Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Lecithin:cholesterol acyltransferase regulation. II. Effect of fluidity of egg phosphatidylcholine vesicles

The regulation of human plasma lecithin:cholesterol acyltransferase (LCAT) by changes in bilayer fluidity of substrate egg phosphatidylcholine (egg PC) unilamellar vesicles was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity was decreased by adding up to 20% cholesterol or increased by adding up to 10% egg 2-lysophosphatidylcholine (lysoPC). The fluidizing effect of lysoPC was suppressed by the addition of cholesterol. LCAT activity with 10% cholesterol vesicles was decreased by adding 5% lysoPC, yet activity with 5% cholesterol vesicles was unaffected by adding 5% lysoPC. This difference may be explained by a balance between the known LCAT inhibitory effect of lysoPC and its ability to increase bilayer fluidity and thereby increase LCAT activity. LCAT esterification of up to 37% of vesicle cholesterol failed to alter the lysoPC/cholesterol balance sufficiently to influence activity in this system. The findings of our studies are in keeping with modulation of LCAT activity by bilayer fluidity, but fluidity changes caused by enzyme action are not sufficient to regulate that activity.     

Radiation-induced changes in permeability in unilamellar phospholipid liposomes

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of alpha-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to gamma radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.     

Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K⁺ so that the membrane voltage approaches the Nernst potential for K⁺. With constant external K⁺ concentration, the time course of internal K⁺ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K⁺ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.     

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.     

Cholesterol in sarcoplasmic reticulum and the physiological significance of membrane fluidity.

Vesicles of sarcoplasmic reticulum from rabbit muscle can be loaded with cholesterol to at least 20 mol% with respect to endogenous sarcoplasmic-reticulum phospholipid without effect on the ATPase activity at 32 degrees C. This applies both to sarcoplasmic-reticulum vesicles in which the ATPase activity is stably coupled to Ca2+ accumulation, and to sarcoplasmic-reticulum vesicles in which the sarcoplasmic-reticulum ATPase is activated severalfold by fully uncoupling the enzyme from net Ca2+ accumulation. Since the incorporation of cholesterol causes a large decrease in fluidity of sarcoplasmic-reticulum phospholipid bilayer, these results for sarcoplasmic reticulum raise the more general question of whether bilayer fluidity is important in modulating the function of membrane proteins under physiological conditions as is widely assumed, or whether the function of membrane proteins may be effectively buffered under normal operating conditions against changes in bilayer fluidity due to extraneous agents.     

Lipid organization regulates annexin A2 Ca2 +-sensitivity for membrane bridging and its modulator effects on membrane fluidity

Annexin A2 (AnxA2) is a phospholipid binding protein that has been implicated in many membrane-related cellular functions. AnxA2 is able to bind different acidic phospholipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI2P). This binding is mediated by Ca^2 +-dependent and Ca^2 +-independent mechanisms. The specific functions of annexin A2 related to these two phospholipids and the molecular mechanisms involved in their interaction remain obscure. Herein we studied the influence of lipid composition on the Ca^2 +-dependency of AnxA2-mediated membrane bridging and on membrane fluidity. Membrane models of ten different lipid compositions and detergent-resistant membranes from two cellular sources were investigated. The results show that the AnxA2-mediated membrane bridging requires 3 to 50 times less calcium for PS-membranes than for PI2P-membranes. Membrane fluidity was measured by the ratiometric fluorescence parameter generalized polarization method with two fluorescent probes. Compared to controls containing low phospholipid ligand, AnxA2 was found to reduce the membrane fluidity of PI2P-membranes twice as much as the PS-membranes in the presence of calcium. On the contrary, at mild acidic pH in the absence of calcium AnxA2 reduces the fluidity of the PS-membranes more than the PI2P-membranes. The presence of cholesterol on the bilayer reduced the AnxA2 capacity to reduce membrane fluidity. The presented data shed light on the specific roles of PI2P, PS and cholesterol present on membranes related to the action of annexin A2 as a membrane bridging molecule during exocytosis and endocytosis events and as a plasma membrane domain phospholipid packing regulator.     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

Lipid composition and fluidity in the jejunal brush-border membrane of spontaneously hypertensive rats. Effects on activities of membrane-bound proteins

The lipid composition and fluidity of jejunal brush-border membrane vesicles (BBMV) have been studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. The activities of both Na⁺-dependent D-glucose cotransport and Na⁺-H⁺ antiport have also been determined. A significant increase in the level of free cholesterol was observed in jejunal BBMV from SHR compared to WKY rats. Since phospholipid values did not change in either group of animals, a significant enhancement in the free cholesterol/phospholipid ratio was observed in SHR. A decrease in the levels of phosphatidylethanolamine together with an increase in the values of phosphatidylserine was observed in hypertensive rats. Although the content of phosphatidylcholine (PC) and sphingomyelin (SM) was not singificantly altered in SHR, the ratio PC/SM significantly increased in these animals when compared to WKY rats. The major fatty acids present in bursh-border membranes prepared from SHR and WKY rats were palmitic (160), stearic (180), oleic (181, n-9) and linoleic (182, n-6), and the fatty acid composition was not modified by the hypertension. A decreased fluorescence polarization, i.e., increased membrane fluidity, was observed in SHR, which was not correlated to the increased ratio of cholesterol/phospholipid found in the brush-border membrane isolated from these animals. These structural changes found in SHR were associated to an enhancement in both Na⁺-dependent D-glucose transport and Na⁺-H⁺ antiport activity in the jejunal BBMV of SHR.Abbreviations BBMV brush-border membrane vesicles - DPH 1,6-diphenyl-1,3,5-hexatriene - FC free cholesterol - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin - SHR spontaneously hypertensive rat - p steady-state fluoroscence polarization - r_s steady-state fluorescence anisotropy - WKY Wistar Kyoto     

Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers

To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant was then labeled with acrylodan, an environment-sensitive fluorescence probe. The fluorescence increased and blue-shifted upon binding to lipid vesicles, consistent with a change into a more hydrophobic, i.e., lipid, environment. This acrylodan-labeled cholesterol oxidase was used to explore the pH, ionic strength, and headgroup dependence of binding. Between pH 6 and 10, there was no significant change in binding affinity. Incorporation of anionic lipids (phosphatidylserine) into the vesicles did not increase the binding affinity nor did altering the ionic strength. These experiments suggested that the interactions are primarily driven by hydrophobic effects not ionic effects. Using vesicles doped with either 5-doxyl phosphatidylcholine, 10-doxyl phosphatidylcholine, or phosphatidyl-tempocholine, quenching of acrylodan fluorescence was observed upon binding. Using the parallax method of London [Chattopadhyay, A., and London, E. (1987) Biochemistry 26, 39-45], the acrylodan ring is calculated to be 8.1 +/- 2.5 A from the center of the lipid bilayer. Modeling the acrylodan-cysteine residue as an extended chain suggests that the backbone of the loop does not penetrate into the lipid bilayer but interacts with the headgroups, i.e., the choline. These results demonstrate that cholesterol oxidase interacts directly with the lipid bilayer and sits on the surface of the membrane.     

Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids

The polyphenolic structures of flavonoids and isoflavonoids confer them with the ability to scavenge free radicals and to chelate transition metals, a basis for their potent antioxidant abilities. Another possible contributory mechanism toward their antioxidant activities is their ability to stabilize membranes by decreasing membrane fluidity. In this study, the effects of representative flavonoids, isoflavonoids, and their metabolites on membrane fluidity and their preferential localization in the membrane were investigated using large unilamellar vesicles (LUVs) as the membrane models. These results were compared with those of cholesterol and alpha-tocopherol. Changes in fluorescence anisotropy values for a series of n-(9-anthroyloxy) fatty acid probes (n = 6, 12, 16) upon addition of the test compounds were used to monitor alterations in membrane fluidity at graded depths in lipid bilayer. The results of the study suggest that the flavonoids and isoflavonoids, similar to cholesterol and alpha-tocopherol, partition into the hydrophobic core of the membrane and cause a dramatic decrease in lipid fluidity in this region of the membrane. Localization of flavonoids and isoflavonoids into the membrane interiors and their resulting restrictions on fluidity of membrane components could sterically hinder diffusion of free radicals and thereby decrease the kinetics of free radical reactions.     

消炎痛对猪胃H~ /K~ -ATP酶质子转运功能的抑制

作为猪胃H~ /K~ -ATPase的非竞争性抑制剂,消炎痛明显抑制H~ /K~ -ATPase泡囊的质子转运功能,造成质子泄漏。在0.15 mg/ml蛋白浓度下,4%的消炎痛结合于H~ /K~ -A- TPase泡囊上。它能渗入膜脂相并显著降低膜的流动性,并使H~ /K~ -ATPase内源荧光受到淬灭。从实验结果看来,消炎痛对猪胃H~ /K~ -ATPase质子转运功能的抑制来自对酶蛋白和膜结构影响两个方面,而非仅抑制酶蛋白本身的功能。     

Lecithin:cholesterol acyltransferase regulation. Effect of fluidity of dimyristoylphosphatidylcholine vesicles

The regulation of lecithin:cholesterol acyltransferase by changes in phospholipid bilayer fluidity was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity of dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles was decreased by the addition of up to 20% (mol/mol) cholesterol and increased by the addition of up to 10% (mol/mol) lysoDMPC. When both cholesterol and lysoDMPC are present in the bilayer, their individual effects on fluidity are altered. These changes can be explained by complex formation between cholesterol and phospholipid as in the model of Presti et al. (Presti, F.C., Pace, R.J. and Chan, S.I. (1982) Biochemistry 21, 3831-3335). Lecithin:cholesterol acyltransferase activity with these vesicles as substrates was measured to determine whether activity can be modulated by the fluidity changes of the bilayer on which the enzyme acts. When 10% lysoDMPC, a known lecithin:cholesterol acyltransferase inhibitor, is added to the vesicles, inhibition of activity is observed. When 7.5% lysoDMPC is added to vesicles which contain either 5 or 10% cholesterol, lecithin:cholesterol acyltransferase activity increases. This increase in lecithin:cholesterol acyltransferase activity due to vesicle-fluidity increase is sufficient to overcome the decrease in activity due to lecithin:cholesterol acyltransferase inhibition. This is the first report of the ability of lysoDMPC to increase lecithin:cholesterol acyltransferase activity.     

Coenzyme Q-3 as an antioxidant. Its effect on the composition and structural properties of phospholipid vesicles.

Coenzyme Q-3 incorporated into the lipid bilayer at physiological concentration provided an 80% inhibition of the lipid peroxidation induced by ferrous ions. In coenzyme Q-containing vesicles, the fluorescence lifetime and the fluorescence anisotropy decay of the probe, 1,6-diphenyl-1,3,5-hexatriene, were measured in order to find out if the presence of the quinone can cause variations in the membrane organization. Our data show that two distinct populations of the probe were present and that both populations were available to quenching by coenzyme Q. The overall effects of coenzyme Q on the static and dynamic properties of the model membranes were: a very small effect in the ordering of the fatty acid chain, and a more noticeable decrease of the probe correlation time and, therefore, an increase in membrane fluidity at increasing quinone concentration. When vesicles were peroxidized in the absence of the coenzyme Q, the fluidity markedly decreased; in its presence, the fluidity was nearly unchanged. The results suggest that the antioxidant properties of coenzyme Q can be ascribed to its ability to react with free radicals. The effect on the fluidity of the lipid bilayer might imply that a requisite for a molecule to act as an efficient antioxidant could be its ability to readily diffuse within the membrane.     

Relationship of alcohol-induced changes in Mg2+-ATPase activity of rabbit intestinal brush border membrane with changes in fluidity of its lipid bilayer

We examined the effects of seven n-alkyl alcohols (from n-butyl to n-undecyl alcohol), isoamyl alcohol and benzyl alcohol on the activity of membrane enzyme Mg²⁺-ATPase of the rabbit small intestinal brush border membrane. Their relationships with the changes in the fluidity of the membrane lipid bilayer were examined through studies on the fluorescence anisotropies of diphenylhexatriene (DPH) and its ionic derivatives. Good linear correlations were found both between the partition coefficients of the alcohols and their concentrations causing similar decreases in the activity of Mg²⁺-ATPase and between their partition coefficients and the alcohol-induced changes in fluorescence anisotropies. Within the concentration range of the alcohols tested, including isoamyl alcohol and benzyl alcohol, the decreases in activity of the membrane enzyme Mg²⁺-ATPase clearly corresponded with the decreases in fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane. From these findings, one possible explanation is that inhibition of this enzyme by the alcohols is due to perturbation of the lipid bilayer of the brush border membrane.The authors thank M. Takano, PhD and Y. Tomita, PhD, Department of Pharmacy, University Hospital of Kyoto University, for instruction in preparation of the brush border membrane vesicles. This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture (05671795 and 06304044) and Takeda Science Foundation.     

Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox

Cholesterol is a key lipid in mediating the enzyme activity or signaling pathway of many proteins on the plasma membrane in mammalian cells. In this report, we demonstrate for the first time that after overexpressing caveolin-1, the plasma membrane cholesterol level was decreased by about 12% and 30% for doxorubicin-sensitive and doxorubicin-resistant Hs578T breast cancer cells, respectively. However, the total cholesterol level in both cell lines was increased by about 10%. By measuring fluorescence and flow cytometry using the fluorescence dyes 1,6-diphenyl-1,3,5-hexatriene and Merocyanine 540, we found that overexpressing caveolin-1 resulted in a similar increase in membrane fluidity and loosening of lipid packing density as cholesterol depletion by 1 mM methyl-beta-cyclodextrin (MbetaCD) or 2-hydroxypropyl-beta-cyclodextrin (HbetaCD). Moreover, we found that the transport activity of P-gp was significantly inhibited by 1 mM MbetaCD or HbetaCD, which is also similar to the inhibitory effect of caveolin-1 overexpression. Our data demonstrate for the first time that the reduction of the plasma membrane cholesterol level induced by overexpressing caveolin-1 may indirectly inhibit P-gp transport activity by increasing plasma membrane fluidity.     

Effects of bilayer cholesterol on human erythrocyte hexose transport protein activity in synthetic lecithin bilayers

In this study, we describe the effects of altered bilayer cholesterol content on reconstituted, protein-mediated sugar transport. The system used was the human erythrocyte sugar transporter (band 4.5) reconstituted into the bilayers of large unilamellar vesicles. Vesicle preparations were formed from synthetic lecithins whose bilayer cholesterol content ranged from 0 to 50 mol %. Transport was measured by microturbidimetric analysis over the temperature range of 0-65 degrees C while bilayer physical state was characterized by differential scanning calorimetry. Reconstituted transport activity was irreversibly lost between 62 and 65 degrees C. The Km for reconstituted transport was found to increase only slightly with increasing temperature and was not systematically affected by bilayer cholesterol content. The most striking observation of this study is that over certain critical cholesterol concentrations, as little as a 2.5% change in bilayer cholesterol can result in as much as a 100-fold change in Vmax per reconstituted protein. Our findings run counter to the view that increasing bilayer cholesterol content monotonically transforms a membrane into a state of "intermediate fluidity". Abrupt, cholesterol-induced bilayer reorganizations occurring at 15-20 and 30 mol % bilayer cholesterol are markedly reflected in altered sugar transport rates. Increasing the cholesterol content of crystalline distearoyllecithin bilayers inhibits the activity of the reconstituted transporter. It is apparent from these studies that bilayer "fluidity" is neither the sole nor a major determinant of the Indeed, we find the effect of cholesterol on transport activity is independent of its ability to fluidize membranes.     

Use of phospholipid transfer protein as a probe to study the lipid dynamics and alkaline phosphatase activity in the brush border membrane of human term placenta

Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver-Burk plot, which showed decreased V(max) and K(M). The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreased in the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences.