dansylated lipid product. Thus, it is possible with $\text{n-}\text{alkanols}$, e.g. $\text{n-}\text{butanol}$, to perturb lipid-translocase interactions resulting in an increase in fluidity in the microenvironment of the enzyme. This change in fluidity correlates with a stimulation of enzymic activity.     

Release and purification of micrococcus lysodeikticus ATPase from membrane extracted with n-butanol

The ATPase associated with the membranes of Micrococcus ysodeikticus has been released into the aqueous phase (i.e. solubilized) by extracting the membranes with $\text{n-}\text{butanol}$ in a two-phase system modified from the procedure of Maddy, A.H. (164) Biochim. Biophys. Acta 88, 448–449. A procedure for the release and purification of the ATPase from the membranes extracted with $\text{n-}\text{butanol}$ is described as an alternate method to that previously used for the shock-wash ATPase. Upon extracting the membrane suspensions with $\text{n-}\text{butanol}$ the soluble ATPase released into the buffer phase no longer exhibits stimulation by trypsin in contrast to the shock-wash type of ATPase. As shown by Salton, M. R. J. and Schor, M. T. (1972) Biochem. Biophys. Res. Commun. 49, 350–357, the shock-wash ATPase possesses associated protein(s) as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis whereas these are absent from the purified ATPase released by the $\text{n-}\text{butanol}$ method. The specific activities of the purified ATPase released by the two methods were generally similar, the $\text{n-}\text{butanol}$ type being consistently somewhat higher.     

The transverse organisation of ubiquinones in mitochondrial membranes as determined by fluorescence quenching

The transverse organisation of ubiquinone in mitochondrial membranes was investigated by quenching a set of fluorescent fatty acids. We show that the fluorescent moiety of the probes is located at a graded series of depths in the mitochondrial membrane. The probes sense the characteristics of the lipid phase and do not significantly perturb mitochondrial function as measured by the respiratory control ratio and the ADP/O ratio. The anthroyloxy fatty acids are readily quenched by ubiquinone-10. A recently developed method in the analysis of quenching data was used to obtain the subvolume of the membrane within which the quenching interactions are confined. The results indicate that ubiquinone-10 is restricted to two sites in the transverse plane of the membrane: one near the surface and the other close to the bilayer centre. The implications of these findings for the two-pool model of ubiquinone organisation are discussed.Abbreviations n-AS n-(9-anthroyloxy) stearic acids (n=6,9,12) - n-AP n-(9-anthroyloxy) palmitic acids (n=2,16) - n-AF n-(9-anthroyloxy) fatty acids (n=2,6,9,12,16) - n nitroxide stearic acids (n=5,16) - UQ _n ubiquinone-n (n=4,6,10) - HBHM heavy beet heart mitochondria     

The transverse location of tryptophan residues in the purple membranes of Halobacterium halobium studied by fluorescence quenching and energy transfer

Fluorescence quenching by a series of spin-labelled fatty acids is used to map the transverse disposition of tryptophan residues in bacteriorhodopsin (the sole protein in the purple membranes of Halobacterium halobium). A new method of data analysis is employed which takes into account differences in the uptake of the quenchers into the membrane. Energy transfer from tryptophan to a set of $\text{n-(9-}\text{anthroyloxy)}$ fatty acids is used as a second technique to confirm the transverse map of tryptophan residues revealed by the quenching experiments. The relative efficiencies of quenching and energy transfer obtained experimentally are compared with those predicted on the basis of current models of bacteriorhodopsin structure. Most of the tryptophan fluorescence is located near the surface of the purple membrane. When the retinal chromophore of bacteriorhodopsin is removed, tryptophan residues deep in the membrane become fluorescent. These results indicate that the deeper residues transfer their energy to retinal in the native membrane. The retinal moiety is therefore located deep within the membrane rather than at the membrane surface.     

Transverse location of anthracyclines in lipid bilayers. Paramagnetic quenching studies

Quenching of anthracycline fluorescence by a series of spin-labeled fatty acids was used to probe the transverse location of the drug in phosphatidylcholine bilayers in the form of small unilamellar vesicles. Stern-Volmer plots of the quenching data indicate that the fluorophore moiety of the anthracycline is intercalated into the hydrocarbon region of the bilayer, with deeper penetration observed in fluid-phase than in solid-phase vesicles. 31P-NMR parameters (T1 and nuclear Overhauser enhancement (NOE] are unaffected by the presence of drug, consistent with a binding site removed from the interfacial region. Comparison of intensity (F0/F) plots with lifetime (tau 0/tau) data shows that the predominant mechanism of anthracycline quenching by membrane-bound nitroxides is static. Since the membrane-bound drug is also accessible to quenching by I-, the binding site in the membrane must create a channel which is accessible to solvent. Two other fluorescent probes, 12-(9-anthroyloxy)stearate (12-AS) and diphenylhexatriene (DPH), were employed to confirm the results obtained with the anthracyclines, giving quenching data representative of their location in the bilayer.     

Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C₁₆ as compared to the C₂ region, while the rotational motion appeared to be the most hindered at the C₇–C₉ level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C₁₆ region. The extent to which the acyl chain reacted to this perturbation at the C₁₂ level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the $\text{3-}\text{oxo}\text{-Δ}{}^4\text{-}\text{steroids}$ in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant ($\text{2–5}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy ($\text{20}\text{kcal}\text{·}\text{mol}{}^{\mathrm{?1}}$) was observed at temperature below 16°C at pH 7.5. A correlated change of the $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}$ as function of temperature was detected; at 36°C $\text{p}\text{K}{}_{\mathrm{<mtext>ES</mtext>}}{}^{\mathrm{<mtext>app</mtext>}}\text{= 8.3}$ while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the $\text{3β-}\text{hydroxy}\text{-Δ}{}^5\text{-}\text{steroid dehydrogenase}$, the $\text{3-}\text{oxosteroid}\text{Δ}{}^5\text{?Δ}{}^4\text{-}\text{isomerase}$ was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.     

Interaction of DDT (1,1,1-trichloro-2,2-BIS(p-chlorophenyl)-ethane with liposomal phospholipids

The influence of $\text{1,1,1-}\text{trichloro}\text{-2,2-}\text{bis}\text{(p-}\text{chlorophenyl}\text{)}\text{ethane}$ (DDT) and several other pesticides on the physical state of membrane phospholipids was investigated using model lipids. The thermal dependence of fluorescence intensity of the probe parinaric acid in dipalmitoylphosphatidylcholine liposomes and lipid vesicles of mixed composition were recorded. DDT was incorporated into the liposomal bilayer. The insecticide lowered the phase transition temperature and broadened the temperature range of the transition. The effects were concentration-dependent.The results may be interpreted as a sort of blurred and facilitated phase transition of bilayer lipids caused by intercalation of DDT between fatty acyl chains of membrane phospholipids.     

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

The transverse location normal to the bilayer surface of a series of n-(9-anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 greater than or equal to 3 greater than 6 greater than or equal to 7 greater than 9 greater than 12 greater than 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells.     

13C NMR studies on fluorescent probes: 13C chemical shifts and longitudinal relaxation times of n-hydroxy-fatty (n=2,6,9,12) acids and n-(9-anthroyloxy)-stearic (n=6,12) acids

¹³C NMR has been used to confirm the structure of two fluorescent probes, n-(9-anthroyloxy)-stearic acids (n=6,12), and the series of n-hydroxy-fatty acids (n=2,6,9,12) from which the set of fluorescent fatty acids may be synthesised. ¹³C longitudinal relaxation times and correlation times of the individual carbon atoms in 12-hydroxy- and 6- and 12-(9-anthroyloxy)-stearic acids show differences in motional properties between these derivatives and the parent stearic acid in chloroform(d) solution. The correlation times of the substituted carbons in 6-, 9-, and 12-hydroxy-stearic acids are longer than the corresponding carbons in stearic acid. The change in correlation times at the substituted carbons reflects the increase in motion along the acyl chain. Attachment of the bulky anthracene ring causes greater restriction of motion at the substituted carbon atom but the gradient of motion along the chain is preserved. These results are discussed in terms of the types of motion which lead to fluorescence depolarization when the fluorescent fatty acids are used as fluidity probes in biomembranes.     

Fluorescent probe studies of mixed micelles of phospholipids and bile salts. Effect of cholesterol incorporation

The binding of the fluorescent alkylamines, $\text{N-(2-}\text{aminoethyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide, $\text{N-(5-}\text{aminopentyl}\text{)-5-}\text{dimethylamino-1-naphthalene}$ sulfonamide (dansyl cadaverine) and $\text{N-(10-}\text{aminodecyl}\text{)-5-}\text{dimethylamino-1-napthalene}$ sulfonamide with phospholipid and phospholipid-deoxycholate micelles, has been shown to increase with the length of the alkyl spacer chain. The probes bind more effectively to micelles containing unsaturated phospholipids and do not interact strongly with bile salt solutions at low concentrations. Cholesterol incorporation into mixed micelles results in a quenching of probe fluorescence due to displacement of probe molecules. The enhanced rigidity of the mixed micelles on solubilizing cholesterol is established by a decrease in pyrene excimer fluorescence and by the less effective perturbation of the micellar structure by 1-anilino-8-naphthalene sulfonate. The anionic probe 1-anilino-8-naphthalene sulfonate is also displaced from the mixed micelles when cholesterol is incorporated, suggesting a dominant role for packing and hydrophobic effects in binding both positively and negatively charged probes.     

Fluorescence anisotropy decays of n-(9-anthroyloxy) fatty acids in dipalmitoyl phosphatidylcholine vesicles. Localization of the effects of cholesterol addition

The localization of the effects of cholesterol addition on the dynamic structure of the fatty acyl chains of dipalmitoyl phosphatidylcholine vesicles has been investigated by the time-resolved fluorescence anisotropy technique with a set of n-(9-anthroyloxy) fatty acids probes. The major effect of cholesterol is observed in the 7–9 carbon region where both parameters of the anisotropy decays, the residual anisotropy (r_∞) and the correlation time, are greatly enhanced whatever the temperature (21, 37 and 47°C). In the 12–16 carbon region, the r_∞ values are lowered upon addition of cholesterol in the gel phase, in agreement with the effect monitored by the 1,6-diphenyl-1,3,5-hexatriene probe. Only slight perturbations on the r_∞ values are observed in the 2-carbon region whatever the temperature.