Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes

Lipid Composition, Membrane Structure Relationships inLens and Muscle Sarcoplasmic Reticulum Membranes

DOUGLAS BORCHMAN,1 DAXIN TANG,1 MARTA C. YAPPERT2

1 Department of Ophthalmology & Visual Sciences, Kentucky Lions Eye Research Institute, School of Medicine,University of Louisuille, Louisville, Kentucky 40202

2 Department of Chemistry, University of Louisville, Louisville, Kentucky, 40202

ABSTRACT: Membrane lipid composition varies in different tissues and species. Since adefined lipid composition is essential to the function of many membranes, the relation-ship between membrane lipid composition and structure was determined using infraredand Raman spectroscopy in four membranes containing a calcium pump: rabbit fast andslow twitch muscle sarcoplasmic reticulum and human and bovine lens fiber cellmembranes. We found that membrane sphingolipid and phosphatidylcholine contentwere correlated to a decrease and increase, respectively, in the infrared lipid CH2

symmetric stretching band frequency. We interpret the change in frequency as achange in lipid hydrocarbon chain structural order. This was confirmed by Raman orderparameters. The high degree of hydrocarbon chain saturation found in the variableamide chains of sphingolipids is likely to account for this correlation. Lipid phasetransition temperature and cooperativity also correlated to sphingolipid and phosphati-dylcholine content, and are the forces defining the order in at physiological temperaturein the samples studied. Ca21-ATPase caused an increase in the CH2 symmetric stretch-ing frequency in fast twitch muscle sarcoplasmic reticulum (interpreted as an increasein hydrocarbon chain disorder), but had no effect on slow twitch muscle sarcoplasmicreticulum lipid hydrocarbon chain structure. In the natural systems studied, we findthat it is the lipid hydrocarbon chain saturation that defines lipid hydrocarbon chainorder. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 151–167, 1999

Keywords: membrane lipid composition; membrane structure; ocular lens mem-brane; muscle sarcoplasmic reticulum membrane; infrared spectroscopy; Raman spec-troscopy

INTRODUCTION

All biological membranes have a well-definedlipid composition and membrane structure. Devi-ation from this well-defined composition andstructure can result in altered membrane func-tion and disruption of cellular homeostasis. The

changes in lipid composition and structure re-ported in human lenses with age and cataractsuggest that lipids play a significant role in main-taining human lens clarity (see references inBorchman 1993 et al.1).

Membranes from the sarcoplasmic reticulum(SR) of fast twitch and slow twitch muscles andthe plasma membranes from the ocular lens werechosen in this study because these three mem-branes have different phospholipid composi-tions2–4 and they all serve as a matrix for theCa21-ATPase, whose activity is affected by phos-pholipid composition.5–12

The SR plays an essential role in the control ofintracellular calcium concentration in skeletalmuscle and thus regulates muscle relaxation and

Correspondence to: D. Borchman, Kentucky Lions Eye Re-search Institute, 301 E. Muhammad Ali Boulevard, Louisville,KY 40202.

Contract grant sponsor: USPHS; contract grant numbers:EY07975, EY11657.

Contract grant sponsors: Kentucky Lions Eye Foundation;Research to Prevent Blindness, Inc.Biospectroscopy, Vol. 5, 151–167 (1999)© 1999 John Wiley & Sons, Inc. CCC 1075-4261/99/030151-17

151

contraction. The magnesium-dependent calcium-stimulated ATPase is responsible for the trans-port of calcium in this organelle and is the pre-dominant protein found in fast twitch muscle SRmembrane. The Ca21-ATPase protein is interest-ing because the composition of the surrounding 30lipid annuli has been shown to effect the activityof the Ca21-ATPase.5–12

The lens is a transparent, avascular organ,held by suspensory ligaments between theaqueous and vitreous humors of the eye. Thefunction of the lens is to focus light onto theretina in the back of the eye. It is comprised oftightly packed cells, referred to as lens fibers,with a single layer of epithelial cells at theanterior surface. The fiber cells have virtuallyno intracellular organelles. Thus, proteins andlipids do not turn over. Cataract, the only dis-ease of the lens, is defined as a clouding of thelens. Correction by surgical removal is the onlytherapy and is the major surgical procedureperformed in the United States.

Using spectroscopic techniques we found inlens membranes that higher levels of hydrocar-bon chain order were correlated to the content ofsphingomyelin (SM) and phospatidylcholine(PC).13 Our previous study2 showed that fasttwitch muscle SR has more PC (64%) than slowtwitch muscle SR (37%), whose PC content isgreater than that in bovine lens (25%) followed byhuman lens fiber membrane (ca. 3%)3 (Table I).The opposite trend is observed for sphingolipidcontent. This leads us to postulate that slowtwitch muscle SR lipid must be more ordered thanfast twitch SR. The relationship between the ratioof SM to PC content and hydrocarbon chain order

has been reported for lens lipids by our laborato-ry.13 In this study, the correlations between lipidcomposition and hydrocarbon chain order is ex-tended to relationships in fast and slow twitchmuscle sarcoplasmic reticulum.

To investigate these membranes, Raman andinfrared spectroscopy were used as nonperturba-tive probes for determining lipid conformationand phase transition parameters. This studyserves as a basis for future studies designed toestablish relationships between lipid composi-tion, structure and Ca21-ATPase activity rela-tionships in lens and muscle SR membranes.

EXPERIMENTAL PROCEDURES

Preparation of Sarcoplasmic Reticulum Membrane

Caudofemoralis (fast twitch), and soleus (slowtwitch) muscles were excised from 2.3–3.6 kg,white, male New Zealand rabbits.

For each of the muscle preparations, the SR (R1washed) preparation procedure of MacLennan14

was used with modifications suggested by Banerjeeet al.15 Muscle masses were measured to 60.1 gprecision and the procedure was scaled proportion-ally by weight for each muscle group. Protein con-centration, where required in MacLennan’s proce-dure, was measured by Spector’s refinement of theCoomassie blue method.16

We found, as did Banerjee et al.,15 that thispreparation can be stored for at least severalmonths at 270°C without loss of ATPase activity.Protein and lipid composition studies were per-formed within 1 week of SR preparation.

Table I. Composition of Membranes

CaudofemoralisMuscle SR

SoleusMuscle SR

BovineLensa

HumanLensb

Phosphatidylcholine (mol %) 64 6 1 37 6 8 25 2.5Phosphatidylethanolamine (mol %) 18 6 2 30 6 3 34 8.4Phosphatidylserine (mol %) 9 6 1 10 6 1 13 Not detectedSphingolipid (mol %) 5 6 1 15 6 4 24 64.5Phosphatidylinositol (mol %) 3.8 6 0.3 8 6 2 3 Not detectedCholesterol mol % of phospholipid 7.7 6 0.6 15 6 1 46 78 6 6CH2/AC 12 6 2 28 6 4 31 40.8 6 9.8Lipid/Ca21-ATPase (mol/mol) 104 6 20 132 6 24 Unavailable Unavailable

a Bovine lens phospholipid content was taken from Ref. 34. Bovine lens cholesterol content was taken from Ref. 35. Bovine lenslipid saturation was calculated from Ref. 36.

b Human lens phospholipid content was taken from Ref. 3. Human lens cholesterol content was taken from Ref. 37. Human lenslipid saturation was averaged from Refs. 38–40. All other data determined experimentally from this study.

152 BORCHMAN, TANG, AND YAPPERT

Lipid Extraction from Sarcoplasmic ReticulumMembranes

Lipids were extracted by adding 3 mL CHCl3/MeOH solution (1 : 2 by volume) to 1 mL R1washed SR suspension (5–15 mg/mL) and vortex-ing for one min. CHCl3 (1 mL) was added and themixture was again vortexed for 1 min. H2O (1 mL)was added and the sample was vortexed for 1 min.The two solvent phases were separated by centrif-ugation.17 Care was taken not to disturb thewhite, denatured protein interface while thelower phase was removed. The lower CHCl3phase was then washed with 4 mL water, andused immediately for phospholipid and choles-terol analyses or for spectroscopic studies.

Purification of Muscle SR Ca21-ATPase Vesicles

Purified Ca21-ATPase vesicles were obtained fromR1 washed SR by the octylglucoside purificationprocedure of Banerjee et al.15 This procedure wascarried out to remove the extrinsic calsequestrinand Ca21 binding protein found in SR.

Contamination of SR with other membranefragments is possible. Transverse T tubules havean identical phospholipid, cholesterol, and pro-tein composition18 as that reported for slowtwitch SR.2 Large-scale contamination of the SRwith T-tubules only in slow twitch muscle prepa-rations is unlikely since the T system diameter,size, and frequency of T tubules are not signifi-cantly different in any of the muscle fiber types.19

Contamination by mitochondria and sarcolemmalmembranes were not detected. No sarcolemmalouabain-sensitive Na-K-ATPase was detected,14

and mitochondria were not visible in electron mi-crographs.20,21

Preparation of Delipidated Ca21-ATPase

To determine the influence of Ca21-ATPase on theorder of the 30 lipids in direct contact with theCa21-ATPase pump, delipidated samples from aprevious study22 were used and lipid order versustemperature was measured using infrared spec-troscopy as described below.

Raman Spectroscopy Measurements ofSR Lipid Extracts

One mL lipid in CHCl3 solution, prepared as de-scribed above, was washed with 4.0 mL water,and the resulting phases were separated by cen-trifugation. The lower phase (CHCl3/lipid extract)

was removed and the CHCl3 was evaporated by astream of N2 (g). The dried lipid was lyophilizedfor 3 h in a culture tube to remove residual CHCl3and water. One mL D2O (99.8% pure Aldrich) wasadded, and the tube was warmed at 30°C for 15min to disperse the lipid. The dispersion was cen-trifuged at 150,000 3 g for 1 h at 4°C, the super-natant was decanted, and 1 mL D2O was added tothe pellet. In order to remove exchanged D2O, thesample was vortexed for 2 min, then centrifugedat 150,000 3 g for 1 h in vacuum at 4°C. Thepellet was transferred to a glass capillary tubewhich was sealed. The sample was compacted inthe tube with the use of a capillary centrifuge at11,000 3 g, 4°C for 15 min.

The capillary tube was placed in a homemadebrass block temperature-control cell, throughwhich a glycerol/water mixture from a NeslabEndocal RTE-5 circulating water bath was circu-lated. Water temperature in the bath was con-trolled to 60.2°C. The openings for incidental andscattered light were sealed by quartz windows,and positive pressure was maintained in the cellsample holder with a N2 stream. Temperaturewas measured by a thermistor placed within thecell. The sample was initially equilibrated at 25°Cfor one h. Before spectral measurements werebegun, the temperature was lowered to 25°C at arate of 0.1°C per min. Between scans, sampletemperature was increased 2°C by warming at arate of 0.1°C per min. The temperature was mon-itored continuously during each scan and found toremain constant within 60.5°C at the cell.

An 8-W argon ion laser was used as a source for514.5-nm monochromatic light. The light was fo-cused on the sample at a power of 180 mW for allSR lipid samples except the soleus SR lipid sam-ple for which 100 mW power was used. Laseremission lines were eliminated by placing a nar-row bandpass interference filter between the la-ser and the sample. The scattered light was fo-cused onto the slits of a Spex 14018 four-slitdouble monochromator. Photons were counted bya Spex DPC-2 digital photometer equipped withan RCA C31034 photo multiplier tube housed in athermoelectric cryostat. The spectral band passwas set at 5 cm21.

Raman Spectroscopy of PurifiedSR Ca21-ATPase Vesicles

A pellet of purified Ca21-ATPase dispersed inD2O was prepared for infrared analysis. The pel-let was transferred to a glass capillary tube whichwas then sealed. In order to compact the sample,

LIPID COMPOSITION, MEMBRANE STRUCTURE FUNCTION RELATIONSHIPS 153

the tube was centrifuged at 11,000 3 g at 4°C for15 min in an IEC MB hematocrit micro centri-fuge. The capillary tube was placed in the tem-perature-controlled cell described above, and Ra-man spectra at varying temperatures weremeasured as described for Raman lipid spectra.

Measurements of ATPase Activity to Test SampleEndurance Through Spectroscopic Measurements

ATPase activity of combined muscle SR was mea-sured at 37°C in the reaction medium containingcalcium buffered with EGTA, and magnesium.23

Liberated phosphate was determined using themethod of Fiske and Subbarow.24 Protein concen-tration was measured by the method of Peter-son.25 The activity of our preparations at 37°Caveraged 3.8 IU/mg 6 0.8 S.D., comparing favor-ably with the 2.9 IU/mg Ca21-ATPase activity at37°C with basal ATPase activity subtracted, re-ported by Hidalgo et al.23 Activity as a function ofincubation time was measured at various temper-atures. Purified muscle SR Ca21-ATPase vesicles(same preparation used for spectroscopic mea-surements) were incubated for 6 and 16 h at var-ious temperatures. Then the activity of each sam-ple was measured at 37°C yielding 6.7 mmolphosphate/mg protein min after each of the fol-lowing equilibration conditions: 0 h or 6 h at 10°C,6 h at 21°C, and 16 h at 4°C.

Equilibrating the Ca21-ATPase for 6 h at 43°Cprior to activity measurement resulted in only a 3%loss of ATPase activity. Sixteen h of equilibration at10°C, 21°C, and 43°C prior to activity measurementresulted in up to 36% loss of ATPase activity. Be-tween 10°C and 43°C the loss of ATPase activity(measured at 37°C) is relatively independent of in-cubation temperature and dependent on incubationtime. These results indicated the integrity of theCa21-ATPase sample was upheld during our spec-troscopic measurements, which required less than16 h for temperature equilibration plus Ramanmeasurements.

Analyses of SR Lipid Composition

Cholesterol (40–400 g) was quantified in lipidextract using the colorimetric Liebermann–Bur-chard reaction at 550 nm.26 Phospholipids wereseparated by two-solvent phase, unidimensionalhigh performance thin layer chromatography onSilica Gel G plates. The solvent phases consistedof chloroform–methanol–(30% aq. ammonia)–wa-ter, 140 : 50 : 7 : 3 by volume and chloroform–methanol–acetic acid–water 160 : 20 : 4 : 5 by

volume. Well-defined bands were visualized withI2 vapor. The identity of the chromatographicbands from our lipid extracts was determined bycomparing the mobility of these bands to the mo-bility of lipid standards determined by Neskovicet al.27 and reconfirmed by our own standard mo-bility determinations. By the method of Parkerand Peterson,28 the amount of phosphate for eachlipid type and the total lipid phosphate in thelipid extract were determined. Approximately100–200 mL of lipid extract containing 5 to 25 mglipid phosphorous were spotted in a 1.5-cm line atthe origin of the silica gel plates. Dipalmi-toylphosphatidylcholine, dipalmitoylphosphati-dylethanolamine, dipalmitoylphosphatidic acid,and mono basic potassium phosphate were usedas phosphorous standards. Sample phosphatemeasurements were approximately corrected forsilica gel background by measuring backgroundphosphate at sites adjacent to sample bands. Cor-rections amounted to less than 3% of the mea-sured sample phosphate.

Analyses of SR Lipid–Protein Ratiosby Chemical Methods

Lipid phosphate was analyzed by the method ofBartlett,29 using nine dilutions of the lipid ex-tract. Protein concentration was determined bythe methods of Lowry et al.,30 Peterson et al.,25

and Spector16 for each sample.

Analyses of SR Protein Composition

SDS polyacrylamide gel electrophoresis was per-formed using 7% acrylamide gels. The procedureis documented by Weber and Osborn.31 Coomas-sie blue dye was used to stain the proteins. AGilford Instrument 2400-S spectrophotometerwas used to measure gel densitometric tracings.The slit width set at 1.464 mm and gels werescanned at 550 and 280 nm. Integrated intensi-ties of the peaks were measured by planimeter.The ratio of 55000 Mr to 465000 Mr protein wasdetermined by cutting and weighing densitomet-ric chart paper.

Spectroscopic Determination of SR LipidHydrocarbon Chain Saturation

Infrared and Raman spectra were used to esti-mate the degree of unsaturation in SR lipids.Muscles were pooled from four rabbits. Washedsarcoplasmic reticulum was prepared and lipidwas extracted as described above. An additional


batch of pooled washed sarcoplasmic reticulum(R1 washed sample, Ref. 14) from six rabbits wasprepared for the soleus SR lipid Raman studybecause of the limited yield of SR lipid from thesmall soleus muscle. Lipid was extracted for Ra-man studies from this additional preparation.

The infrared CH stretching region was used toestimate the degree of lipid hydrocarbon chainunsaturation for soleus and caudofemoralis mus-cle SR lipids. The intensities of the soleus andcaudofemoralis lipid (D2O dispersion, 25°C) 3010cm21 CHACH stretch bands were compared tothe intensities of their respective 2850 cm21 CH2stretch bands. Background at 3010 cm21 was es-timated using the spectrum of dipalmitoylphos-phatidylcholine.

The Raman CH stretch region was also used toestimate the degree of lipid hydrocarbon chainchain saturation for soleus and caudofemoralisSR lipids. The quotient of the Raman 3010 cm21

HCAHC stretch band peak height divided bypeak height of the 2850 cm21 symmetric CH2stretch band was used as a measure of theACH/CH2 molar ratio content of the lipid chains. Lin-olenic acid (18 : 3), oleic acid (18 : 1), and potas-sium oleate (18 : 1) were used as standards.

Infrared Measurements of SR Lipid Extracts

Sample Preparation

Lipid (1.0 mL) in CHCl3 solution was washedwith 4.0 mL water. The two phases were thenseparated by centrifugation. The lower phase(CHCl3/lipid) was removed and an aliquot of thislipid solution was layered on a AgCl window.CHCl3 was evaporated by a stream of N2 (g). Thedry lipid was lyophilized on the AgCl window for3 h to remove residual CHCl3 and water. The lipidwas dispersed in 10 mL of D2O (99% pure, Aldrich)and added to the AgCl window. A 0.10-mm Teflonspacer and a second AgCl window were added tocomplete the sample holder. The sample holderwas then placed in a Precision Cell Inc. variabletemperature cell (model No. 21.000), which waspurged with dry filtered N2 (g).

Temperature Control

Temperature was measured by a thermistortouching the AgCl sample cell window. Our initialinvestigation began by studying lipid extractedfrom combined muscle SR. After placing the sam-ple into the variable temperature cell, the tem-perature was controlled using a Neslab PCB-4-II“cold finger” operated against two heating plates.

Temperature was controlled 60.1°C using aNeslab PCB-4-II “cold finger” operated againsttwo heating plates.

The sample cell was equilibrated at 50°C for 15min to insure complete hydration of the lipid. Thesample was then brought to room temperatureover a period of 15 min and then allowed to equil-ibrate at 25°C for 3 h. IR temperature studiesstarted at 25°C. Cooling studies were carried outfrom 25°C to 225°C for caudofemoralis and soleuslipid samples. The cooling studies were followedby heating studies to 50°C.

Lipid dispersion spectra were measured with 2cm21 resolution by a Nicolet DX-20 FTIR spec-trometer. One hundred fifty scans (approximately5 min) were signal averaged for each spectrum.Temperature was allowed to stabilize 2–3 minprior to spectral measurement.

Curve Fitting of Infrared Temperature Studies

Nonlinear regression analysis1 was used to fit thelipid phase transition curves to the function be-low:

Frequency 5 P1 1 ~P2/~~1 1 P3/T!P4!! (1)

P1 is the minimum frequency of the CH2 symmet-ric stretching vibration in wavenumbers for thephase transition and represents the most orderedstate of the transition. P2, the magnitude of thephase transition, is the net change in wavenum-bers and is related to the change in the number oftrans-to-gauche rotomers (see the Discussion sec-tion for details). P3 is the transition temperatureand indicates the temperature at which half thelipid molecules have undergone a phase change. Tis the absolute temperature. P4 is the relativecooperativity. This term is similar mathemati-cally to the Hill coefficient which was first used todemonstrate cooperative nature of hemoglobin–oxygen binding. The broader the phase transitionthe smaller the value for P4.

The percent disorder at 36°C was calculatedusing Eq. (2) below.13

% disorder 5 100 3 ~n# 2 2849 cm21!/5.5 cm21

(2)

where n# represents the frequency (in wavenum-bers) interpolated from Eq. (1) above. There isevidence that suggests that the wavenumber ofthe CH2 symmetric stretching band and the num-ber of gauche rotomers in the hydrocarbonchain(s) are related,32 but correlations between


the absolute wavenumber and number of gaucherotomers is unavailable (see discussion and Fig. 5caption).

Temperature Studies of PurifiedSR Ca21-ATPase Vesicles

The purified SR Ca21-ATPase vesicle samples tobe examined by IR temperature studies were notstored in buffer, but rather were suspended inD2O (pD 5 7.8, 10 mg/mL protein, 4°C) after thefinal wash (pH 7.8). No pD adjustment was nec-essary because of the natural buffering capacityof the protein at a pH of 7.4, and perhaps due to,at most, 5 mm HEPES buffer pH 7.4, carried overfrom the step prior to resuspension in D2O. Thesample was vortexed at 4°C for 5 min, then cen-trifuged at 100,000 3 g for 30 min in vacuum at4°C. The supernatant was decanted, and D2O wasagain added (10 mg/mL protein, pD 5 7.8, 4°C).The mixture was vortexed and centrifuged at100,000 3 g for 30 min in vacuum at 4°C. Thesupernatant was decanted and the pellet wasspread under N2 (g) on an AgCl IR window pre-viously cooled to 1°C.

After 30 min equilibration at 1°C, IR spectra ofcaudofemoralis and soleus purified SR Ca21-ATPase vesicle IR spectra were measured with a2-cm21 resolution Nicolet 20DX FTIR spectrome-ter. One hundred fifty scans were signal-aver-aged, requiring 5 min for each spectrum. Duringeach spectral measurement, temperature re-mained constant to 60.3°C.

For caudofemoralis and soleus samples, afterchanging the temperature approximately 2°Cover a period of 20 min, the temperature wasallowed to stabilize 2–3 min prior to spectral mea-surement.

The cooling cycle was carried out from 1°C to230°C, and was followed by heating studies from245°C to 50°C. Between spectra measurements,temperature was changed approximately 2°Cover a period of 20 min.

RESULTS

In previous publications we established theunique lipid composition of membranes from slowtwitch and fast twitch muscle SR2 and humanlens lipids.3,4,33 The compositions were confirmedin this study and shown in Table I. The followingspectroscopic studies were designed to determinelipid structural compositional correlations be-tween all four tissues, that is, slow twitch and fast

twitch muscle SR, bovine, and human lens fibermembranes.

SR Lipid Raman Studies, Lipid Raman BandAssignments

The 700–1800 cm21 Raman spectrum of caud-ofemoralis lipid (pD 7.4) at 25°C is displayed inFigure 1. The spectrum is typical for lipids in theliquid–crystalline state. Because PC composesabout 60% of the caudofemoralis SR membrane,the choline (H3C)N1 symmetric stretch band at717 cm21 with a bandwidth of 20.6 cm21 is quitepronounced. This frequency is representative of ahydrated disordered bilayer head group region inwhich electrostatic interactions between neigh-boring head groups are minimal.41 The phospho-lipid head group OOPOO symmetric and anti-symmetric stretches appear at 775 and 806 cm21,respectively.42 Headgroup COC stretch bands areobserved at 975 and 927 cm21.42 The intensity ofthe carbonyl band near 1730 cm21 is too low foraccurate evaluation of this band(s).

The hydrocarbon chain acyl C2OC1 stretchingmode appears as a broadband, centered at about875 cm21, indicating a disordered acyl interfaceregion.43 The hydrocarbon chain COC opticalacoustic region (1050–1150 cm21) is characteris-tic of a membrane in the liquid crystalline state.The 1130 cm21 and 1066 cm21 COC stretchingintensities of all-trans segments of the backboneare very weak compared to the strong 1090 cm21

COC stretching vibration of gauche rotamers.The OOPOO symmetric stretch band putatively

Figure 1. Raman spectra 514.5 nm incident light, ofaqueous lipids at 25°C, pH 7.4, from rabbit (a) caud-ofemoralis muscle (fast twitch) SR and (b) soleus mus-cle (slow twitch) SR.


lies under the 1090 cm21 band. At 1300 cm21 and1440 cm21 are the CH2 twisting and CH2 defor-mation modes, respectively. The considerable de-gree of unsaturation of the lipid acyl chains isevident from the strong 1656 cm21 cis CACstretching band.44 The intense 1270 cm21 band isdue to aACH in plane deformation.45 The inten-sity ratio I1300/I1270 cm21 is equal to 1.1, whichcorresponds to about 10 CH2 groups per CACgroup.46

Band assignments for the Raman CH2 stretch-ing region are also discussed in the following sec-tion.

SR Lipid Hydrocarbon Chain Saturation

We find that by three separate spectral measure-ments, the degree of unsaturation for caudofemo-ralis SR is twice as unsaturated as the soleus SRlipid. The infrared and Raman CH stretch regionwas used to estimate the degree of lipid acyl chainunsaturation for soleus and caudofemoralis SRlipids. The intensities of the soleus and caud-ofemoralis lipid (D2O dispersion, 25°C) 3010 cm21

CHACH stretch bands were ratioed to the inten-sities of their respective 2850 cm21 CH2 stretchbands. We estimate the molar ratio CHACH/CH2to be 1/13.2 (determined by infrared spectroscopy)and 1/16 (determined by Raman spectroscopy) forcaudofemoralis and 1/28 (determined by infraredspectroscopy) and 1/30 (determined by Ramanspectroscopy) for soleus lipid.

The larger degree of caudofemoralis SR lipidunsaturation is also evident from the RamanCAC modes near 1655 cm21 (Fig. 1) in the SRlipid dispersion spectrum of caudofemoralis andsoleus lipid extracts. The peak height of theCHACH stretch band was normalized to the peakheight of the 1450 cm21 CH2 deformation band toestimate lipid saturation. We estimate the molarratio CHACH/CH2 to be 1/10 for caudofemoralislipid and 1/17 for soleus lipid.

The ratio CHACH/CH2 was estimated usingthree different spectral parameters and were de-termined to be: 1/16, 1/10, and 1/13 averaging1/13 6 2.0 for caudofemoralis; and 1/30, 1/17, and1/28 averaging 1/24 6 4 for soleus (Table I). Thevalue for caudofemoralis saturation averaging1/13 6 2.0 was in excellent agreement with thevalue of 1/12.3 calculated using chromotogra-phy47 and a value of 1/10 calculated using theRaman 1300 cm21 region bands (see the bottom ofthe previous section for details). The degree ofunsaturation for the soleus SR lipid was com-pared to the degree of unsaturation for the caud-

ofemoralis SR for each of the three spectral pa-rameters used. The ratio of the degree ofunsaturation for caudofemoralis SR lipid com-pared to that for soleus SR lipids is 1.9 from theRaman I3010/I2850 data, 1.7 from the Raman I1655/I1450 data and 2.1 from infrared I3010/I2850 data.The average value for these ratios is 1.9 6 0.2.The degree of unsaturation for caudofemoralis SRlipid is therefore shown by three separate spectralmeasurements to be about twice as unsaturatedas the soleus SR lipid.

Caudofemoralis SR Acyl Chain Order

Both the hydrocarbon chain backbone COCstretching region (1050–1150 cm21) and the CH2stretch region were used to monitor lipid acylchain order and phase transitions.48 The inten-sity of the 1128 cm21 band assigned to chain COCall-trans segments decreases with increasingtemperature while intensity of the 1090 cm21

band assigned to COC chain gauche rotamers(with some PO2

2 symmetric stretch contribution)increases. The peak height ratios I1128/I1090 areplotted against temperature in Figure 2. Fromthe plot we see the intensity ratio I1128/I1090 de-creases continuously from 0.85 at 217°C to 0.3 at0°C. The peak height ratio I1130/I1090 reported foregg lecithin (PC) in water at 266°C is 1.4. Thecaudofemoralis SR lipid COC stretch peak heightratio values indicate the bilayer is already partly

Figure 2. Raman COC stretching band intensity ra-tios for an aqueous dispersion caudofemoralis (fasttwitch) muscle SR lipids. A lower band intensity ratioindicates a lower hydrocarbon chain order.


disordered at 216°C. The peak height ratio foregg lecithin dissolved in CHCl3 is reported to be0.23.45 The COC stretch peak height ratio of 0.3for caudofemoralis SR lipids above 0°C indicatesthe hydrocarbon chains are highly disorderedabove the ice–water transition.

Six bands were resolved in the CH2 stretchingregion: the 2846 cm21 CH2 symmetric stretchband, the 2886 cm21 Raman-active Fermi reso-nance CH2 stretch band, the 2928 cm21 antisym-metric infrared-active CH2 stretch band, the 2958cm21 out-of-plane chain end asymmetric CH3stretch vibration, the 2935 cm21 chain end CH3symmetric stretch, and the 3010 cm21 unsatur-ated ACH stretch band.

The 2886 cm21 Raman Fermi resonant band issensitive to intra- and interchain interactions. Theintegrated intensity and peak height of the banddecreases as intra molecular chain disorder (trans-gauche COC bond isomerization) increases.49 Also,as lateral packing interactions between chains aredisrupted, the intensity of the 2886 cm21 band de-creases.49 Except for transitions in crystal packing,there is relatively little change observed in the 2850cm21 band in lipid transitions. Therefore, the peakheight ratio I2886/I2550 is used to monitor the changein the peak height of the 2886 cm21 band.49 Thepeak height intensity ratio, I2886/I2850, indicative ofintramolecular chain disorder and lateral packingbetween lipid acyl chains, is plotted against temper-ature in Figure 3. The decrease of the ratio from1.15 at 27°C to 0.97 at 0°C is indicative of a phasechange from a loosely packed gel phase to a typicalliposome liquid crystalline phase.

For comparison purposes, the I2886/I2850 ratiomeasured for a dipalmitoyl–phosphatidylcholinedispersion at about 35°C (just below the maintransition) is about 1.25. The value for a liquid-crystalline (or melted) liposome phase dipalmi-toyl–phosphatidylcholine is about 1.0.

The peak height of the 2756 cm21 terminalCH3 asymmetric stretch band is representative ofthe deepest part of the hydrophobic bilayer inte-rior. This ratio decreases sharply over the sametemperature range at which the 2886 cm21 CH2antisymmetric stretch band decreases (data notshown), providing evidence that melting at theinterior of the bilayer proceeds simultaneouslywith disordering of the acyl chains as a whole.

Soleus SR Acyl Chain Order

As discussed in the section above, the CH2stretching region may be used to monitor lipidgel-to-liquid crystal phase transitions. The CH2

stretch peak height intensity ratio, I2886/I2850, isplotted against temperature in Figure 3. The ini-tial value at 222°C is 1.43. This ratio is high andis characteristic of a liquid crystalline phase. Theratio decreases from 1.43 at 222°C to about 1.07at 3°C, indicating a broad transition to the meltedliquid crystalline phase of a multilamelar suspen-sion or liposome. The background was subtractedbefore peak height measurements were made;however, it is possible that the broad transitionfrom 222°C to 3°C may consist of multiple tran-sition for which resolution was not possible be-cause of increased background interference forsoleus SR lipids. The I2886/I2850 ratio remainsabove 1.0 for the temperature range studied.

The I2886/I2850 ratio is lower at all tempera-tures for caudofemoralis lipids (Fig. 3) than forsoleus lipids thus, the extent of lipid chain disor-der is far greater for caudofemoralis lipids thanfor soleus lipids at all temperatures.

Infrared Spectroscopy of SR Lipid ExtractDispersion; Temperature Induced CH2 StretchFrequency Shifts for Lipids Extracted fromCaudofemoralis SR

The infrared CH2 stretching frequency was usedto measure changes in the order of lipid hydrocar-bon chains (see Methods section). There were tworegions where changes in hydrocarbon chain or-

Figure 3. Raman CH2 band intensity ratios for aque-ous dispersions of (E) soleus muscle (slow twitch) SRlipid and (F) caudofemoralis muscle (fast twitch) SRlipid. A lower band intensity ratio indicates a lowerhydrocarbon chain order.


der were detected in lipid extracted from fasttwitch caudofemoralis muscle SR [Fig. 4(a) andTable II]. A major transition was found to occur at

24°C and was twice the magnitude of the broadchange in hydrocarbon chain order centered at21°C. We believe the change in order centered at

Figure 4. Lipid gel to liquid crystalline phase transitions for aqueous dispersion of (a)caudofemoralis muscle SR lipid; (b) (E) soleus muscle SR lipid, (- - -) caudofemoralis SRlipid fit from (a), for comparison; (c) (F) caudofemoralis muscle SR membrane containingCa21-ATPase pumps, (- - -) caudofemoralis SR lipid fit from (a), for comparison; (d) (E)soleus muscle SR membrane containing Ca21-ATPase pumps, (- - -) soleus SR lipid fit from(b); (e) nonlinear regression analysis fit of data from (—) soleus SR muscle membrane in (d)and (- - - -) caudofemoralis SR muscle membrane from (c). The lines through the datarepresent the nonlinear regression analysis fit as described in the Methods section.


21°C is real because (1) it has been observed byother investigators for SR lipid extracts measuredusing Raman spectroscopy,50 and an increase inthe infrared CH2 symmetric stretching frequencyrising between 10 and 30°C with a less steep riseabove 30°C has been reported51; (2) a single sig-moidal curve characteristic of a single lipid phasetransition does not fit all of the data, but twosigmoidal curves provide an excellent fit; (3) thefrequency does not shift (there is a plateau) be-tween 2 and 9°C in two other samples which alsoexhibited a broad transition centered near 21°C(data not shown). One broad 2.1 cm21 (2851.0–2853.1 cm21) frequency shift commencing at211°C and ending at 20°C is observed for the2850 cm21 symmetric CH2 stretching band fromsoleus SR lipid spectra [Fig. 4(b) and Table II].Soleus lipid was more ordered than caudofemora-lis lipid above 210°C [Fig. 4(b)].

The Effect of Ca21-ATPase on Caudofemoris SRand Soleus SR Lipid Phase Transition

In comparison with pure caudofemoralis vesicles,Ca21-ATPase disordered the lipids (calculatedfrom CH2 symmetric stretching frequency) andbroadened the phase transition measured for pu-rified SR membranes from the caudofemoralis[Fig. 4(c), Table II]. A delipidated sample (54moles of phospholipid/mole Ca21-ATPase) con-taining mostly lipids in contact with proteinshowed Ca21-ATPase pump had the same effect(Table II). There was no significant difference be-tween the phase transition parameters for soleuslipids compared to soleus membranes containingCa21-ATPase pumps [Fig. 4(d), Table II].

SR vesicles containing predominantly Ca21-ATPase pump were more ordered (had lower CH2symmetric stretching band frequencies), at alltemperatures, purified from the soleus musclecompared to the caudofemoralis muscle [Fig. 4(e),Table II].

Sphingolipid/Phosphatidylcholine MembraneComposition and Lipid Structure Correlations

Membrane sphingolipid and phosphatidylcholinepercent molar composition were found to be cor-related to lipid order [Fig. 5(a)] in human andbovine lens fiber membranes; slow twitch and fasttwitch muscle SR. SR had the highest percent PCand lowest SM. From Figure 5(a), one may ob-serve that the lower the PC and the higher theSM, the higher the membrane lipid hydrocarbonchain order at 36°C. The gel (ordered) to liquid

crystalline (fluid) phase transition temperature[Fig. 5(b)] and the cooperativity [Fig. 5(c)] werealso found to be correlated to the PC and SMcontent of the membranes.

Lipid Saturation, Membrane Composition, andLipid Structure Correlations

The sphingolipid content correlated inverselywith the average number of variable hydrocarbonchain cis double bonds [Fig. 6(a,b)]. The oppositewas seen between glycolipid content and unsat-uration. Lipid saturation correlated with lipid hy-drocarbon chain conformational order [Fig. 6(c)]and phase transition temperature [Fig. 6(d)].

DISCUSSION

The major finding of this study is that the infra-red hydrocarbon chain conformational order wascorrelated to membrane sphingolipid and PCmembrane content. We also found that the cal-cium pump elevated the CH2 symmetric stretch-ing band frequency which we interpret as a dis-ordering of the caudofemoralis SR lipidhydrocarbon chains.

Measurements of Lipid Order

The fluid mosaic membrane model52 for biologicalmembranes indicates that the membrane is a dy-namic system. Membrane “fluidity” is a term thatdefines the dynamic motions of the whole mem-brane system. The dynamics of a lipid may bedefined by bond rotations, trans–gauche isomer-izations, torsional oscillations, bond rotations,lateral diffusions, rigid body motions (wobbling),and flip-flop type motions, to name a few.53 Noone technique can measure all of the dynamiclipid motional parameters. A number of spectro-scopic techniques have been used to measure thedynamics of lipid motion: infrared, Raman, fluo-rescence, nuclear magnetic resonance, electronspin resonance. Other techniques have been usedto measure static lipid structure: X-ray crystal-lography, Langmuir trough pressure–volumemeasurements, and electron microscopy. Differ-ential scanning calorimetry has been used tomeasure enthalpy and entropy changes. In gen-eral, all of the techniques are complimentary andhave been successfully applied to measure the gelto liquid crystallin phase transition tempera-ture.53

Dynamic data obtained from studies using


probes to measure lipid motion, (i.e., anisotropy,lateral diffusion, rotation) are useful in providingour group54–57 and others58 with information onlipid–protein interactions. However, the resultsshould be interpreted with caution since the dy-namics of the probe indirectly infer the motion ofthe lipids; the probe molecule may perturb thesystem; and the location of the probe moleculewithin the membrane is often not accuratelyknown. However, many of the dynamics reportedby probe techniques have been confirmed by non-perturbing techniques which indicates that theconcerns discussed above may be small.58

In this study Raman and infrared spectroscopywere used to measure lipid hydrocarbon chainconformational order. Raman and infrared spec-troscopy are nonperturbing techniques that pro-vide a “snapshot” of the lipid structure in theorder of 10214 seconds.59 In this study we used,among other parameters discussed in the resultssection, the lipid CH2 symmetric stretching fre-quency to estimate the trans, gauche rotomer ra-tio, a measure of conformational order. Lipids inthe conformationally disordered state may be de-fined as having all gauche rotomers. Those in theconformationally ordered state may be defined ashaving all trans rotomers. This definition of con-formational order was based on the empirical andexperimental results of Snyder et al.,32 who usedn-paraffins as models. They found a change fromthe all trans conformation to the all cis conforma-tion is accompanied by many spectral changesincluding a 6-cm21 increase in the frequency ofthe CH2 symmetric stretching band. Frequencyshifts in the CH2 symmetric stretching band havebeen used by a number of investigators to monitor

phase transitions. A classic study involving liveAcholeplasma laidlawii B bacteria was publishedin 1983.60 Changes in the CH2 symmetric stretch-ing band frequency with the state of the lipid,corresponded well with the changes in the CH2symmetric stretching band half bandwidth andabsorbance.61 Dluhy et al.62 provide empirical ev-idence that the relationship between the % transrotomers and the frequency of the CH2 symmetricstretching band is not linear. These calculationswere based on a two-state model in which lipidsdid not interact with one another and cannot beapplied to the complex systems used in this study.Mendelsohn and Senak63 quantified unequivo-cally infrared bands due to gauche and transrotomers. Although this technique is excellent forwell-defined model systems, application of thistechnique to our complicated native systems wasnot possible due to the diverse lipid composition ofour native lipid samples. Because of the largefrequency differences between our samples (seediscussion below) and because all of the trends weobserved were expected based on model studiesusing a variety of biophysical techniques (see dis-cussion below), we believe the infrared and Ra-man frequency shift data accurately representschanges in lipid conformational order.

Membrane Lipid Composition, StructureRelationships

In a previous study, we have shown that the sar-coplasmic reticulum (SR) purified from fasttwitch muscle contained more PC (64%) and lessSM (5%) compared to SR extracted from soleus(slow twitch muscle) which contained 37% PC and

Table II. Lipid Phase Transition Parameters

PhaseTransition

Temperature(°C)

Order at36°C

(% trans)Cooperativity

(Relative)

Caudofemoralis SR lipid 24 6 1 8.4 63 6 1221 6 3 56 6 34

Caudofemoralis SRpurified membrane

1 6 1 7.9 49 6 6

Soleus SR lipid purifiedmembrane

7 6 1 24 37 6 6

Delipidated SR purified(mixed) membrane

1 6 1 8.9 44 6 9

Human lens 39–72 44–66 22 6 7

a Lipid hydrocarbon chain order was calculated from the infrared CH2 symmetric stretching frequency [see Eq. (2) andExperimental Procedures and Discussion sections].


15% SM.2 Since the sphingolipid to phosphatidyl-choline ratio of a membrane is believed to beinversely related to membrane fluidity,64 we pos-tulated that the lipids in the slow twitch SRshould be more ordered than those of the fast

twitch SR. We found that the lipids in fast twitchSR are indeed more ordered than slow twitch SRlipids at all temperature studies [Fig. 4(b,e)].

In a later article,13 we found that membraneSM and PC content correlated to lipid structural

Figure 5. Correlation between (E) phosphatidylcholine and (F) sphingolipid contentof lipids and (a) hydrocarbon chain order at 36°C. Lipid order as used in this figure wascalculated directly from the CH2 symmetric band frequency as outlined in the Methodssection. One hundred percent order on the y axis corresponds to a CH2 symmetric bandfrequency of 2849 cm21, while 0% order corresponds to a CH2 symmetric band fre-quency of 2854.5 cm21. It is likely this order parameter relates to the number of transrotomers (see Discussion section); (b) lipid gel to liquid crystalline phase transitiontemperature; (c) lipid phase transition cooperativity. (—) Linear regression linethrough phosphatidylcholine data in (a) and (b) and sphingolipid in (c). Sarcoplasmicreticulum data was obtained from this study. Human lens lipid order information wasobtained from the FTIR and FTRaman studies of Borchman et al.13 Human lens lipidcomposition was obtained from Borchman et al.13 Bovine lens lipid composition wasobtained from Broekhuyse et al.34 Bovine lens lipid order was obtained from Borchmanet al.35


order and disorder, respectively. As these previ-ous studies were done in membranes of a singletissue, this study extended the correlations to avariety of tissues and species. Raman and infra-red spectroscopy were used to measure lipidstructure and composition. The two techniquesare complimentary in that bands that appear inRaman spectra often do not appear in infraredspectra and vice versa. We found that the hydro-carbon chain order (based on the CH2 symmetricstretching frequency) of the lipids extracted fromfast twitch muscle and slow twitch muscle SR,bovine lens nuclear and cortical lipids and humanlens lipids were correlated with PC and SM con-tent. This correlation appeared to be a linear oneespecially for PC [Fig. 5(a)]. The increase in mem-brane order measured at 36°C was a consequenceof a decrease in phase transition temperature[Fig. 5(b)] with a decrease in PC and increase in

phase transition temperature with an increase inSM percentage. The inverse relationship with SMpercentage and phase transition cooperativity[Fig. 5(c)] and direct correlation between PC andphase transition cooperativity also contributed tothe correlation between SM and PC and lipidorder at 36°C.

Sphingolipids may contribute to a less fluidlipid membrane because the variable amidelinked hydrocarbon chain is more saturated thanthe acyl linked glycerolipids and the sphingosinebase chain contains a trans double bond in the 4position which would be expected to order themembrane but does not contribute to lipid fluidityas much as a double bond located in otherpositions deeper in the hydrocarbon chain. Sphin-golipids have tighter interface regions due to hy-drogen bond interactions,65 however, sphingomy-elins have the same phase transition temperature

Figure 6. Correlation between hydrocarbon chain saturation and (a) sphingomyelinmembrane content, (b) phosphatidylcholine membrane content, (c) hydrocarbon chainorder (see Fig. 5 for details), (d) phase transition temperature. Lipid composition datawas obtained as stated in Table I.


as analogously substituted variable chain phos-phatidylcholines,66 suggesting that lipid satura-tion contributes to a greater degree to hydrocar-bon chain order than the phospholipid backbonestructure. These considerations are discussed inthe following section.

Hydrocarbon Chain Saturation and Lipid StructureInteractions

Introduction of a cis double bond into an acylchain induces a bend in the hydrocarbon chainthat cannot be compensated for by rotationalisomers.67 As a consequence of the bend, phos-pholipids with unsaturated hydrocarbon chainspack less compactly, and in general have lowerphase transition temperatures as a result ofdiminished van der Waal’s interactions com-pared to phospholipids possessing hydrocarbonchains of the same chain length that are satu-rated. In general, an increase in lipid unsatura-tion increases membrane fluidity.67– 69 The cor-relation between increased hydrocarbon chainunsaturation and lipid order is complex. Doublebonds located near the center of the hydrocar-bon chain have a greater influence on fluiditythan those located in other positions.70 Thenumber of cis double bonds beyond the firstdouble bond does not result in a proportionalincrease in fluidity.68,71,72

Since cis double bonds have a profound effecton lipid fluidity, we explored the possibility thatthe ordering effect of the sphingolipids, as dis-cussed in the section above, could be a result ofthe sphingolipids being more saturated than thecorresponding glycerolipids. In this study wefound that the membrane concentration of sphin-golipids inversely and glycerolipids directly corre-lated to the average number of variable hydrocar-bon chain cis double bonds [Fig. 6(a,b)]. In humanand bovine lens membranes we found that thesphingolipids are indeed more saturated than theglycerolipids. Sphingomyelin, composing 7 to 13%of the total phospholipid of the human lens3 con-tains 1 cis double bond per 68 CH2 groups.73 Di-hydrosphingomylein,4 a rare sphingolipid foundonly in quantity (54.5%) in the human lens3 con-tains 1 cis double bond per 56 CH2 groups.74 Incomparison to the total human lens lipid satura-tion,38–40 we calculate that the glycero phospho-lipids are much more unsaturated containingonly 1 cis double bond per 4.7 CH2 groups. Thesphingomyelin is more saturated in the bovinelens also, containing 1 cis double bond per 31.3CH2 groups compared to the saturation of phos-

phatidylcholine, ethanolamine, and serine whichcontain a normalized mole percent average of 1cis double bond per 23.4 CH2 groups.36 An in-crease in the average saturation of the membraneas a result of an increase in sphingolipid contentcould account for the inverse correlation betweensphingomyelin and variable chain saturation[Fig. 6(a)]. As expected, lipid saturation corre-lated with lipid hydrocarbon chain conforma-tional order [Fig. 6(c)] and phase transition tem-perature [Fig. 6(d)].

Hydrocarbon Chain Length and Lipid Structure

Hydrocarbon chain length is directly related to lipidhydrocarbon chain order.66 The tissues used in thisstudy all have about the similar average hydrocar-bon chain lengths 17.3, 17.7, and 17.3 for humanlens,38–40 bovine lens36 and fast twitch muscle sar-coplasmic reticulum lipids,47 respectively. Hydro-carbon chain length is therefore not a factor regu-lating lipid order in the tissues studied.

Cholesterol, Lipid Structure Interactions

It has been established that SM concentration iscorrelated to the cholesterol content of a mem-brane.74 We found this to be the case as shown inFigure 7(a), where the content of cholesterol andsphingolipid were correlated. Furthermore, wefound that the content of cholesterol in the mem-brane correlated well with lipid hydrocarbonchain order as shown in Figure 7(b). Althoughcholesterol and hydrocarbon chain order were cor-related, we do not believe cholesterol contributessignificantly to the large differences we found inlipid order between that of the sarcoplasmic re-ticulum and the human lens. In a previous studywith bovine lens lipid, we found that completeremoval of cholesterol only caused a small modu-lation of hydrocarbon chain order and by nomeans brought the hydrocarbon order of the bo-vine lipids to that of the fast twitch muscle sar-coplasmic reticulum even with no cholesterolpresent.35 We believe that cholesterol serves tomodulate lipid order in lens membranes within10–20%. Conversely adding cholesterol to fluidSR lipids would be expected to order the hydro-carbon chains, but the ordering would not beenough to bring the order of the SR lipids to thatobserved in lens membranes.48


Effect of Ca21-ATPase on LipidHydrocarbon Chain Order

We found that the fast twitch muscle contained104 moles phospholipid/mole Ca21-ATPase (TableI). Slow twitch muscle SR contained 132 molesphospholipid/mole Ca21-ATPase (Table I). As-suming an annulus of 30 moles of lipid for eachCa21-ATPase,5 we calculate that about one thirdof the lipids are in direct contact with the calciumpump. Since it has been shown that Ca21-ATPaseimmobilizes lipids, the influence of the hydrocar-bon chain structure of the lipids was investigated.In this study, we found that the pump decreasedcaudofemoralis lipid order below physiologicaltemperature [Fig. 4(c); Table II]. The same resultwas found for the hydrocarbon chain order of a

delipidated sample containing mostly lipids incontact with the pump (Table II). These resultsare similar to the results of Jaworsky and Men-delsohn75 who showed that in ternary syntheticlipid complexes the Ca21-ATPase pump disordersthe lipid hydrocarbon chains broadening thephase transition and shifting it to lower temper-atures. Soleus lipid hydrocarbon chain order wasnot changed significantly by the Ca21-ATPase[Fig. 4(d)]. This result is in agreement with ourprevious observations for lens membranes whichcontained over 50% by weight protein.76 In thesemembranes, no change in lipid hydrocarbon chainorder was observed in the presence of either ex-trinsic or intrinsic membrane proteins. The dif-ferences between lipid protein interaction be-tween the slow and fast twitch muscle SR may bedue to differences in lipid composition2 and theisoform composition of the calcium pump. TheSERCA2 isoform of the calcium pump is ex-pressed mainly in slow twitch muscle.77 TheSERCA1 isoform is expressed mainly in fasttwitch muscle SR.77

Lipid Order, Ca21-ATPase Activity Relationship

Our spectroscopic studies of the isolated lipidsfrom the two SR types show that lipid order isgreater in slow twitch muscle compared to fasttwitch muscle [Figs. 3 and 4(b,e)]. In slow twitchSR, dystrophic fast twitch muscle,78 and in cata-ractous lens,38 cholesterol, protein, and sphingo-myelin content are higher and Ca21-ATPase ac-tivity is lower.79 The higher amounts of thesecomponents are expected to contribute to a moreordered membrane and hence lower Ca21-ATPaseactivity. We have shown previously that theCa21-ATPase activity in the lens was correlatedto lipid order.80,81 SR Ca21-ATPase, reconstitutedinto ordered (about 40% trans) lens lipids, werefive times less active than when reconstitutedwith native SR lipids.81 It has been establishedthat in synthetic lipid systems, the mobility of aprobe in the lipid bilayer (except in the case oflipids completely in the gel or liquid crystallinephase) does not correlate with Ca21-ATPase ac-tivity.82 Studies using ordered native lipid sys-tems reconstituted with the Ca21-ATPase pumpmay be useful for determining the influence ofhydrocarbon chain structure near the gel phaseon Ca21-ATPase activity and could be used toevaluate the contribution of lipid compositionaland structural differences to functional changesin the membrane. Changes in lipid composition

Figure 7. Correlation between cholesterol and (a)sphingolipid content and (b) hydrocarbon chain order.(—) Linear regression line. See Figure 5 for details.


and/or order with oxidation, age, and/or diseasesuch as cataract or muscular dystrophy would beexpected to influence the function of the mem-branes studied and could contribute to the diseasestate.

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Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes