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     

Mobility of fluorescent probe molecules in lipid bilayer vesicles as studied by steady-state and time-dependent nuclear Overhauser effect measurements in 1H-nuclear magnetic resonance spectroscopy

In order to elucidate the mobilities of the fluorophores of fluorescent 2- and 16-(9-anthroyloxy)palmitic acids (16-AP and 2-AP, respectively) in lipid bilayer vesicles, the steady-state and time-dependent nuclear Overhauser effects in ¹H-NMR spectroscopy, but not the fluorescence depolarization in fluorescence spectroscopy, have been measured. The steady-state nuclear Overhauser effect measurements showed an appreciable magnitude of negative nuclear Overhauser effects between the resonances due to the fluorophores of the two fluorescent probes and lipids. These results definitely mean that in lipid bilayers, the fluorophores (anthroyloxy ring) of the fluorescent probes experience other types of motions with much longer correlation times than those detected by the fluorescence depolarization measurements, since at the correlation time showed by the fluorescent method (1–2 · 10⁻⁹ s or less), no such transfer of the negative nuclear Overhauser effects is expected to occur. The correlation times of the fluorophores, as calculated from the cross-relaxation rates of the anthroyl ring protons of 16-AP and 2-AP, were 3.8 · 10⁻⁸ and 1.1 · 10⁻⁷ s, respectively. These values, respectively, compare favorably with those of the terminal methyl of acyl chains and the choline methyl carbons which were estimated by ¹³C T₂ relaxation times. Thus, it is concluded that the fluorophores of both 16-AP and 2-AP have a slow form of motion which moves with a similar time scale to those of lipids in addition to the faster one that causes fluorescence depolarization.     

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 pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-¹⁴C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [¹⁴C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-¹⁴C]20:5n-3 to [3-¹⁴C]22:6n-3 was more efficient than that of [1-¹⁴C]20:4n-6 to [3-¹⁴C]22:5n-6. At low substrate concentration (4 μM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 μM). The conversion of [1-¹⁴C]22:5n-3 to [1-¹⁴C]22:6n-3 was 1.7 times more efficient than that of [1-¹⁴C]22:4n-6 to [1-¹⁴C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-¹⁴C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-¹⁴C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-¹⁴C]20:4n-6 or [1-¹⁴C]22:4n-6 to [¹⁴C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-¹⁴C]20:5n-3 and [1-¹⁴C]22:5n-3 to [¹⁴C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

High resolution nuclear magnetic resonance spectroscopy of glycosphingolipids. I: 360 MHz 1H and 90.5 MHz 13C NMR analysis of galactosylceramides

The high resolution ¹H and ¹³C nuclear magnetic resonance (NMR) spectra of galactosylceramides containing n-fatty acids and α-hydroxy fatty acids were recorded in dimethylsulfoxide solution with and without addition of D₂O. From the coupling constants of the sugar ring protons, a ⁴C₁ conformation can be deduced. In contrast to the conformation in aqueous solution, the C⁶ hydroxymethylene group is freely rotating around the C⁶C⁵ bond. In the ceramide residue all signals produced by protons linked to carbons bearing electronegative substituents could be attributed. The large difference in coupling constants of the methylene protons of C^1′ to the C^2′ methine proton of the sphingosine indicates a restricted rotation around the C^1′C^2′ bond. The assignments of the hydroxy and amino protons follow from the decoupling of the corresponding methine protons.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

二价金属离子对嵌有线粒体H~+-ATP酶的脂酶体不同层次脂质流动性的影响

本文报道用荧光偏振及顺磁共振两种方法研究Mg~(2+)及其它二价金属离子对嵌有H~+-ATP酶的脂酶体不同层次脂质流动性的影响。 (1)顺磁标记探剂5-、12-、16-氮氧基硬脂酸测定结果表明Mg~(2+)和其它二价金属离子都能降低膜脂双分子层表层的流动性。降低流动性的顺序为Mg~(2+)=Ca~(2+)>Sr~(2+)>Cd~(2+)。较深层脂则无明显变化。 (2)荧光探剂7-、12-(9-蒽酰)硬脂酸及16-(9-蒽酰)棕榈酸的测定结果也表明Mg~(2+)和其它二价金属离子降低了膜脂表层的流动性,尤以Mn~(2+)、Ca~(2+)降低流动性最显著,流动性降低的顺序为;Mn~(2+) Ca>Sr~(2+) Mg~(2+) Cd~(2+)。除Mn~(2+)、Ca~(2+)还能影响膜脂深层的流动性外,其它与对照无明显差异。     

An assessment of the fluidity gradient of the lipid bilayer as determined by a set of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12, 16).

The rotational behavior of a set of n-(9-anthroyloxy) fatty acid fluorescent probes is examined in two liquid paraffins and in liposomes composed of dipalmitoyl phosphatidylcholine. As has been observed with other membrane fluorescent probes (Hare, F., and Lussan, C. (1977) Biochim. Biophys. Acta 467, 262-272), the degree of fluorescence depolarization for a given solvent viscosity is dependent on the solvent standard employed. In addition, when the anthroyloxy group is in the terminal position of the acyl chain, it has more rotational freedom than when it is conjugated to positions 6, 9, or 12 where the rotational motion of the fluorophore is similar. When incorporated into lipid bilayers, values of fluorescence polarization reflect the gradient of "fluidity" which extends from the surface to the center of the membrane. The nature of this polarization gradient is discussed in relation to the intrinsic differences between the probes and the anisotropic rotations responsible for depolarization.     

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.     

Dienoic acids,synthesis and 13C NMR spectral analysis

A preparation of E,E-2,4-dienoic acids, together with the assignments of their ¹³C NMR signals and the shifts observed after transformation into their sodium salts, is described. The stereochemistry of the double bonds of 3,5-dienoic esters, obtained from 2,4-dienoic acids, on the basis of ¹³C NMR data, is also presented.     

The use of 13C spin relaxation to investigate molecular motion in liquid tristearin

Longitudinal and transverse ¹³C spin relaxation times have been used to investigate the molecular motion of tristearin over a range of temperatures in the melt. Overall molecular rotational diffusion rates have been obtained as well as the diffusion rates about successive bonds in the stearoyl chains. The data can be explained using an anisotropic rotor model in which the fast and slow molecular diffusion rates are $\text{? 8 x 10}{}^9\text{sec}{}^{\mathrm{?1}}\text{and}\text{0.018 (2) x 10}{}^9\text{sec}{}^{\mathrm{?1}}$ respectively The alignment of the fast diffusion axis is close to the long chain axis of the ‘tuning fork’ model and the existence of such a configuration in the melt is supported by the observation of different relaxation times for the two chemically equivalent primary glyceryl carbons.The low flexibility gradient and high end group mobility of the acyl chain found at low temperatures in the melt is similar to that observed in lipid vesicle studies and suggest that the chains are aligned parallel. A break down of this short range order is apparent above 150°C.     

Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

The interaction between the plant hormone, 3-indoleacetic acid (IAA), and some phospholipids in CDCL₃ has been studied by ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidylcholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (K_d). In all cases, K_d values were approx. 10^?2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of 1AA.     

Interactions of phenytoin with rat brain synaptosomes examined by fluorescent fatty acid probes

Phenytoin (PHT) modified the fluorescent characteristics of anthroyloxy-fatty acids in synaptosomal membranes. Association of PHT with synaptosomal membranes caused the greatest change when the fluorescent probe was located at the 6-carbon position of N-(anthroyloxy)stearic acid and was incorporated into the membranes. Phenytoin and 6-(anthroyloxy)stearic acid compete for high affinity binding regions which are probably lipid domains within the membrane. Phenytoin has a weaker association with the sites than the fluorescent fatty acids. Divalent cations, e.g. Mg²⁺ or Ca²⁺, are required to observe maximal change in polarization of fluorescence of fatty acid probes in the presence of PHT. It is proposed that the membrane lipid bilayer reorganizes to accommodate exogenous compounds, such as phenytoin or the fatty acid probe in order to permit the most efficient packing of lipids. This reorganization of the lipid bilayer may influence membrane enzyme activities and ion channels.     

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.     

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.     

Comparative study of the lateral motion of extrinsic probes and anthracene-labelled constitutive phospholipids in the plasma membrane of Chinese hamster ovary cells

9-(2-Anthryl)-nonanoic acid is a new photoactivatable fluorescent probe which has been designed for the study of the lateral diffusion and distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. This anthracene fatty acid can be incorporated metabolically into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of Chinese hamster ovary (CHO) cells in culture. The diffusion coefficient of intrinsic lipids in the plasma membrane of these eukaryotic cells can thus be measured using the fluorescence recovery after a photobleaching technique, since illumination of the fluorescent anthracene groups yields non-fluorescent photodimers. For the sake of comparison, the extrinsic lipophilic probes 5-(N-hexadecanoyl)-aminofluorescein, 12-(9-anthroyloxy)-stearic acid, 9-(2-anthryl)-nonanoic acid and a synthetic anthracene-phosphatidylcholine were also used to label the plasma membrane of CHO cells. The diffusion coefficients for the extrinsic and intrinsic probes ranged over 1 - 2 x 10(-9) cm2/s. Small but significant differences were observed between the various probes reflecting differences they exhibit in size and polarity. All the extrinsic probes were free to diffuse, with a mobile fraction close to 100%. In contrast, a fractional recovery of only 75% was observed for the intrinsic anthracene-labelled phospholipids, suggesting that the anthracene fatty acid was metabolically incorporated into membrane lipid regions which were inaccessible to the extrinsic probes.     

Formation of (n-9) and (n-7) cis-monounsaturated fatty acids in seeds of higher plants

The relative abundance of (n-9) and (n-7) isomers in the monounsaturated fatty acids of seed lipids has been determined for selected plants in order to assess the biosynthetic reactions involved in their formation. 9 Desaturation of stearic acid to (n-9) octadecenoic acid is almost exclusively operative in the formation of monounsaturated fatty acids in the seeds of Helianthus annuus, Glycine max and Brassica napus, cv. Quinta and Erglu, in which chain elongation of monounsaturated fatty acids terminates at the level of an 18 carbon chain. 9 Desaturation of palmitic acid is a minor yet significant pathway in the seeds of Sinapis alba and Brassica napus, cv. Rapol and Tira, in which chain elongation of monounsaturated fatty acids occurs extensively beyond the 18 carbon chain. In each of these seeds, both (n-9) and (n-7) octadecenoic acids formed are subsequently elongated to icosenoic acids. However, elongation of the (n-7) isomer is terminated at the level of a 20 carbon chain, whereas the (n-9) icosenoic acid is selectively elongated to docosenoic acid and even up to tetracosenoic acid in Sinapis alba. 9 Desaturation of palmitic acid followed by elongation to (n-7) octadecenoic acid occurs to a minor extent in the seeds of Tropaeolum majus. Only the (n-9) octadecenoic acid, and not its (n-7) isomer, is elongated to icosenoic and docosenoic acids.     

High-resolution 13C nuclear magnetic resonance studies of small unilamellar vesicles containing glycophorin A

Glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, has been incorporated in small unilamellar vesicles containing phosphatidylcholine and phosphatidylethanolamine in varying proportions. Hydrocarbon chains of these two lipids have been selectively enriched with ¹³C and ¹³C-NMR spin relaxation parameters have been monitored in the presence and absence of protein. Perturbations to ¹³C line-widths and spin-lattice relaxation times are found to be small and consistent with relatively weak interactions. The perturbations, though small, show some specificity. The carbonyl carbons in both phosphatidylcholine and phosphatidylethanolamine are broadened, but in addition the olefinic carbons in phosphatidylethanolamine are broadened.     

Changes in the oceanic 13C/12C ratio during the last 140 000 years: High-latitude surface water records

Detailed records of the carbon and oxygen isotopic ratios of Neogloboquadrina pachyderma are compared between nine high-latitude sediment cores, from the Northern and Southern Hemispheres, covering the last 140 000 yrs. The strong analogies between the δ¹³C records permit to define a δ¹³C stratigraphic scale, with three clear cut transitions simultaneous with the oxygen isotopic transitions 6/5 (125 kyrs.), 5/4 (65 kyrs.), and 2/1 (13 kyrs.). The δ¹³C records of N. pachyderma in the high-latitude cores, which follow the changes in δ¹³C of the surface water TCO₂ near areas of deep water formation present trends similar to the benthic foraminifera δ¹³C records in cores V19–30 and M12-392, although amplitudes of the isotopic shifts are different. This implies that a large part of the observed variations represents global changes in the carbon distribution between biosphere and ocean.The ¹³C/¹²C ratios of N. pachyderma in the North Atlantic cores display larger regional variations at 18 kyrs. B.P. than at present. To explain these differences, we have plotted the 18 kyrs. B.P. δ¹³C values of N. pachyderma from 17 cores distributed N of 40°N. Comparison with published surface water temperature distribution at 18 kyrs. B.P. indicates that a strong divergent cyclonic cell, centered approximatively 55°N and 15°W, was active during most of the last ice-age maximum This hydrology, analogous to the present Weddell Sea, explains the published evidences of bottom water formation, if located on the northern flank of the gyre, and the strong polar front on the southern flank, probable location of intermediate water formation.