Membranes of Animal Cells

[T’. LIPIDS OF THE L CELL AXD ITS SURFACE MEXIBRA~E*

(Kecrived for publirat,ion, February 28, I’&%)

DAVID B. IIT~~~~~~~~ AND JULIAX B. MARSH

From the Department of Biochemistq, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

x4RY CATHERINE GLICK ASD LEOXARD W.~RREN

From the Department of Therapeutic Research, School of Xedicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

SUMMARY

Surface membranes were isolated by the fluorescein mer- :uric acetate and Tris procedures from mouse fibroblasts (L :ells) grown in suspension culture. Membranes isolated by :he fluorescein mercuric acetate method contained approxi- nately 4.7% of the protein and 13.8 % of the lipid of the whole :ell. Approximately 95 % of the plasma membrane lipid was accounted for. Comparison of lipid-protein ratios for whole :ells and surface membranes shows a d-fold enrichment of ipid in the surface membrane. Neutral lipids make up 40 % )f the total membrane lipid, with cholesterol and triglycerides accounting for 20 and 13%, respectively. The high choles- erol content of the surface membrane is reflected in a :holesterol-phospholipid molar ratio of 0.69 while the cor- ,esponding ratio for total cell lipid is 0.26. Fifty-nine per :ent of the membrane phospholipids contain choline, and the ;urface membranes contain more sphingomyelin and less ecithin and phosphatidylethanolamine than the total cell ipid. Phosphatidylserine and sphingomyelin show a Z- to L-fold enrichment of unsaturated and long chain fatty acids ,elative to other membrane phospholipids. The lipid com- josition of the surface membranes of L cells was compared o the composition reported in the literature for surface mem- cranes of animal cells. The comparison indicates that the ipid compositions of L cell and liver cell surface membranes Lre basically similar. Lipid composition may be useful in lassifying surface membrane preparations.

Iierent ljrogress in our understanding of the structure and unction of cell rncmbrallev has come from both physical and hemica data. Many of the physicochemical properties of rtificial binlolecular lipid membranes are similar to those of ~;~tural membranes (1). Studies on the interaction of lipids

* This work TV~S supported by United States Public Health iervice Grants 1.ROllID-005201 and IIE-05285 and by American [~IKY~ Society Grant PNP-2S.

and proteins have indicated that the organization of metnbrane structure may be dependent upon the lipid composition of metnbranes (2). Correlation of data from the study of model

. . systems an vztro and of natural membranes can be achieved only when precise analytical data on membrane components are available.

The wide variation in the lipid composition of membranes (3) is l)robably important in determining the properties and func- tions of a particular membrane. In this respect, the markedly different cholesterol contents of the surface membranes and internal mcmbrnncs of xnirnnl tissues (4-6) may indicate different affinities for the vnrious types of lipids involved in plasma membrane formation. Until recently, studies on lipids of surface membranes lrere limited to t’he erpthrocyte mem- brane, which could be readily isolated and purified in large quantities. Emmelot et al. (7) prepared a fraction from rat liver which consisted of lhs~n:~ mctnbrane fragrnent,s, but the lipids ivere only partially characterized. Takeuchi and Tera- yarn;~ (8) and Skipski et crl. (0) attempted a complete characteri- zation of the lipid components of similar preparations of mern- branes from rat liver. Both investigations were complicated by technical dificulties which do not alloy direct comparison with the more yuantitative studies of T)od and Gray (10) and Pfleger, Anderson, and Sn>-der (11).

Warren, Glick, and Nash (12) hare described several n&hods for the isolation of surface membranes from mouse fibroblasts (L cells) grown in suspension culture. Preparations of intact surface nlelllbranes are obtained from as much as SOYi of the cells grown under controlled environmental conditions. This study describes the lipid composition of these surface metnbranes and makes a comparison n-ith the lipid composition of the whole cell and with analyses reported by others of the lipids of plasma, membranes of normal animal tissues. -1 preliminary report of this work has been presented (13).

EXPERIRIEA-TAL PROCEDURE

Materinls-All glassware \vas acid-wa*hcd and rinsed with deionized water. Methanol and ether \vere reagent grade and \\-ere redistilled in an all ~I:ISS apparatus. Reagent grade chloro- form was distilled inlmedi:rtely before use or was stored with

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4104 Membranes of Animal Cells. IV Vol. 244, Xo. 15

0.8’ C methanol ndded as a preserx-ntivc after distillation. Hesane (955;) \\ as shaken with conccntrnted sulfuric acid for 15 Illin and leashed twice with distilled water, and the frac- tion boiling at 67..%68.5” was used. Sephadcs G-25 was washed and prepared according to the m&hod of Siakotos and Rouser (14). Reagent grade silicic acid (Mathewn (‘olemnn and Bell) ~1~ washed succwsirel\- n ith 6 s II(‘1, 105; KOII in mrthanol, \vatcr, methanol, and rther and XV:IS air-dried and activated at 110” for 12 to 16 hours before use. Florisil, 60 to 100 mesh (Fisher) l~--ns heated at 110” for 12 hours and JY:V deactil-ated by the addition of ic/,; water before use, as described by Curroll (15). Seutral lipids and phospholipitls uhcd as chromatographic n11(1 quantitnti\-c standards were ~)urch:wxl from Applied Science Laboratories (State College, l’eiirlsglvaili:~), Sigma, and Pierce Chemical Comlxmy (Chicago, Illinois) and were further purified by preparatire thin layer chromatography 011 prcwaahcd plates of Silica Gel G (Brinkmann Instruments). 2, B-Di-tert-butyl- p-cresol was obtained from Eastman; AT-nitroso-N-methyl urea from General Biochemicals; yalmitaldeh~-tic sodium bisulfite compound from Aldrich; and 14 ‘,~c (w/‘v) bo~,on fluoride-meth- anol from Applied Science Laborutorici.

Surface nlenlbrnnes were isolated Iron1 1, cells by the FhIA’ and Tris methods of Warren et nl. (12). SLWOXY was removed fro111 the menlbrane preparations b\- dialysis on :I rocking stage for 24 hours at 4” against a flow of distilled water. L cells were grown and harvested as previously described (12).

Lipid Ezlraction- L cells, 2 X lo* (al)proxinintely 15 mg of total lipid), washed twice with 0.16 N XaCl, and 2 to 4 x IO* surface menlbranes (approximately 1.8 to 3.6 mg of total lipid) \vere extracted by a five-step treatment \+, ilh (‘II(‘l,-C’HJOH (1: 1, v/v). Pivc volunles of cold nlethanol were added to the cells or membranes in a glass centrifuge tube. The extraction proceeded for 15 min at 0” with occasional stirring; an equal volunle of cold chloroform was added to the tube, and stirring lvvas continued for 15 min. The lipid extract, recovered by centrifugation, n-as kept under an atmosphere of nitrogen at -20”. The cell or membrane pellet n-as re-estracted at O”, then

extracted twice at room temperature, and finally at 45” under a flow of nitrogen gas. Conipletenws of litlid extraction was determined by hydrolysis of the residrw n-ith 6 s II(‘1 at 100” for 16 hours followed b\- gas chrolllntogr:lphic estimation of the amount of fatty acids released.

Removal of Nonlipid Jf afcric~l&Two diff went procedures for removal of nonlipid material from L cell lipid extracts were used with comparable results. In tht first procedure, the lipid extracts were washed with 0.2 volunle of water at, 4”. The two phases were allowed t,o separate completely, and the chloroform layer was removed and rewashed with 0.1 volume of n-ater. Only trace amounts of phosph:ltidylseriIle n-erc found in the aqueous mcth:tnolic mashes. In the second procedure, the un- washed lipid extract xas dried under reduced pressure at 10” on a Buchi Rotavapor (Fisher), and the damp residue was dissolved in chloroforn-methanol (19: 1, v/v) saturated n-it,h Ivater and apl)lied to a column of Sephades G-25 (1 x 18 cm) at 4” for quantitative separation of lipids from nonlipids (14). Purification of the lipid extracts by Sephades chromatography resulted in 99.8y0 recovery of lipid and greater than 997; removal of added ‘4C-glucose and ‘4C-lcucine. FMA rnembrane lipids were purified by this technique to eixurc complete remora1 of sucrose.

1 The abbreviation llsed is: FRIA, fluorescein mercuric acetat,e.

Lipid Clnss Scpctrnfi~~~s-~.ln aliquot of the purified lipid extracts W:IS rcmored for determin:ttion of total lil,itl content (we Mo~v). Lipid extra& uwc then .*epar:Ltetl into neutral and polar lil)id fractions b> ,silicic acid chromatography :IS dcscril)ed 1)s \\‘a>~ and Ilanahnn (16). Seutral lipids n-ere eluted with freshl?- dktilletl chloroform, and polar lipids were elutcd with nlcth:mol containing 0.005c, 2, A-cliitcrt-but!-l-p- cresol t,o act as an :mtiositlnnt (1’7). The fin:d concentration of 2, B~di~tert-bllt-1-p-cre,ol in the polar lipid fraction n-as kept at 5 pg per mg of lipid. The 3 to 47$ 0 f lipid phosphorus remaining on the silicic acid column could be rccorcrcd by the addition of 0.2 s I%(‘1 to the clution yol\-ent. The neutral lil)id fraction was dried untlcr reduced pressure at 10” and apl)lied in hesane to a 55.~111 long four-bore glaw column (1Ied~~‘hem Laboratories, Detroit, Jlichigan) coiltaiiiillg 9 to 10 g of deactil-atcd Floriqil. The sewn neutral lipid cl:weq v\-erc separated b\- the method of Fixher and Knbara (18) lvith increxsing amounts of cthcr in hesanc as the clution solvent. Completeness of separation 17as dcternlinctl by- thin la!-er chromatogr:aI)hy of the nclltral lipid fractions with the double development technique of Freeman and T\.est (19) Polar lipids lvere separated by two-dimensional thin l:Lyer chromatography according to the method of Abramson and I%lecher (20). Spots were visualized by brief exposure to iodine vapor or by spraying with alkaline bromthymol blue and esl)osing the 1)l:ltcs IO ammonia vapors (21). Purified lipids (hpplied Science Laboratories) and rat liver lipid which was freshly extracted were used as reference standards.

~Lnnk~ticnl J/fthods~-Proteins n-err determined 1),x- the nlicro- biurct method of Itzhaki and Gill (22), \I-ith bovine ~crum nlbu- min as a st,aildnrd. Total lipid was determined b>- the charring llirthod of 1Iarsh and J\-einsteill (23). After the initial analyses had determined the composition of n-hole cell and surfacc mem- brane lipids, caharring standards of identical composition were l)repared n ith mixtures of l’urified lipids. These standards were used ill subsequent analyses for determination of total lipid recover)-. Seutrnl lipids were determined after Florisil chromatography by the charring method, with a purified lipid of the same class as II stalldurd. Cholesterol and cholesterol esters lvere also determillcd 1)~. tho method of Zlatkis, Zak, and Boyle (24); glyceritlcs 1,~. tlw method of Van Handel and Zilrersmit (25). l%ospholil)ids \~ert’ determined by scraping the lipid spots off the Silica Gel (: plates and transferring the gel to digestion tubes; 0.15 1111 of concentrated sulfuric acid na+ added, and digestion was carried out in an aluminum he:Lting block at 220” for 90 min. l’hos~~horus content n-:rs measured by the method of Bartlett (26) and phospholipid content n-as calcu- lated by multiplying the phosphorus value by 25. Plasn~nlogen- bound aldehydes lrere detrrmined by the p~nitrophen!-lh!-crazine method of l’ries and Biittcher (2i). Sphingolipid base< were isolated bg methnnolysk of sphingomyelin according to the method of Rosenberg and Stern (28). The long chain bases were separated and nleasured by the gas chromatographic method of Carter and Gaver (29). Lipid fraction> were stored in de- aerated hesane at -20” under a nitrogen atmosphere. All analyses \T-ere performed in duplicate.

F&y _ I cid and _ I ldehyde k-nrrlysis-Fatty acid comlwsition was determined by two methods. (n) The sample was hy drolyzed with constant-boiling HCl in sealed tubes unde, nitrogen for 12 hours at 100”. Fatty acids were extracted n-it1 hesnne, washed n-ith water, and drird. Methyl esters mere prepnrcd by treatment of the free fatty acids with freshly distiller

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dinzomethane generated by alkaline treatment of IL--nitroso-N- FlZIh nlny be a reflection of small anlounls of free mercur\- which methyl urea (30). (b) Lipid s:m~ples were tran~estcrified with niay be fornird from the reagent during lipid storage. BFs-methanol by the procedure of Xlorriaon and Smith (31). Fatty aldeh!.dc dimethglacetals n-we prepnrcd lvith 13F3- RESULTS

methanol and were freed of fatty acid methyl rsters by the Lipid E&tactions-The lipids of the L cell and surface men- saponification technique of Farquhar (32). lIethy esters and branc preparations were completely extracted with chloroform- dimethylacetals were analyzed by gas-liquid chromatograph\- on methanol, 1: 1 (v/v). Phospholipid recovery from L cells was a l’!.(x argon chromatograph equipped with :I strontium ioniza- 3 to 5(,‘, greater with this solvent than with chloroform-methanol, tion detector. A &foot column of 17.5(1 polyethylene glgcol 2: 1 (v/v), or alcohol-ether tnistures. The initial extractions ?uccinate on 80 to 100 mesh (;:I>-(‘hrom I’ (A\pplied Science were performed in the cold to minimize lipid osidation and Lnboratories) operated at 165” and 7.5 pounds of pressure of activation of any hydrolytic enzymes ljresent in the samples. argon TV:W uccd. Cnanturated fatty acids and long chain fatty Esantination of the h!-drolyzed cell and membrane residues acid> lvere also identified b) gas-liquid chrotnatograph\- on a revelled the presence of 1 to 2 pg of fatty acid per mg of protein. -l-foot column of loi, Apiezon L on 100 to 120 mesh C‘elite. These trace amounts of pnlmitic and stearic acid are not solvent- Fatty acid nlcth\-1 ester identification and detector response estractable and may represent fatty acids col-alently linked to wrc dctermincd with S.T.H. Type Misturc KD and KF (Xl)- protein (33). The use of lipid mistures as standards for the plied Science Laboratories), total lipid determination was tested by preparing a grnrimetric

Contakzafion of Lipid Extracts with Ileavg Jletccls and Lipid standard of L cell lipid and a lipid mixture of the same composi- ;lu/oxidafior2-Atternl)ts to analyze quantitatively the lipids tion. The charring densities of equal quantities of the t1v-o of pln~ma membranes prel)ared by the %nClt l)rocedrtre of Warren standards agreed within +1.552. The total recovery of lipid et nl. (12) failed because of the presence of quantities of zinc in from I, cell and surface membrane lil’id extracts afler separation the lil)id esttwt sufficicllt to C:IUSC major changes in the chro- and measurement by the methods described above can be matographic propeltics of the l)olar lipids. The heavy tnetal readily estimated from the total lipid value calculated from the could not be removed by Scphadcs chloinat,ograp2i?-. The use charring assay. Uy this nlcthod 95.7 =t 0.4Gj, of the lipids of of an orgaltometallic agent (FJI;\) in the isolation of surface the whole cell and 94.1 + 0.45, of the surface membrane lipids membranes requires special precautions to l)rcvellt lipid autosida- can be accounted for. tion due to the presence of heavy metals. Only small amounts Neufral Lipid Compositio~l~Table I su~nmarizes the data on of nlembrane-bound FI\IA could be solubilized by chloroform- the lipid composition of five samples of whole L cells, five samples methanol (1 :l) giving the lipid extract a yelloworange tint. of surface membranes isolated by the FMA procedure, and one Sephades chromatography of the extract did not remove the sample of surface membranes prepared by the Tris procedure. FJIA. Subsequent silicic acid chromatography gave an FMA- L cells grown in medium containing 107; fetal bovine serum were free neutral lipid fraction, but the phospholipid fraction found to contain 0.225 mg of lipid per mg of protein. EightJ remained contaminated with FMX. The addition of 2, B-di-tert- per cent of the cell lipid is phospholipid; cholesterol and glycer- but\-l-p-cresol (5 pg per mg of lipid) to the membrane phos- ides comprise 11 and 8y0, respectively, of the remaining lipid. pholipid fraction completely protected the lipid; when it was Free cholesterol accounts for half of the neutral lipid of the cell,

omitted, extensive autoridativc changes were observed similar triglycerides an additional 30%, and cholesterol esters only 2% of to those reported by Dodge and Phillips (17) for erythrocyte the cell neutral lipid. Gas chromatographic analysis of the cell

lipid e\;tracts contaminated with hemoglobin derivatives. Xo sterol fraction indicated that cholesterol accounted for at least autosidativc artifacts were observed in the neutral lipid fraction. 98’;; of the sterol present,. No other steroid peaks were found.

T\~o-dirncnsional thin la?-er chromatograph\- of F1I&mem- FMX membranes contain 0.662 t11g of lipid per mg of protein.

brane phospholipids revealed that FI\L1 was not bound to any The neutral lipid fraction constitutes twice as much (41y0) of lipids although traces of FM;1 were occasionally observed at the the total lipid of the surface membrane as of the whole cell lipid origin. FMA streaked acro.ss the top of the thin layer chroma- (20y0). Free cholesterol accounts for half of the neutral lipid

tography plate and did not interfere in separation or analysis and 2074 of the total membrane lipid. The glyceride and free

of the lhospholipids. However, 2,6-di-tert-butyl-p-cresol fatty acid content of membrane neutral lipid is essentially identi-

interfered n-ith the cholesterol assay and caused the appearance cal with that, of whole cell neutral lillid, with triglycerides making

of artifacts during gas-liquid chromatographic analysis of fatty up over 12cT, of the tot31 membrane lipid. The molar ratio of

acids (li). Samples f or cholesterol and fatty acid analysis cholesterol to phospholipid is 0.26 for whole cells and 0.69 for

~~erc taken before the addition of 2,6-di-tert-but-l-p-cresol. FM=1 membranes, indicating the relatively large enrichment of

Loss or degradation of lipids due to the presence of F1Ih was neutral lipid in the surface membrane.

tested by treating whole cells with F1IA in the same manner in Phospholipid Composition-Analysis of the phospholipids of L

\vhich cells were prepared for membrane isolation. The cells cells (Table II) reveals that 54yb are choline-containing lipids.

ww extracted, and the lipids were esomined and compared with Lecithin (43’;;) and phosphntidylethanola~~li~~e (23 ‘%) make up

untreated cell lipids. FRlh had no effect on the fatty acid, the bulk of the cell phospholipid and account for more than half

aldehyde, neutral lipid, or phospholipid composition of the cells. of the cell lipid. The more acidic phospholipids (phosphatidyl-

L cell lipids which remain ill the presence of FXIA and 2,6-di- serine, phosphutidylinositol, and phosphntidic acid) make up 16yo,

tert-bllt!-lop-cresol for several days at -20” begin to show in- and cardiolipin less than 1 yc, of the cell phospholipid. The high lysophosphatide content (756) is a chromatogrsphic artifact

creased lysolecithin and decreased plnsmalogcn content. Since since lipid extracts not separated on silicic acid columns do not 0.005 M H&l:! can cleave l)las~~~:~logen-bouiid aldehydc (17)) the contain more than 1 :‘, lysophosphatide. decrease in plasmalogen content of L cell lipid in the presence of (‘omparison of plasma membrane and cell phospholipids re-

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4106 Membranes of Animal Cells. IV

TAIILE 1

Lipid composition of L cells and surjace membranes

Vol. 244, No. 15

Lipids of whole L cells and srlrface membranes were separated by silicic acid and Florisil column chromatography. Total lipid content was determined by the sulfuric acid charring assay after removal of nonlipid material from the extract. Individual lipid classes were assayed by specific calorimetric procedures and by the charring assay. The data represent the mean +S.E. for duplicate analyses of five lipid extracts of whole cells and FMA surface membranes and one analysis of Tris surface membranes.

- L cells FMA surface membrane Tris surface membrane

Total lipid Neutral lipid Total lipid Neutral lipid Total lipid

%

20.0 z!z 0.7 0.2 -Ir 0.1

10.2 + 0.9 0.4 f 0.1

6.1 zk 1.2 1.4 l 0.4 0.5 f 0.1

1.2 + 0.3 80.0 * 0.7

6

1.0 51.0

2.0 30.5

i.0

2.5 G.0

%

41.3 i 0.2 0.5 f. 0.0

19.6 i 0.2

1.4 zk 0.1 12.6 zk 0.4 4.0 f 0.2

0.5 * 0.2 2.7 f 0.2

58.7 * 0.2

%

1.2

47.5 3.4

30.5

9.7 1.2 G.5

v ,o

42.3 0.2

2O.G

1.3 12.8

4.i

0.9 1.8

57.7

Neutral lipid

0.5 ‘is.7

3.1 30.3 11.1

2.1 4.2

Lipid class I-

-I-

Neutral lipid ....................... Hydrocarbon .................... Cholesterol. ...................... Cholesterol esters. ...............

Triglyceride. ..................... Diglyceride ....................... Monoglyceride. ...................

Fatty acid. ...................... Phospholipid. ......................

Cholesterol to phospholipid molar ratio. .........................

Lipid concentration, mg/mg of pro- tein. ...........................

Total lipid recovered, y0 ...........

0.26 + 0.03 0.09 + 0.02 0.74

0.225 + 0.008 0.662 zk 0.005 O.G75 95.7 zt 0.4 94.1 * 0.4 95.5

TABLE II

Phospholipid composition of L cells and surface membranes

L cell andsurface membrane phospholipids were separated by two-dimensional thin layer chromatography. The phospholipidspots were scraped from the plates, and the gel was digested with sulfuric acid and analyzed for phosphorus content. Phospholipid content was calculated by multiplying the phosphorus value by 25. The data represent the mean +S.E. for duplicate analyses of five lipid

samples of whole L cells and FMA membranes and one analysis of Tris membranes. -

FMA membranes Tris membranes L cells Phospholipid -

Total phospholipid Total lipida -

Material at origin. ................. Lysolecithin. ......................

Sphingomyelin ..................... Lecithin ............................ Phosphatidylethanolamine. .........

Lysophosphatidylethanolamine ...... Phosphatidylserine ................. Phospha.tidylinositol.. .............. Phosphatidic acid. .................

Lysophosphatidic acid .............. Cardiolipin .........................

Plasmalogen” .......................

Choline. ......................... Ethanolamine. ...................

Recovery of lipid P %C .............

%

0.7 + 0.2 2.1 f 0.5

9.3 zk 0.9 43.0 + 1.3 23.3 f 1.4

3.9 + 0.5 6.9 zk 0.8 3.8 It 0.5

4.5 f 0.9 0.8 f 0.3 0.7 f 0.2

12.3

8.2 4.1

99.0 + 0.2

- T

_ /I .~ ‘otal phospholipid Total lipid

%

1.2

5.0 24.4 32.2 10.4

2.6 4.0

5.1 12.8

1.3

I- %

0.7 2.9

14.1 18.6

6.0 1.5 2.3

2.9 7.4 0.8

8.1

5.1 3.0

99.0

Total lipid

%

1.0 3.3

13.6

18.0 G .6 1.5

2.6 3.2 6.6 1.1

%

0.6 1.7

7.4 34.4 18.6

3.1 5.5 3.0

3.6 0.G 0.G

Total phospholipid

%

1.7 zk 0.2 5.7 zt 0.2

23.1 + 0.5

30.G 3~ 0.5 11.3 * 0.3

2.6 zk 0.2

4.4 f 0.5 5.4 + 0.3

11.2 * 0.3

1.8 31 0.3

7.2 5.0 2.2

97.8 f 0.3

-

- a Total lipid determined by charring assay. * The data represent the average of three determinations. c Expressed as percentage of total lipid phosphorus applied to thin layer chromatography plates.

veals several significant differences. Although the content of the total cell lipid but only 8% of the total surface membrane choline-containing phospholipids is only slightly higher (59 as lipid. The acidic phospholipids account for 13% of both the against 54a/,) in surface membrane, total membrane lipid has cell and membrane lipid, with the membrane containing more almost twice as much sphingomyelin and significantly less phosphatidic acid but less phosphatidylserine than the whole lecithin + lysolecithin (21 as against 36%) compared to the cells. total cell lipid. Ethanolamine phospholipids make up 22% of Cerebrosides and sulfatides were not detected in L cell lipid

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Issue of August 10, 1969 Weinstein, Xarsh, Glick, and Wawes 4107

TABLE III

Plasmalogen altlehycle composilion”

Lipid samples were treated w-ith 145; BF3-methanol for 1 hour

at 100”. Aldehyde dimethylacetals were freed of fatty acid methyl esters by saponification with 0.5 z XaOH in 90’,;. metha- nol at 85” for 4 hours. Dimethylacetals were extracted with

hexane, washed twice with 3 N NaOH in 507, ethanol, and ana- lyzed by gas-liqllid chromatography on a 4-ft CO~LII~I~ of 17.5c/;

polyethylene glycol srlcrinate at 165” :illd 5 to 10 lb of argon pres-

stlre. Decomposition of dimethylxcetals dllring chromatography wm not observed.

Aldehyde Fetal

bovine sermn

1Iyristnldehyde (14:0)b.. ./ Palmitaldehyde (16:O).

stenrnldehyde (18:O). _. Ott adecenaldehyde (18: 1) Ott adecadienaldehyde (18:2).

$4

1.4 54.7

30.4 10.9 2.5

L cell

Phos- Phos-

@a- pha-

tidal- :h’$t;, ethanol

amine

5%

57.0

28.1 11.9

2.9

c-

OY8

62.5

22.2 8.1 6.3

FAl.4 surface membrane

-

Phos- pha-

tidal- choline le

cc

63.1

22.4 11.7

2.G i

pphhoas tidal- thanol- amine

5%

1.2

55.8 29.2 11.3

2.4

a Expressed as percentage of plasmdogen aldehyde recovered

from two samples of each material analyzed. b Nomenclature indicates the carbon length and Ilrlmber of

double bonds present in each aldehyde.

extracts and cardiolipin was not detected in plasma membrane preparations. The absence of cardiolipin (a major mitochon- d&l lipid (34)) in the membrane lipid extracts indicates that the plasma membrane preparations are not contaminated with mitochondria. This is supported by the inability to detect cytochromes a, a3, and c in the plasma membrane prcparations.2

The snxdl amount of lipid phosphorus which does not migrate on thin layer chromntogral)hp plates contains small quantities of inositol (determined by gas-liquid chromatographic analysis)

and probably represents traces of 1)olyphosphoinositides as well

as SOI~F phospholipid degradation products. X0 major non- phosphorus-containing polar lipids were found by tw-dimen- sionnl chromatographic analysis. Glycolipids accounted for approximately O.TC/, of L cell lipid.3 Small amounts of ceramide diheroside were found in the polar lipid fraction, and several gangliosides were identified after separation from the lipid es- tract by Sephndex chromatography or the aqueous washing. Gangliosides were also found in the surface membranes.

Approximately 12 y0 of the L cell phospholipids and 5 71 of the membrane phospholipids occur in the form of plasmalogens. Two-thirds of the cell and rnernbrane plasmalogen is found in the lecithin fraction and the remainder in the phosphatidyl- ethanolamine fraction. Analysis of the plasmalogen-bound aldehydes of bovine fetal serum and L cells (Table III) reveals that the L cell probably utilizes the aldehydes which are present in the serum-supplemented media. Palmitaldehydc (59 %), stearaldehyde (26c/,), and octadecenaldehyde (115;;) make up

2 These analyses, by low temperature spectroscopy, were per- formed in collaboration with I>rs. David Wilson and Britton Chance of the Johnson Research Foundation, University of Penn- sylvania.

3 I>. B. Weinstein, L. Warren, J. 13. Marsh, and &I. C. Glick, data to be published.

nearly all of the plasmnlogen nldehyde found in the cell. The distribution of aldehydes found in the surface membrane lipid is essentially identical.

Gas chromatographic analysis of the trirnethglsilyl et,her derivatives of the sphingolipid bases of the whole L cell revealed that C18-sphingosine accounted for 59.6$& of the bases. C18- dihydrosphingosine (31.07;) and (‘20.sphingosine (9.4%) were the only other bases found.

The total lipid and phospholipid composition of surface mem-

brancs prepared by the Tris procedure shows excellent agreement with that of the surface nwnbrnnes prepared by the FN.1 pro- cedure. XIenlbrnne preparations which had been stored frozen in dilute sucrose solutions showed significant loss of lipid to the aqueous medium upon defrosting. Membranes stored for 2 IT-eeks to several months showed losses of 20 to 45% in cholesterol, lecithin, and I)hosphatid~lethnaol,zlllirle content. Therefore, all of the analytical work reported was performed on freshly pre- pared membranes.

Fatty Acid Paftcms~Table 1V presents a comparison of the fatty acid patterns of L cells and surface membrane preparations.

L cells grown in the presence of serum utilize the long chain polyunsaturated fatty acids which arc present in the serum (35, 36). The fatty acid composition of the lipids of the L cell most probably reflects to a large extent the fatty acid composition of

the fetal bovine sew111 in which they were grown. Pulrnitic,

stcnric, and oleic acids make ul) about iic?;m of the total fatty acids of the cell, :md long chain fatty acids (20 to 21 carbon atoms) make up an additional 9 yc. -1pprosirn:ttely half of the cell fatty

TABLE IV

Fall!/ acid composition oJ‘ L cells and their surface membranes

Fatty acid compositions were determined by gas-liquid chroma- tography of the methyl esters prepared by treatment of the lipid with BFs-met.hanol or treatment of the isolated fatt,y acids with

fresh diazomethane. Fatty acids were separated on a 4.foot column of polyethylene glgcol succinate at 167” and 7.5 polrnds of argon pressure.

I Percentage of total fatty acids

Major fatty acids

Media”

Myristic (14:0)d. 0.8 Palmitic (16:O). 28.6 Palmitoleic (16:l). 2.2

Stearic (18:O). _. 11.1 Oleic (18: 1) 44.8

Linoleic (18:2). 5.0 Linolenic (18:3). 1.9 Behenic (22:O). 0.6

Arachidonic (20:4) 2.4 Lignoceric (24:O). 0.8

Lnsnturated fatty acids m , ,c 56 48 48 45

L cellsb FMA membranes”

Tris mem- branesC

2.4 i 0.6 2.6 + 0.4 I- 2.4 9.5 dz 1.821.4 rt 1.2 21.3

3.4 z?z 0.4 6.6 * 1.1 7.0 !4.7 rt 2.112.2 f 1.6 14.1 12.7 f 2.814.6 f 1.1 13.4

2.0 l 0.5 1.2 * 0.3 1.7 4.3 + 1.025.4 f 2.2 22.6 2.5 zk 0.6 1.7 + 0.6 2.1

5.7 & 1.2 0.3 f 0.2 Trace 0.9 + 0.312.0 f 1.1 13.4

u Average composition of three samples of media supplemented with fetal bovine serllm.

b Average composition of five samples expressed as mean +S.E. c Composition of one sample. Performed in duplicate. d Numbers represent the number of carbon atoms and the num-

ber of double bonds present in each fatty acid.

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4108 Membranes of Animal Cells. IV Vol. 244, No. 15

TIIILE V

Fui/~y acid composilion of major phospholipitls and ner~lral lipid of surface membmnes of I, cella

Plasma membrane neutral lipid and phospholipid were sep:t- rated by silicic acid rhromwtogrnph\-. Phospholipids were fur- ther xparated by two-dimensional thin layer chromatography,

and the lipids were visrlalized by spr:tying with bromthymol blue. Ioditle was not Itsed in order to avoid possible losses of rmsatrl-

rated fatty acids. Fatty arid methyl esters were prepared and analyzed as described in Table IV.

Major fatty acid$

14:o 16:O ICI:1 18:O 18:l

18:2 18:3 22:o

20:4 24:o

UnsatLvated fatty acids, c7

Lo:& chain

fatty acids,

5%” PolyLmsatu-

rated fatt,y acids, 0/O

-

n rota1 eutral lipid

4.5 13.1

1.3 4G.2 23.0

0.9 4.1

3.8

2.9

33.1

G.7

8.8

Phos- pha- tidyl- serine

0.5 8.3

0.9 7.5 1.4

1.8 48.8

1.5

28.4

52.9

29.9

50.8

Lipid fraction

phingc- nyelin

Ikace 3.0

2.8 2.3 0.3

59.9 0.8 0.4

30.3

G2.9

31.5

GO.6

-

L ecithil

0.9 30.9

2.1

20.1 5.1

2G.8

0.2 13.G

34.2

13.8

27.0

n et

.-

-

Phos- pha- tidyl- .hanol imine

hospha- tidyl-

inositol

P hospha- ti die acid

1.2 29.2

8.0 10.3 13.6

2.0 24.6

0.3

10.7

48.2

0.6 23.7

9.0 12.8 lG.3

2.5

19.7 1.5

Trace 13.3

40.8 4.6

15.1

1.7 0.8

27.4 1.0

Trace 8.0

47.5 34.5

11.0 14.8 9.0

2G.G 22.2 28.2

a Data represent the average composition of fatty arids present in the lipids of two FMA membrane preparations.

b Fatty acid nomenclature is the same as that of Table IV.

c Long chain fatty acids are defined <as fatty acids containing 20 or more carbon atoms.

acids are unsaturated, l\-ith the polyunsaturated acids account- ing for 127c of the total.

The fatty acid pattern of the plasma membranes shows several differences. (a) Palmit’ic, stearic, and oleic acids make up slightly less than 5070 of the total fatt,\- acids, with long chain acids accounting for 147, of the total. (b) The stearic and oleic acid contents of the membranes are usually half that of the whole cell. (c) Linolenic (18:3) and lignoceric (24:0) acids, xvhich make up only 56 of the cell fatty acids, account for over 357, of the membrane fatty acids. (d) Approximately half of the surface membrane fatty acids are unsaturated, with the poly- unsaturated fatty acids accounting for 27 y0 of the total.

In Table V is seen a comparison of the fatty acid composition of the major phospholipids and the total neutral lipid fraction of surface membranes prepared by the PMA procedure. The follolving features can be pointed out. (a) The neutral lipid fraction contains less long chain fatty acids (7%) than any of the major phospholipids (9 to 32%). (b) The neutral lipid fraction cont’ains less polyunsaturated fatty acids (9$&) than any of the major phospholipids (22 to 61 yO). Arachidonic acid (20:4) is a

notable exception since it appears to be concentrated in the neutral lipid fraction. (c) The major phospholipids can be separated into two groups. I’hos1)hatid?rlseriic and sphingo- my&n both cont,ain npprosimately 30 TTO long chain fatty acids while the remainilig phospholipids contain only 9 to 15%. Simi- larly, phosphwtidylserine and sphingom!-elin contain 51 and 615; of polyunsaturated fatty acids \vhilc the remaining phospholipids contain only 22 to 28 “C. (d) L inolenic acid (18 : 3) and lignoceric acid (24:0) make up if and 9OC;b of the total f:ltty acids of phos- phatidylscrine and sphingom,wlin, respectively.

DISCGSSIOS /

L cells grown in suspension culture provide an esccilent source of surface membranes for the study of lipid composition. Bailey, Gey, and Gey (3’7) have shown that cultured mammalian cells derive most of the lipids required for growth from the lipids of the medium. Bailey (38) has shown that lipid synthesis de nova is inhibited up to 95’; m L cells grown in the presence of serum lipid. The operation of metabolic controls of this type may be important in the mnilltenance of the remarkably constant lipid composition of the cells observed throughout the course of this and other investigations. V:m Ileenen et al. (39) have sho\vn that despite large variations in diet the proportions between the major lipid classes of the erythrocyte membrane did not change, although the fatty acid patterns of the individual membrane lipids were subject to marked alterations. *Uthough 95y0 of the cell lipid can be derived from serum lipid, the composition of the cell 1il)id is markedly different from that of the serum lipid (36). The i;pccific utilization of serum lil)id by the L cell can be readily seen by comparing the fatty acid composition of the serum and the cells (Table IV). This combination of specific utilization of lipids by the cell and the constant over-all lipid composition allows one to predict that the differences observed between whole cell lipids and the lipids of the isolated surface membrane \vill be significant and no>- represent specaific lipid requirements for surface membrane biosynthesis.

.\t, present there are several rel)orts in the literature \vhicl! supply data on the liljids of plasma nlembranes of rat, liver iso. lated by the variations of the method of Xerille (40) and or membranes isolated from other sources. Table VI presents B summary of the data taken directly or calculated from these reports. Emmelot et al. (Line 1) and Ashworth and Green (Lint 2) examined only the sterol and phospholipid components and reported low values for the cholesterol-phospholipid molar ratio The results of Takeuchi and Terayama (Line 3) are difficult tc interpret owing to large discrepancies in the data on the twvc membrane samples which were analyzed. The low content 01 cholesterol and phospholipid and the very high lipid-protein ratic probably indicate gross contamination of the preparations witl- smooth endoplasmic reticulum (41). Skipski et al. (Line 4) re- ported that 40% of the dry weight of rat liver surface membrant was lipid-extractable, but they were unable to account for 26% of the lipid and suggested that this missing lipid was glycolipid although no glycolipid spots were found in the chromatographic analysis. Dod and Gray (Line 5) have isolated surface mem branes from rat liver and reported a lipid composition which ir in good agreement with the lipid cornposit,ion of L cell surface membranes reported in this paper. Pfleger et al. (Line 6) haw isolated rat liver l~lasma membranes by tonal centrifugation These preparations h:ave a much lower lipid to protein ratio ant lower neutral lipid and phospholipid content than previousl!

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Issue of August 10, 1969 Weinstein, .Marsh, Glick, and Warrejz

TABLE VI

Comparison of lipid composition of surface membrane preparations

Source of membranes Method of preparation

1. Rat liver 2. Itat liver 3. Itat liver

4. Rat liver 5. l&t liver G. ltat liver

7. L cells 8. IleLa cells

9. Rat intestinal

microvilli

10. Ox brain mgelin

11. Rat erythrocytes 12. Influenza virus

propagated in oco

Yeville (40)

Ncville (40) Neville (40) Neville (40)

Keville (40) Neville (40) FMA

Dotmcc homoge- nization

EDTA fractiona-

tion of brush borders

Isopycnic ccn-

trifugation Hemolysis Isolation of in-

fluenza virus

Lipid concentration

mg/mg pvotein 0.4w

0.4%

1.01

O.G9 0.58

0.44

0.66

0.72

O.Gl

3.45

-

Seutral lipid

Me 9 34

47 21 53

34e 17 59

27 22 47 41 21 50

57e 24 43

20 14 26

28 28 42

28' 28 61

47 4oi 53

Percentage of recovered lipid

Total choles- , tero1a

--

Total phospho-

lipid

Choline- ntainiq >hosphc-

lipid&

50

48

59

74

59 59

29

40

70

61

A-

-

GlyCO- lipids

‘if

<I

54h

30

ll/

“holesterol- hospholipid( molar ratio

rota1 lipid actually

recovered

7,

0.38

0.26

0.52

0.81

0.60

0.748

0.69

1.05

100 74

100 91 94

100

1.2G 100

1.32 100

0.92 98 1.5 81

-

4109

Reference

7

4

8

9 10 11

This report

41

42

43

44

45

a The average molecular weight of cholesterol esters is assumed to be 670. b Percent age of tot al phospholipid.

c The average molecular weight of a phospholipid is assumed to be 775. d Lipid weight includes only cholesterol and phospholipid. e Xeutrnl lipid content calculated as weight of total lipid minus weight of phospholipid.

f Glycolipid content calculated as weight of total lipid minus weights of neutral lipid and phospholipid Q lteported as micromoles of free cholesterol per rmoles of membrane phospholipid phosphorrls. n The high glycolipid content ma.y be due to the filamentoas surface coat projections of the membranes, which may be rich in car-

bohydrate (42). i The high cholesterol content of the viral envelope was determined after extraction with boiling ethanol hadsolubilized lipid which

was not soluble in 2:l (v/v) chloroform-methanol (45).

reported by others for rat liver membranes (Lines 4 and 5). An unidentified phosphorus-free lipid makes up 12% of the total

membrane lipid of their preparations.

The cholesterol-phospholipid molar ratio for L cell surface

membranes is 0.69 while the ratio for the whole cell is 0.26.

Therefore, the surface membrane of the cell contains much

greater amounts of cholesterol than do the internal membranes.

Free cholesterol accounts for 95y0 of the total cholesterol found

in the surface membrane of L cells. Recent investigations (4,

46) on subcellular organelles from a variety of animal tissues have indicated that free cholesterol is the predominant or only

form of cholesterol in biological membranes. No other sterol

was found in L cell surface membranes. Free and esterified cholesterol constitutes 21 y0 of the total lipid of the L cell men-

brane. This value can be compared to 17 to 22% of the total

lipid in rat liver membranes (9-11). L cell membrane neutral lipid contains 51 y0 cholesterol, 3Oa/,

triglyceride, and 6% free fatty acid. Skipski et al. (9) found

that 45y0 of the total neutral lipid recovered from rat liver mem- branes was cholesterol and that free fatty acids and triglycerides

made up 20 and lo%, respectively, of the remaining neutral lipid. Dod and Gray (10) found that 50% of the neutral lipid was cholesterol and that fatty acids accounted for a large part of the remaining neutral lipid. Pfleger et al. (11) report’ed a low neutral

lipid content (27 ‘,o of the total lipid), of which SOY0 was ac-

counted for by cholesterol. The high content of neutral lipid in surface membrane is

accompanied by a decrease in the relative proportion of phospho-

lipid. While SOY0 of rat liver and L cell lipid is phospholipid,

the rat liver membrane lipid contained 47 to 59%, phospholipid (Y-11) and the L cell membrane, 59% phospholipid. Choline- containing phospholipids account for 48 to 74 70 of rat liver mem-

brane phospholipid (7-11) and 59% of L cell membrane phospho- lipid. Sphingomyelin, which normally makes up only 7% of L cell lipid and about 3C10 of rat liver lipid (9), makes up 14 and 20% (10) of the lipid of the surface membranes of these cells. Both plasma membranes show corresponding decreases in lecithin and phosphatidylethanolamine content compared to whole cell lipid. Dod and Gray (10) did not find phosphatidic acid in their rat liver membranes; however, Skipski et al. (9) and Pfleger et al. (11) found “polyglycerophosphatide” fractions which

account for 7 and 3%, respectively, of the membrane phospho- lipids. The high phosphatidic acid content of L cell surface membranes in our study does not appear to be a degradative artifact since the fatty acid pattern of this phospholipid is quite different from that of any of the other major phospholipids (Table V)

13osmann, Hagopian, and Eylar (41) have isolated surface

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4110 Membranes of Animal Cells. IV Vol. 244, No. 15

membranes from HeLa cells grown in suspension culture. These membranes have approximately the same lipid to protein ratio as rat liver and L cell surface membranes (Table VI, Line 8). HeLa cell surface membranes surprisingly have been reported to have a low phospholipid content (437; of the total lipid) and therefore a high cholesterol-phospholipid molar ratio of 1.05. The neutral lipid and phospholipid contents are quite different from that of L cell and rat liver surface membranes. Since a high ratio of cholesterol to phospholipid appears to be a common feature of all surface membranes which have been studied at the present time, Bosmann et al. (41) have suggested that the very high ratio found for HeLa cell membranes is a good criterion of the purity of the preparations. We suggest that the published figures for HeLa cell membrane lipids indicate that HeLa cell surface membranes may belong to a different class of surface membrane than do those of liver and L cell.

The IIeLa cell membrane appears to be more closely related to microvilli and erythrocyte membranes and myelin (Table VI, Lines 9 to II), all of which have cholesterol-phospholipid molar ratios of 0.9 or higher. Purified fragments of plasma membranes from an avian source (influenza virus lipoprotein envelopes (Table VI, Line 12)) have also been shown to have a cholesterol- phospholipid molar ratio greater than 1.

It can be seen from Table VI that surface membranes which have high cholesterol-phospholipid molar ratios may also have high glycolipid contents. Dod and Gray (10) found at least fire alkali-stable components of rat liver plasma membranes which were chromatographically identified as glycolipids. The glyco- lipid content of the plasma membranes was estimated to be 7y0 by gravimetric analysis. This number was calculated by sub- traction of the weights of neutral lipid and phospholipid from the weight of the total lipid. So information is available concerning the presence of glycolipids in the HeLa cell membranes. We have found by nongravimetric means that glycolipid accounts for less than 170 of the lipid of L cell surface membranes pre- pared by the FJIA procedure.3 It may be possible to classify surface membranes into groups based upon their glycolipid toll- tent since, as seen in ‘Table VI, some membranes are rich in glycolipids and others arc quite poor.

The relatively constant composition of the FMA membrane lipids and the escellent agreement with the composition of L cell membranes prepared by treating cells with Tris-Cl buffer (pI1 7.4) are good indications that extensive degradation of lipids did not occur during the isolation of surface membranes. FAIL1 membranes are isolated as whole ghosts, and it can be estimated that these ghosts contain approsimately 4.7% of the protein of the whole cells from which they are prepared. Similarly, it can be estimated that 13.8 To of the lipid of the L cell is in the surface membrane as isolated by the FMA method.

Coleman (6) and Skipski et al. (9) found that the rat liver plasma membranes contained less unsaturated fatty acids than the whole cells from which they were isolated. L cells and theil surface membranes both contain 4SL% of unsaturated fatty acids. However, inspection of the fatty acid composition of L cell mem- brane lipids points out several interesting features. (a) L cells grown in media containing linolenic acid (18 :3) appear to concen- trate this fatty acid selectively in the surface membrane, account- ing for 2570 of the total fatty acids of the membrane. (b) There can probably be a certain amount of latitude in the degree of unsaturation of the surface membrane lipid without affecting its structural integrity and function. L cells which are grown in

the absence of endogenous lipid do not make polyunsaturated fatty acids (35) and under these conditions the total degree of unsaturation of the surface membrane would be much lower. Although differences in the chemical and quantitative distribu- t,ion of lipids may be related to differences in the surface proper- ties, such as permeability (44), no definitive data concerning this type of relationship are currently available. (c) Compared to the whole cell total lipid there is almost a 2-fold enrichment of sphingomyelin in the surface membrane of the L cell. Ninety per cent of the fatty acids of the membrane sphingomyelin are accounted for by linolenic acid (18 : 3) and lignoceric acid (24 : 0). This accounts for a large part of the concentration of these fatty acids in the surface membrane. (d) All of the membrane phos- pholipids contain relatively large amounts of polyunsaturated and long chain fatty acids (Table V). Analyses of whole cell phosphatidylserine and sphingomyelin fatty acids3 also indicate enrichment with polyunsaturated fatty acids, in contrast to most other mammalian cells which have been examined (47, 48). These fatty acid patterns indicate the operation of selection processes involving the utilization of fatty acids by L cells in tissue culture.

Dodge and Phillips (47) have reported that the fatty acid distribut,ion of sphingomyelin from human erythrocytes is very different from that of the major ” glvcerophospholipids of the erythrocyte. Sphingomyelin contained less unsaturated fatty acids than the other phospholipids but contained most of the lignoceric (24:0) and nervonic (24: 1) acid found in the erythro- cyte phospholipids. Pfleger et nl. (11) found that the fraction from rat liver plasma membranes which contained phosphatidyl- serine and phosphatidylinositol had a higher arachidonic acid (20:4) content than any other lipid fraction. They also found that sphingomyelin was the only membrane lipid that contained fatty acids longer than arachidonic acid. It would seem, then, that the fatty acid patterns of surface membrane sphingomyelin and phosphatidylserine are quite different from those of other lipids.

In summary, several characteristic features of animal cell membranes have emerged from analytical studies of membrane lipids. (a) The lipid composition of the surface membrane of a cell is quite specific in that it differs from that of other membrane systems of the cell (4, 41, 46). (b) Except for variations in acyl chains due to environmental factors (39), the lipid compositions of the plasma membranes of animal cells show characteristic features such as greater cholesterol and neutral lipid content than other membranes from the same source. (c) Surface mem- branes from rat liver (10, ll), erythrocytes (44), and cultured mouse fibroblasts (Table II) show a greater sphingomyelin con- tent and lower lecithin and phosphatidylethanolamine contents than internal cell membranes. (d) Individual membrane lipids may contain very specific fatty acid patterns. (e) Lipid param- eters may be useful in the classification of surface membrane preparations.

Acknowledgments-We wish to thank Mrs. M. R. Andrews, Mrs. R. Koser, and Miss A. Klein for excellent technical assist- ance.

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David B. Weinstein, Julian B. Marsh, Mary Catherine Glick and Leonard WarrenSURFACE MEMBRANE

Membranes of Animal Cells: IV. LIPIDS OF THE L CELL AND ITS

1969, 244:4103-4111.J. Biol. Chem. 

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