能化态线粒体内膜脂流动性与能量偶联活性

本文从观察温度的影响出发,探讨了鼠肝线粒体内膜体,在琥珀酸氧化建立跨膜质子电化学梯度（ΔμＨ＾＋）时,膜脂双分子层中ＤＰＨ荧光偏振值（ｒ）的变化与膜能量偶联活性之间的相互关系。结果表明,１５￣３５℃温度内,能化引起ｒ值变化趋势相似,ｒ值变化速率随温度升高而增加,但与温度对ｒ值影响相比只是在较小的范围内变动。另一方面,１５￣３０℃温度内,随温度升高质子回漏速率加快,ＲＣＲ值和ＡＤＰ／Ｏ比值下降,但跨     

用荧光素磷脂酰乙醇胺直接测定线粒体内膜外表面pH

由磷脂极性头部基团和结合水分子组成的氢键网络有利于质子沿膜表面侧向快速扩散。因而在线粒体氧化磷酸化过程中,与呼吸链电子传递相偶联的跨膜转运质子是否滞留于线粒体内膜外表面即成为一个值得探讨的课题。本文采用荧光素磷脂酰乙醇胺标记于线粒体内膜外表面,首次建立了直接测定线粒体内膜外表面ｐＨ的方法。标记后,线粒体内膜体呼吸控制率,呼吸链电子传递驱动的质子跨膜转移活性及ＡＴＰ合成活性下降了近２８．０％,１１．     

心磷脂和线粒体内膜

心磷脂是构成线粒体内膜的主要磷脂之一,约75～90%的心磷脂分布在线粒体内膜脂双层的基质面,是线粒体内膜的特征性磷脂。心磷脂使线粒体内膜具有良好的流动性,利于呼吸链各复合物在膜脂双层中的侧向扩散。呼吸链的复合物与心磷脂特异结合才能表现其活性。在一定的条件下,心磷脂亦能形成六角形(?)相,这种多形性特点对离子转运和电子传递有重要意义。     

耗竭性运动对大鼠骨骼肌线粒体内膜的影响

观察SD大鼠一次急性运动至力竭后骨骼肌线粒体内膜流动性、NADH-CoQ还原酶及ATP酶活性变化．结果显示,大鼠骨骼肌线粒体内膜微粘度较安静时显著增高,线粒体内膜NADH-CoQ还原酶和ATP酶活性分别较安静时下降34．2％和46．2％．研究提示,耗竭性运动后大鼠骨骼肌线粒体呼吸链内膜分子动力学和呼吸链酶组分活性变化,可能是运动性疲劳重要的膜分子特征．     

运动性疲劳状态下大鼠心肌线粒体内膜变化的研究

采用递增负荷力竭性运动模型,观察了Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠急性运动至力竭后心肌线粒体内膜流动性、ＮＡＤＨ－ＣｏＱ还原酶及ＡＴＰ酶活性的变化。结果表明,大鼠心肌线粒体内膜荧光偏振值较安静时显著增高（Ｐ＜０．０１）,示膜流动性降低。线粒体内膜ＮＡＤＨ－ＣｏＱ还原酶和肌线粒体内膜功能改变,其膜流动性和呼吸链酶活性变化,可能是运动性疲劳的重要膜分子制之一。     

能化态与线粒体及其内膜体膜表面局部脱水

荧光探针ＤＰＥ（Ｄｉｐａｌｍｉｔｏｙｌ－Ｎ－ＤａｎｓｙｌＰｈｏｓｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ）标记于鼠肝线粒体及其内膜体膜表面,以测定其膜表面介电常数（ε）的变化与膜能化态的关系．在含有鱼藤酮的线粒体或其内膜体悬液中,加入琥珀酸,使膜处于能化态,可使膜表面。值分别下降１１％和２０％,分别再加ＫＣＮ,ＣＣＣＰ或Ｎｉｇｅｒｉｃｉｎ均能使ε值回升９％;若在上述悬液中,先加ＫＣＮ或ＣＣＣＰ,然后加入琥珀酸,则膜表面ε值无变化。若在线粒体或其内膜体悬液中,先加入ＣＣＣＰ或Ｎｉｇｅｒｉｃｉｎ,两者可使膜表面ε值升高１２％,其后加入鱼藤酮或琥珀酸等,则ε值均无变化,说明呼吸链抑制剂或解偶联剂抑制或解除膜的能化态,均可使膜表面ε值不发生变化。在另一组实验中,先加ＫＣＮ后,再加ＡＴｈ,使线粒体内膜处于能化态,表面ε值下降８％,此时再加寡霉素,则膜表面ε值相反上升１０％。这些实验事实均证明,线粒体膜处于能化态时,ε值下降,解除给化态,使ε值又上升。为能化态与线粒体膜表面水化力下降的相互关系提供了新信息．也为质子泵引起膜表面质子化,进而引起表面脱水,驱动膜融合的理论模型提供了新的证据。     

呼吸链电子传递与线粒体内膜流动性

本文报告用稳态荧光各向异性研究呼吸链底物氧化启动电子传递时线粒体内膜的流动性变化。结果表明呼吸链底物使内膜流动性增大,磷脂分子脂酰链的活动度增加(从2位碳到12位碳)。FCCP(p-trifluoromethoxycarbonylcyanide phenylhydrazone)取消H⁺梯度时底物仍可使内膜流动性增加,提示流动性的增加与底物氧化启动的电子沿呼吸链的传递过程密切相关。     

呼吸链底物和抑制剂对线粒体内膜流动性的影响

用DPH和ANS标记大鼠肝线粒体内膜,以稳态荧光偏振法,研究了呼吸链底物和抑制剂对内膜流动性的影响。1.苹果酸+谷氨酸、琥珀酸分别为底物,均能引起内膜流动性增加。2.琥珀酸对含心磷脂的脂质体的膜流动性无影响。3.在鱼藤酮存在的条件下,苹果酸+谷氨酸对内膜流动性的增加作用消失,但琥珀酸的作用仍然存在。有氰化钾时则琥珀酸的作用消失。4.不论外加底物存在与否,鱼藤酮使内膜的流动性下降,而氰化钾则使之增加。抗霉素A亦可使内膜的流动性增加。上述结果表明:线粒体内膜流动性与其功能密切相关。电子沿呼吸链传递使线粒体内膜流动性增加,这种变化可能与呼吸链成分的氧化还原态有关。     

大鼠急性应激时肝线粒体质子跨膜转动活性的调控

大鼠烫伤早期（烫伤后３０ｍｉｎ）,肝线粒体质子加电子传递速度均加快,线粒体能化态跨膜电位降低（均以琥珀酸为底物）,线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。     

大鼠急性应激时肝线粒体质子跨膜转运活性的调控

大鼠烫伤早期(烫伤后30min),肝线粒体质子和电子传递速度均加快,线粒体能化态跨膜电位降低(均以琥珀酸为底物),线粒体膜脂流动性降低。皮下注射去甲肾上腺素后也有上述现象发生。推测急性应激通过儿茶酚胺类作用于肝细胞,导致线粒体内膜有序性增强所致。     

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).     

Influence of DPH on the structure and dynamics of a DPPC bilayer

We have conducted extensive molecular dynamics (MD) simulations together with differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments to quantify the influence of free 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes on the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer. Atomistic MD simulations show that in the membrane-water interface the influence of DPH is minor, whereas in the acyl-chain region DPH gives rise to major perturbations. In the latter case, DPH is found to influence a wide range of membrane properties, such as the packing and ordering of hydrocarbon tails and the lateral diffusion of lipid molecules. The effects are prominent but of local nature, i.e., the changes observed in the properties of lipid molecules are significant in the vicinity of DPH, but reduce rapidly as the distance from the probe increases. Long-range perturbations due to DPH are hence not expected. Detailed DSC and (2)H NMR measurements support this view. DSC shows only subtle perturbation to the cooperative behavior of the membrane system in the presence of DPH, and (2)H NMR shows that DPH gives rise to a slight increase in the lipid chain order, in agreement with MD simulations. Potential effects of other probes such as pyrene are briefly discussed.     

The membrane fluidity concept revisited by polarized fluorescence spectroscopy on different model membranes containing unsaturated lipids and sterols

Quantitative analysis of time-resolved anisotropy measurements of DPH or TMA-DPH in lipid vesicles yields more than one mathematically correct solution. The solutions differ with respect to the average orientation and to the reorientational dynamics of the probe molecules in the bilayer. This leads to quite opposite results regarding the effects of cholesterol on membrane fluidity. One solution predicts an increase in fluidity, the other a decrease. Angle-resolved fluorescence depolarization (AFD) measurements of probes in oriented lipid bilayers enable determination of the average orientation of the probes in the bilayer and, if the fluorescence decay function is known, of the reorientational dynamics. Analysis of AFD measurements of DPH and TMA-DPH show that increasing unsaturation leads to a decrease in molecular order and a decrease in reorientational dynamics (= fluidity) of the probes. At temperatures above the phase transition of the lipids, the addition of cholesterol causes an increase in molecular order and an increase in reorientational dynamics (= fluidity). The plant sterol stigmaterol, which is structurally closely related to cholesterol, has different effects than cholesterol. The effects vary with the structure of the surrounding lipids. The membrane fluidity concept as it was originally proposed by Chapman attempts to describe the structural and dynamic properties of lipids in a membrane using one single parameter indicated as 'membrane fluidity'. Our results show that it is necessary to distinguish between structural parameters describing molecular order and motion parameters describing molecular dynamics, thus supporting a similar suggestion by Seelig and Seelig. In order to be useful, the membrane fluidity concept has to be limited to the parameters describing molecular dynamics.     

Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures

To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.     

Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

We derive equations that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of electron spin resonance (ESR) nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. We make use of an assumption used by us in an earlier paper. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. We consider cases where the bilayer is either in a single homogeneous phase or in a two-phase region. We apply our equations to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase, both for T greater than Tc. The following conclusions were derived: (i) Ca2+-ATPase oligomers are "randomly" distributed, for the concentrations studied, in the fluid phase. (ii) There is no fixed stoichiometric ratio of "boundary" lipids and oligomers. (iii) Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. Finally, we apply our equations to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T less than Tc. We find that the results reported are in accord with the predictions of the model. In the Appendix, we show that an analytical expression for probabilities used by us is in very good agreement with the results of computer simulation.     

The interaction of cannabinoid receptor agonists, CP55940 and WIN55212-2 with membranes using solid state 2H NMR

Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using (2)H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ(9)-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2' and 16' positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer. Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-(2)H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime ((2)H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type (2)H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.     

Interactions of the Anticancer Drug Tamoxifen with Lipid Membranes

Interactions of the hydrophobic anticancer drug tamoxifen (TAM) with lipid model membranes were studied using calcein-encapsulated vesicle leakage, attenuated total reflection Fourier transform infrared (FTIR) spectroscopy, small-angle neutron scattering (SANS), atomic force microscopy (AFM) based force spectroscopy, and all-atom molecular dynamics (MD) simulations. The addition of TAM enhances membrane permeability, inducing calcein to translocate from the interior to the exterior of lipid vesicles. A large decrease in the FTIR absorption band’s magnitude was observed in the hydrocarbon chain region, suggesting suppressed bond vibrational dynamics. Bilayer thickening was determined from SANS data. Force spectroscopy measurements indicate that the lipid bilayer area compressibility modulus K_A is increased by a large amount after the incorporation of TAM. MD simulations show that TAM decreases the lipid area and increases chain order parameters. Moreover, orientational and positional analyses show that TAM exhibits a highly dynamic conformation within the lipid bilayer. Our detailed experimental and computational studies of TAM interacting with model lipid membranes shed new light on membrane modulation by TAM.     

Modulation of reconstituted pig kidney Na+/K(+)-ATPase activity by cholesterol in endogenous lipid vesicles: role of lipid domains

Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme.     

Effect of aminophospholipid glycation on order parameter and hydration of phospholipid bilayer

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.