DETERMINATION OF HUMAN EYE LENS MEMBRANE STRUCTURE · PDF fileSTRUCTURE BY X-RAY DIFFRACTION ANALYSIS ROBERT F. JACOB, ... this study indicate that the lens fiber cell plasma membrane

Introduction

The ocular crystalline lens is a transparent tissuethat by means of altering its shape provides theability for visible light to be transmitted unimpededinto the eye and focused onto the retina for propervisual sensation. The lens is an encapsulatedstructure consisting almost entirely of a largenumber of rigid, elongated cells known as lens fibers or fiber cells. These cells are produced via theterminal differentiation of a monolayer of epithelialcells located just beneath the anterior lens capsule[1]. This process begins early in embryogenesis andcon-tinues throughout life, resulting in thedeposition of one layer of fiber cells upon another.As new layers of fiber cells are produced, existinglayers are displaced toward the center of the lens.Fiber cell layers that are compacted into the center of the lens during embryonic development and through

adulthood comprise a region known as the lensnucleus; fiber cells peripheral to the nucleus,including the newly formed and metabolically active fiber cells, define the lens cortex.

During their progressive displacement towardsthe lens nucleus, fiber cells lose all subcellularorganelles which would scatter light and thus impairvision [2]. Consequently, plasma membranebecomes essentially the only organelle of the lens[1]. The fiber cell plasma membrane is unique in that it contains only trace amounts of polyunsaturatedfatty acid [3], very high concen-trations of a 26kDawater channel protein known as main intrinsicprotein (MIP or MP26) [4], and, in the human lens, aphospholipid composition of over 50% sphingo-myelin and sphingomyelin derivatives [5, 6]. Inaddition, human lens fiber cell plasma membranecontains a relative concentration of cholesterol thatis the highest found in nature [7]. The cholesterol tophospholipid (C/P) mole ratio ranges from 1 to 2 in

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The Rigaku Journal

Vol. 17/ number 1/ 2000

CONTRIBUTED PAPERS

DETERMINATION OF HUMAN EYE LENS MEMBRANESTRUCTURE BY X-RAY DIFFRACTION ANALYSIS

ROBERT F. JACOB, RICHARD J. CENEDELLAH, AND R. PRESTON MASON*

Membrane Biophysics Laboratory, Cardiovascular and Pulmonary Research Institute, MCP Hahnemann University School of Medicine, NeuroscienceGraduate Program, Allegheny Campus, Pittsburgh, PA 15212-4772

HDepartment of Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, MO 63501

The molecular structure of human ocular lens fiber cell plasma membranes was directlyexamined using small angle X-ray diffraction approaches. A distinct biochemical feature of thesemembranes is the high relative levels of free cholesterol; the mole ratio of cholesterol tophospholipid (C/P) measured in these membranes ranges from 1 to 4. The organization ofcho lesterol in this membrane system is not well understood, however. In this study, the structure ofplasma membrane samples isolated from nuclear (3.3 C/P) and cortical (2.4 C/P) regions of humanlenses was evaluated with X-ray diffraction approaches. Meridional diffraction patterns obtainedfrom the oriented membrane samples demonstrated the presence of an immiscible cholesteroldomain with a unit cell periodicity of 34.O A, consistent with a cholesterol monohy drate bilayer.The dimensions of the sterol-rich domains remained constant over a broad range of temperatures (5-20EC) and relative humidities (31-97%). In contrast, dimensions of the sur rounding sterol-poorphase were significantly affected by experimental conditions. Similar structural features wereobserved in membranes reconstituted from fiber cell plasma mem brane lipid extracts. The results ofthis study indicate that the lens fiber cell plasma membrane is a complex structure consisting ofseparate sterol-rich and -poor domains. Maintenance of these separate domains may be required forthe normal function of lens fiber cell plasma membrane and may interfere with the cataractogenicaggregation of soluble lens proteins at the membrane surface.

*Send correspondence to: R. Preston Mason, Ph.D., Al legheny GeneralHospital, 320 East North Ave., 2-ST, Pitts burgh, PA 15212-4772; E-mail: [email protected]

the cortex to 3 to 4 in the nucleus [8, 9]. In contrast,plasma membranes of typical eukaryotic cells haveC/P mole ratios between 0.5 and 1.0 [10].

Despite an advanced understanding of the lipidcomposition of the human ocular lens, there is littleinformation about the structural organization of thelens plasma membrane. How does a membraneaccommodate 3 to 4 cholesterol molecules per onephospholipid? The presence of cholesterol at suchextremely high relative concentrations in lensmembrane has led to the proposal that the fiber cellplasma membrane is “a mosaic of phospholipidbilayer and cholesterol patches” [8]. A number ofindependent studies employing other bio-logicalmembrane systems give credence to this possi-bility. Using model membrane systems, it has beendetermined that cholesterol tends to aggregate intoclusters at C/P mole ratios in excess of 0.3 [11], andC/P mole ratios greater than 1.0 (i.e., 50 mole%sterol) can yield pure cholesterol phases [12]. Inwell-defined lipid monolayer systems, the additionof cholesterol produces lateral sterol domains, ascharacterized by microscopy approaches [13-15].The formation of distinct cholesterol domains hasalso been observed in various membrane bilayersystems. Using small angle X-ray diffraction, it hasbeen shown that increasing the relative cholesterolcontent to 50% of total phospholipid in modelmembrane bilayers produces an immisciblecholesterol monohy-drate phase with a unit cellperiodicity or d-space of 34.0 D existing within theliquid crystalline lipid bilayer [16]. The periodicityof 34.0 D corresponds to a tail-to-tail cholesterolbilayer as the long axis of cholesterol monohydrateis 17 D in the crystalline state [17]. Recent studies inour laboratories have also provided evidence for theexistence of separate cholesterol domains invascular smooth muscle cell plasma membranesunder diseased, atherosclerotic conditions, a process character-ized by an excessive accumulation ofcholesterol in the vascular wall [18]. In thesediseased membranes, the C/P mole ratio approached1.0, a level that is three-fold higher than in normalmembranes. Small angle X-ray diffraction analysisof these membranes provided evidence for theformation of membranerestricted cholesterol do-mains in vivo under pathologic conditions.

In the present study, small angle X-raydiffraction approaches were used to characterize thestructural organization of cholesterol within fibercell plasma membranes isolated from human ocularlenses. Immiscible cholesterol monohydratedomains were present in lens cortical and nuclearmembrane samples, and remained stable over abroad range of temperatures and relative humidities.Additionally, membrane protein content did notaffect the presence or molecular dimensions of theobserved cholesterol domains. The structurallydistinct cholesterol phase was found to be restrictedwithin the surrounding sterol-poor membranebilayer phase with dimensions that were highlyaffected by temperature, humidity, and proteincontent. These results provide direct evidence for the existence of immiscible cholesterol monohydratedomains in human ocular lens fiber cell plasmamembranes under physiologic-like conditions. Thestructural stability of these domains suggests a well-ordered membrane system that must be conserved tomaintain lens transparency to visible light.

Experimental Procedures

Isolation of Human Lens Plasma Membrane and Lipid

Six normal human lenses were obtained from the National Disease Research Interchange (Philadel-phia, PA). The ages of the lens sample donors ranged from 65 to 77 years. Lenses were decapsulated withconcomitant removal of lens epithelial cells whichadhere to the capsule. The decapsulated lenses wereplaced in a 20 CM2 culture dish containing 8ml of5mM Tris.HCI (pH 7.4), 5 mM EDTA, 10 mM β-mercaptoethanol (buffer A) and stirred on a rotatorymixer at 100 rpm for 2 hr. Under these gentle stirring conditions, the lens cortex separated from thenucleus as clumps of fiber cells. Based on lensweight before and after fractionation, the removedcortex accounted for 48% of total lens volume.

Cortical and nuclear lens regions wereseparately homogenized in 8ml buffer A using aglass Dounce homogenizer. Plasma membraneswere isolated using the methods described byRussell, et al [19]. Briefly, the homogenates werecentrifuged at 10,000 × g for 20min. The pelletswere washed twice with buffer A, extracted twicewith 7M urea in 50 mM Tris"HCI (pH 7.4), and

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extracted twice with 0.1 mM NAOH. The pelletswere then washed once with 5 mM Tris"HCI (pH8.0), 1 mM EDTA, 150 mM NaCl, 0.02% NaN3

(buffer B) and were resuspended in 1.2 to 1.4ml ofbuffer B. Small aliquots were taken for quantitationof protein using a modified Lowry assay [20]. Mainintrinsic protein of 26 kDa and its 22 kDa de-gradation product were overwhelmingly the prin-cipal proteins shown present by SDS-PAGE in thecortical and nuclear membrane preparations (datanot shown). Total lipids from one-half of eachmembrane suspension were Folch extracted aspreviously described [21] and dissolved in chloro-form. Aliquots of these membrane lipid extractswere taken for gas-chromatograph quantitation ofcholesterol [22] and calorimetric assay of phospho-lipid [23]. Compositions of the intact membranesuspensions and reconstituted membrane lipidsamples are shown in Table 1.Preparation of Oriented Intact Lens Plasma MembraneSamples for X-ray Diffraction

Intact lens plasma membrane samples wereoriented for X-ray diffraction analysis as describedpreviously [24]. Briefly, 200µg (phospholipid) ofplasma membrane samples (in buffer B) were loaded into Lucite sedimentation cells. Each sedimentationcell contained an aluminum foil substrate uponwhich the membrane pellets were collected. Themembrane samples were centrifuged in a SorvallAH-629 swinging bucket ultracentrifuge rotor(Dupont Corp., Wilmington, DE) at 35,000 × g for50 min at 5°C. Samples were washed three timeswith diffraction buffer (0.5 mM HEPES, 150 mMNaCl, pH 7.3). After the final washing cycle, thesupernatants were removed and the aluminum foil

substrates, containing the membrane pellets, wereremoved from the sedimentation cells and mountedon curved glass supports. The samples were thenplaced in hermetically sealed brass canisters inwhich temperature and relative humidity werecontrolled during X-ray diffraction experiments.

Preparation of Oriented Lens Membrane Lipid Samplesfor X-ray Diffraction

Aliquots of lens plasma membrane total lipid,initially solubilized in chloroform (see above), were added directly to 13 × 100mm glass test tubes toyield 230µg of phospholipid. The samples werethen dried down under a steady stream of nitrogengas to the sides and bottom of the test tubes whilevortex mixing. Residual solvent was removed undervacuum. A volume of diffraction buffer was addedto each test tube to yield a final phospholipidconcentration of 0.38 mg/ml. Multilamellar vesicleswere formed by vortex mixing the buffer and lensmembrane lipids for 3min at ambient temperature.Volumes yielding the equivalent of 200,ug ofphospholipid for each sample were loaded intosedimentation cells. Oriented membrane multibi-layers were prepared by centrifugation, as describedabove for the intact membrane samples.

Small Angle X-ray Diffraction Analysis

The oriented lens plasma membrane sampleswere aligned at grazing incidence with respect to acollimated, monochromatic X-ray beam (CuKα,λ=1.54 D) produced by a Rigaku Rotaflex RU-200,high-brilliance rotating anode microfocus generator(Rigaku USA, Danvers, MA). The fixed geometrybeamline utilized a single Franks mirror providingnickel-filtered radiation (Kα1 and Kα2 unresolved)at the detection plane. Diffraction data werecollected on a one-dimensional, position-sensitiveelectronic detector (Innovative Technologies, New-buryport, MA), the calibration of which was verified using cholesterol monohydrate crystals. Thesample-to-detector distance used in these experi-ments was 150mm. Representative two-dimensional diffraction patterns for each sample were alsocollected on a two-dimensional Phosphorlmagersystem (Molecular Dynamics, Sunnyvale, CA) at asample-to-detector distance of 70 mm.

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Table 1. Composition of lens membrane samples

Protein (µg/µl)

Cholesterol (µg/µl)

Phospholipid(µg/µl)

C/P(mole)

Intact lens membrane

Cortex 3.07 1.23 1.00 2.42

Nucleus 4.80 1.66 1.00 3.26

Reconstituted lens membrane

Cortex --- 0.263 0.214 2.43

Nucleus --- 0.301 0.181 3.27

The unit cell periodicity, or d-space, of themembrane lipid bilayer is the measured distancefrom the center of one lipid bilayer to the next,including surface hydration. The d-spaces for themembrane multibilayer samples were calculatedusing Bragg's Law:

hλ=2dsin θ

in which h is the diffraction order number, λ is thewavelength of the X-ray radiation (1.54 D), d is themembrane lipid bilayer unit cell periodicity, and θ isthe Bragg angle equal to one-half the angle betweenthe incident beam and scattered beam.

Saturated salt solutions were used to define therelative humidity (RH) levels employed in these X-ray diffraction analyses. The following salt solutions (with associated RH in parentheses) were used in

these experiments: MgCl2A6H20 (33%), Mg(NO3)2A6H20 (52%), K2C4H406AH20 (74%), (NH4)2SO4(79%), NaKC4H4O6A4H20 (87%), Na2C4H4O6A2H20(92%), NH4H2PO4 (93%), K2SO4 (97%).

Results

Analysis of Human Lens Fiber Cell Plasma MembraneStructure

Small angle X-rav diffraction approaches wereused to directly characterize the structural organi-zation of plasma membranes isolated from humanocular lens fiber cells in the presence and absence ofmembrane protein. Representative X-ray diffractionprofiles generated from oriented fiber cell plasmamembranes at 20°C, 92% RH are shown in Figure 1.All samples yielded meridional diffraction patternsconsistent with two structurally distinct membranedomains or phases: a sterol-poor, liquid-crystalline

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Fig. 1. Representative X-ray diffraction patterns obtained from oriented human ocular lens fiber cell plasma membranesamples. Data were collected on a one-dimensional, position-sensitive electronic detector at 20°C and 92% RH. Typicaldiffraction profiles were generated from intact plasma membrane samples (A) and reconstituted lipid membrane samples (B)isolated from the lens cortex, and intact plasma membrane samples (C) and reconstituted lipid membrane samples (D) isolated from the lens nucleus. In each panel, diffraction peaks labeled as 1’ and 2’ correspond to immiscible cholesterol domains(periodicity of 34.0 D); peaks labeled as 1 and 2 correspond to the surrounding membrane lipid bilayer phase.

membrane bilayer phase corresponding to dif-fraction orders 1 and 2, and an immiscible choles-terol monohydrate domain defined by diffractionorders l' and 2'. Calculation of d-space valuescorresponding to these diffraction orders revealeddistinct structural features of these separate lipiddomains. The width of the sterol-poor membranebilayer region varied with each sample, with intactplasma membranes from the cortex and nucleus(Fig. 1A and 1C, respectively) yielding d-spacevalues of 80.6 D and 88.8 D, respectively. In theabsence of membrane protein (reconstituted mem-brane samples), the cortical and nuclear membranelipid bilayer d-space was 79.1 D (Fig. 1B and 1D,respectively). In contrast, the d-space for thecholesterol domain remained unchanged at 34.0 D.

Effects of Temperature on Membrane Structure

Intact and reconstituted cortical and nuclearplasma membranes were examined over a tem-perature range of 5°C to 40°C. Diffraction peakscorresponding to an immiscible cholesterol domainwere observed at every temperature level. Thecalculated width of the cholesterol domains wasunaffected by temperature changes, with a repro-ducible d-space of 34.0 D. In contrast, the surroun-ding lipid membrane bilayer phase was significantlyaffected by sample temperature. Consistent with adisordering effect with increasing temperature,eleva-ting the temperature from 5°C to 40°C causedan overall decrease in membrane unit cell perio-dicity. Intact cortical and nuclear plasma membranebilayer d-spaces decreased by 5.2 D (7%) and 3.3 D(5%), respectively.

Effects of Relative Humidity on Membrane Structure

As observed for temperature, changes in relativehumidity did not affect the presence of cholesterolmonohydrate phases in each of the samples tested.Over a range of 33% to 97% RH, the cholesteroldomains remained highly organized and stable witha consistent d-space value of 34.0 D. However, thesurrounding membrane bilayer phase for eachsample was significantly altered as a function ofrelative humidity. With increasing relativehumidity, d-space values increased by 28.2 D (54%)and 30.9 D (60%) for intact and reconstituted lens

cortical plasma membrane bilayer phases, respec-tively. Over the same relative humidity range,nuclear sample d-space values increased by 40.1 D(79%) and 24.0 D (44%) for intact and reconstitutedsamples, respectively.

Discussion

Cholesterol is asymmetrically distributed withintypical eukaryotic cells, and it is estimated thatgreater than 90% of cellular cholesterol associateswith the plasma membrane [25]. This estimate isprobably low for lens fiber cell plasma membranes,however, since mature fiber cells lack internalorganelles, particularly in the lens nucleus [8].Plasma membrane accounts for only ~1% of totallens volume [26, 27], and surface area calculationshave indicated that phospholipid accounts for only -1/3 of lens plasma membrane [8]. Since essentiallyall lens cholesterol is confined to this small portionof the total lens volume, the C/P mole ratios fornative lens membranes are extremely high. In thesestudies, the C/P mole ratios ranged from 2.42 to 3.27(Table 1), consistent with previous reports [8, 9].These ratios are significantly greater than C/P values of 0.5-1.0 reported for the typical cell membrane[10].

It is well-established that C/P mole ratios inexcess of one (i.e., 50 mole% sterol) can promote the formation of separate cholesterol domains in the cellmembrane [12]. Numerous theoretical and modelmonolayer and bilayer studies have demonstratedthat systematic addition of cholesterol to biologicalmembranes can eventually induce lateral phaseseparation, forming membrane-restricted immisci-ble sterol domains [11-16, 18, 28]. Additionally,recent small angle X-ray diffraction studies in ourlaboratory have provided direct evidence for theformation of separate, membrane-restricted choles-terol domains in vascular smooth muscle cell plasma membranes isolated from animals modelingatherosclerosis, a process characterized by an exces-sive accumulation of cholesterol in the vascular wall[18]. In these diseased membranes, the C/P moleratio approached 1.0, a level three-fold greater thanin normal membranes [18]. These studies confirmthe physicochemical propensity of cholesterol toform separate membrane phases at high relative

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concentrations. Because the C/P mole ratios ofocular lens fiber cell plasma membranes areextremely high, it has been proposed that thesemembrane bilayers are a mosaic of cholesterol-richand cholesterol-poor regions [8]; however, directevidence for the existence of separate cholesteroldomains in lens fiber cell plasma membrane has notbeen previously provided.

Immediately evident from the results of thisstudy is the presence of two structurally distinctmembrane domains within the lens fiber cell plasmamembrane: a sterol-poor, liquid crystalline mem-brane bilayer phase and an immiscible sterol-richmonohydrate bilayer phase. A striking feature of thecholesterol monohydrate phase is its stability over abroad range of temperatures and relative humidities.In contrast, the sterol-poor, liquid crystallinemembrane bilayer phase was significantly in-fluenced by temperature and humidity. The bio-chemical basis for these changes in membrane width may be attributed to the complex headgroup and acyl chain composition of the lens fiber cell plasmamembrane. This question is being systematicallyevaluated in a separate study.

The high levels of cholesterol present in thesemembrane samples may contribute to the stability ofthe lateral cholesterol phases. Cholesterol is presentat levels 2.5- to 3.5-fold greater than that required toproduce sterol domains in previous studies [18]. Inaddition, the phospholipid constitution of thesurrounding membrane bilayer may promote choles-terol domain stability. Sphingomyelin and itsderivative 4,5-dihydrosphingomyelin are the mostabundant phospholipids in human ocular lens,accounting for greater than 50% of totalphospholipid [5]. Cholesterol is known to have fa-vorable molecular interactions with sphingomyelin[29-31], exhibiting greater affinity for sphingo-myelin-enriched plasma membranes than for othercellular membranes [32, 33]. Although the mechan-isms responsible for the preferential interaction ofcholesterol with sphingomyelin are not fullyunderstood, it is believed that an increased proba-bility of van der Waal's forces may contribute to thestrength of their interaction [14, 30, 34]. It should bepointed out that sphingomyelin is not required for

the formation of cholesterol domains since thesehave been observed in systems composed exclu-sively of other lipids [28], including dimyristoyl-phosphatidylcholine [35], dimpalmitoylphospha-tidylcholine [13], N-palmitoylgalactosylsphingo-sine [16], and dimyristoylphosphatidyl-serine [28].However, recent experiments conducted in ourlaboratory suggest that cholesterol domains formmore readily in cholesterol/sphingomyelin binarymixtures and exhibit stability characteristics similarto that of cholesterol domains formed in the lensfiber cell plasma membrane (unpublished data).Slotte has published data suggesting that the high-affinity interactions of cholesterol with sphingo-myelin may reduce the free energy needed to formthe critical nuclei for the growth of cholesteroldomains in cholesterol/sphingomyelin monolayers[14]. In addition, the lateral surface pressurerequired to abolish lateral phase boundaries ofcholesterolrich domains appears to be significantlylower for cholesterol/dipalmitoylphosphatidyl-choline monolayers than for mixtures of cholesterol/ sphingomyelin [14]. These data suggest that thesphingomyelin-rich lens fiber cell membraneprovides the ideal lipid milieu for forming verystable cholesterol domains. It is also interesting tonote that nuclear lens membranes contain greateramounts of sphingomyelin [36] and saturated fattyacids [9] than do cortical membranes. Thisobservation would suggest that cholesterol domainsoccur more readily in the lens nucleus, possiblyexplaining the fact that the diffraction peakscorresponding to the cholesterol domains in theintact nuclear samples were more well-defined thanin the intact cortical samples (compare Figure 1Aand B).

Protein content did not affect the presence ofimmiscible cholesterol domains within the lens fiber cell plasma membrane. However, the intensity of the cholesterol diffraction peaks was greater in theabsence of membrane protein (reconstituted sam-ples). This may be due to partial protein interferencewith diffraction of the cholesterol domains. It is alsoclear that the formation of cholesterol domains doesnot require lens membrane protein since cholesteroldomains were present in reconstituted samples.Thus, lateral cholesterol domains occur within the

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phospholipid bilayer regions of the lens fiber cellplasma membrane.

These data support a model that is consistentwith the existence of separate cholesterolmonohydrate bilayers within the plane of the cellmembrane (Fig. 5). Individual cholesterol molecules appear to align in a tail-to-tail fashion as previouslydescribed in model bilayers and atheroscleroticvascular smooth muscle cell membranes [18, 37]. X-ray crystallography approaches have determinedthat the long-axis dimension of an individualcholesterol monohydrate molecule is 17 D [17];thus, a tail-to-tail orientation in the cholesterolbilayer yields a periodicity of 34.0 D. The formationof cholesterol domains appears to be supported bydirect interaction of cholesterol with the acyl chainsof surrounding membrane phospholipids, indepen-dent of molecular interaction with membraneprotein. This conclusion is supported by theobservation of distinct cholesterol phases in bilayersreconstituted solely from lens fiber cell plasmamembrane lipid.

The functional significance of cholesteroldomains within ocular lens fiber cell plasma mem-brane is not completely understood. However, theessential function of the lens fiber cell plasmamembrane is to maintain lens transparency to visible light throughout life, and the unusually high

membrane concentrations of cholesterol appear tobe critical for supporting this function. Usinginfrared spectroscopy approaches, it has beendetermined that the progressive increase in mem-brane cholesterol concentration moving from thelens cortex toward the lens nucleus is necessary tobuffer the structural order of these two regions tosimilar fluidity levels [38]. This membrane orderingeffect of cholesterol may be essential to maintaininglens transparency and is only achieved by signifi-cantly higher concentrations of cholesterol innuclear versus cortical membranes. In addition tocontaining the highest relative levels of membranecholesterol, the lens also contains high concentra-tions of soluble protein, known as lens cyrstallins.Association of crystalline primarily α-crystallin,with the lens membrane has been shown to accom-pany the development of human and experimentalanimal cataracts [39-41]. This association may bepromoted by reductions in membrane cholesterol asthe inhibition of cholesterol biosynthesis in the lenshas been shown to induce the development ofcataracts in rats [42], dogs [43], and humans [44,45]. Maintenance of high membrane concentrationsof cholesterol may attenuate the interaction of α-crystallin with the lens fiber cell membrane [46], butthe mechanism is not understood. Based on thefindings from this study, it is proposed that theformation of separate sterol-rich and -poor domains

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Fig. 2. Schematic model of the proposed lipid organization of the human ocular lens fiber cell plasma membrane. The lensfiber cell plasma membrane is characterized by separate cholesterol domains with a width of 34.0 D surrounded by asterol-poor liquid crystalline lipid membrane bilayer.

may interfere with the ability of extrinsic proteins toaggregate at the membrane surface. This hypothesisis currently being investigated in our laboratory andmay lead to new insights into the effects of fiber cellplasma mem-brane lipid organization on cataractformation. The results of this study indicate thatsmall angle X-ray diffraction methodology is veryeffective in characterizing the molecular organi-zation of biologic membranes of the human eye.

Acknowledgements

The authors acknowlege support from PPGHL22633 (R.P.M.) and EY02568 (R.J.C.).

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Fig. 1 and Fig. 2 were reprinted from the Journalof Biological Chemistry, Vol. 274, No. 44 (pp.31615 and pp. 31617, respectively) by courtesy ofthe issuer of the journal, The American Society forBiochemistry and Molecular Biology, Inc.

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