Proc. Nat. Acad. SC{. USAVol. 73, No. 1, pp. 173-177, January 1976Cell Biology

Analysis of membrane halves: Cholesterol(cell surface/freeze-fracture/membrane splitting/human erythrocytes/cholesterol quantitation)

KNUTE A. FISHERCardiovascular Research Institute, University of California, San Francisco, Calif. 94143

Communicated by D. E. Koshland, Jr., October 28,1975

ABSTRACT Membrane splitting by freeze-fracture hasbeen used as a preparative tool for chemical analysis of outerand inner "hal]"-membranes. In a previous report I showedthat monolayers of human erythrocytes, bound to cationizedglass, fracture nonrandomly, producing membrane fractionssubstantially enriched in outer or inner "halves." For thepresent study cells were used in quantities compatible withmicroanalysis. For quantitation the total amount of mem-brane present and the fractional enrichment of outer andinner "half"-membranes were determined. Cholesterol wasexamined by quantitative thin-layer chromatography modi-fied to assay nanogram amounts. Comparison of lipids ex-tracted from intact membranes with lipids from fracturedmembranes indicated that cholesterol was asymmetricallydistributed across the plane of the membrane, more beingpresent on the exterior side than on the interior side.

It is widely accepted that the lipids of many biological mem-branes are arranged in a bilayer (1-3) and that certain lipidsof the erythrocyte membrane are asymmetrically distribut-ed across the bilayer (4-8). Evidence for lipid as well as pro-tein and carbohydrate asymmetry has often been derivedfrom chemical labeling and enzymatic degradation of intacterythrocytes compared to isolated membranes or "ghosts"(4-11). Although individual studies often lack rigorous evi-dence that the label does not penetrate the membrane orthat the method does not perturb its structure (12), the bulkof data points strongly to carbohydrate, polypeptide, andphospholipid asymmetry.

Still in doubt, however, is the transmembrane-distributionof cholesterol even though it accounts for about 24% of thetotal lipid of human erythrocytes (13). Although the sym-metric distribution of cholesterol in erythrocyte "ghosts" hasbeen suggested (14), most investigators exclude cholesterolfrom molecular models of the erythrocyte membrane. Thisuncertainty is in part due to the lack of methods for examin-ing lipids in minimally perturbed membranes.

During freeze-fracture membranes are split, as proposedby Branton (15), along an internal plane between terminalmethyl-groups in the center of the bilayer. Proteins withinthis plane appear as particles that partition asymmetricallybetween the fracture faces, thus providing an electron mi-croscopic marker for the outer or inner "half." Given themeans for identification, if one could collect the separated"halves" of fractured membranes, chemical analysis wouldyield direct information on the transmembrane distributionof components. Although this idea is not new in that prelimi-nary attempts to enrich for "half"-membranes produced byfreeze-fracturing have been reported (16-18). no detailedquantitation of any membrane constituent has been com-municated. I recently described a method for producingmembrane fractions enriched in outer or inner halves, dis-cussed its electron microscope application, and suggested itspotential application to chemical analysis (19). The purposeof this report is to discuss one approach to harvesting half-membranes in quantities suitable for microanalysis, the gen-

173

eral features of transmembrane quantitation, and the specif-ic transmembrane distribution of cholesterol.

MATERIALS AND METHODS

Erythrocyte Preparation. Human blood was drawn intoacid citrate dextrose solution, kept at 0-5°, and washed asdescribed (19). The pellet was diluted with buffer and thenumber of erythrocytes per ml determined by hemacytome-try.

Coverglass/Copper Preparation. Coverglasses, no. 1, 24X 50 mm (Corning Glass Works, Coming, N.Y.), werecleaned and treated with 100 gl of 5 mM polylysine, averagemolecular weight = 2000 (19). Pure copper sheet, 30 X 57 X0.508 mm, flattened by pounding, was dipped into 35% ni-tric acid in distilled water (vol/vol) for 10 sec, then rinsedwith glass-distilled water and dried with N2.

Erythrocyte Application. Two hundred microliters ofbuffer containing 1.2 X 109 erythrocytes were applied toeach polylysine-treated coverglass. If the droplet failed toflow quickly, edge drying and air/buffer interface effectsproduced boundaries of lysed cells and the coverglass wasdiscarded. After 10-15 sec the coverglass was washed at0-5° in three 50-ml changes of buffer (25 sec each change);the back side was wiped dry and free of cells, and thenplaced cell side against the copper sheet. For binding assays,coverglasses were photographed within 30 sec of prepara-tion; for splitting assays, they were frozen within 10 sec.

Binding Assays. Glass-bound erythrocytes were placedagainst another coverglass, and quadrants were photo-graphed at random with a Zeiss photomicroscope. Cover-glass pairs were then scanned with a Cary 14 recording spec-trophotometer equipped with scattered transmission accesso-ry (model 1462). For both photography and spectrophoto-metry, samples were handled rapidly and gently to avoid os-motic or mechanical lysis.

Freeze-Fracture. The copper-erythrocyte-glass laminatewas frozen in partially solidified Freon-22, -150°, andstored under liquid nitrogen. Fracturing was performed atroom pressure with the copper in contact with liquid nitro-gen, using a tool carrying a single-edge razor blade to prythe glass from the copper. A diagrammatic representation ofthe method is shown in Fig. 1. Unfractured controls, eitherbound to glass or free in microcapillaries, were similarly fro-zen and stored under liquid nitrogen.

Fracturing Assays. Fractured coverglasses were transpar-ent except for patches that appeared pink. Residual pinkareas were measured after lipid extraction. Coverglasseswere washed with CHCl3:MeOH (2:1), dried with nitrogen,placed in a photographic enlarger, and "printed" at an en-largement of 4.75X. Prints were placed on a light box andareas measured using a compensating polar planimeter(model 620015, Keuffel & Esser Co., Morristown, N.J.). Con-

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BIND FREEZE FRACTURE

.-.:.:-.:-.:-:.:; . .. 1.copper--w...

FIG. 1. Micropreparative method for half-membrane enrich-ment. Cells are bound to a coverglass, placed against a copperplate, and frozen. The glass is pried from the copper to reveal apreferential fracture through the bound membranes.

trol coverglasses, split and unsplit, were freeze-dried andsimilarly enlarged and measured.

Electron Microscopy. Coverglasses fractured at roompressure were transferred to a cold stage under liquid nitro-gen, covered, and placed in a Varian VE-61 vacuum evapo-rator. The stage was warmed to about -80° and shadowedwith platinum-carbon (20) at an angle of 20 degrees. Repli-cas were floated off the glass using a 1:1 dilution of 48%HF:distilled water in polystyrene culture dishes, given sever-al distilled water rinses, and examined and photographedwith a Siemens 1 electron microscope.

Lipid Extractions were conducted under dry N2 at 220using reagent grade solvents from freshly opened bottles. Allglassware was acid-washed. Lipids were extracted accordingto Bligh and Dyer (21). Because total lipids were in the lowmicrogram range, special care was taken to avoid contami-nation during handling. The cholesterol standard was a giftof Dr. Wayne Hubbell and was chromatographically pure asdetermined by thin-layer chromatography with various sol-vent systems.

Thin-Layer Chromatography. Adsorbosil 5 "Prekotes"(Applied Science Laboratories, Inc., State College, Pa.) werewashed and activated. Samples were spotted with a holdermanufactured to allow critical positioning of a 10-pl Hamil-ton syringe. Plates were run to 4 cm (origin to front) inCHCI3:acetone (90:10), dried, sprayed with 50% sulfuricacid in distilled water (vol/vol), and charred at 1800 for 30min (22). Charred spots were scanned with a Mark III CSdouble-beam recording microdensitometer (Joyce Loebl &Co., Ltd., England). Optical density peaks were integratedelectronically or by planimetry.

RESULTSErythrocyte Binding. Four factors are particularly im-

portant for obtaining a closely packed monolayer of erythro-cytes (Fig. 2). First, the glass must be clean and totally wet-table. Hydrophobic areas bind neither polylysine nor cells.Although lower concentrations of polylysine (e.g., to 0.5mM) and shorter adsorption or wash times also result inlarge numbers of erythrocytes bound to glass, the proceduredescribed was chosen for convenience and reproducibility.Second, the cells must be thoroughly washed and intact. Re-sidual serum protein or hemoglobin released by lysis readilybinds to treated glass and prevents adsorption of cells. Third,the "age" of the cells must be known. Compared to freshcells, erythrocytes stored in acid citrate dextrose solution, inthe dark at 0-5', bind to glass in higher numbers at lowerconcentration (Fig. 3). Although of interest, the "age" effecthas not been pursued. Fourth, the optimum concentration of

FIG. 2. Closely packed monolayer of washed human erythro-cytes flattened against polylysine-treated coverglass. X1100.

cells must be applied (Fig. 3). Under standard conditions atlow concentrations (e.g., 2 X 108 erythrocytes/100 gl ofphosphate-buffered saline per 4.84 cm2 coverglass), too fewfresh cells bind to ensure reproducible nonrandom fractur-ing and enough lipid for quantitation. At higher concentra-tions (e.g., 9 X 108 erythrocytes) cell suspensions are too vis-cous to apply rapidly and evenly, producing inhomogeneousmonolayers. For freshly drawn and washed erythrocytes, asused exclusively for the present cholesterol quantitationstudies, the optimum concentration is 1.2 to 1.4 X 10P eryth-rocytes/200 ,ul of phosphate-buffered or Tris-buffered salineper 12 cm2 glass surface.

Freeze-Fracture. After freeze-fracture the transparentglass contained random patches of pink (Fig. 4). Pink areascould result from any of three possible fractures: through thecenter of the cell, through the unbound membrane, or

2.5 Q.50%

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72 HOURS 3.5 HOURSe%.L 1.0 7 0.2o

0.5m 0.1

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CELLS APPLIED (xIO0)FIG. 3. Adsorption of erythrocytes to polylysine-treated cover-

glass as a function of dilution and duration of storage (3.5 hr and72 hr, buffered, dark, Q-50) of erythrocytes prior to application.Cleaned and dried coverglasses, 22 X 22 mm, were treated with 50,41 of 5 mM polylysine for 30 sec, washed in 50 ml of distilled water,and dried. One hundred microliters of erythrocytes washed sixtimes were applied at various dilutions for 10 sec, then washedthree times for 20 sec in phosphate-buffered saline, pH 7.4, 0-5°.Coverglasses were photographed (areas selected at random) andthen scanned at 415 nm.

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Proc. Nat. Acad. Sci. USA 73 (1976) 175

FIG. 4. Monolayers of erythrocytes bound to 24 X 50 mm cov-erglasses after freezing and lipid extraction. Unsplit control sam-

ple, above. Fractured sample, below. Prior to lipid extraction thetransparent portion (black) of the glass contained "half"-mem-branes; the unfractured portion (grey), intact cells. X1.

through the buffer-copper interface. Several observationssupport the third possibility: i.e., between intact cells and thecopper. First, after fracturing, frozen pink areas on glass re-

semble plateaus that match complementary depressions ex-

tending to copper. Second, when the frozen copper side is

washed with room temperature solvents, the pink erythro-cyte layer melts first to reveal an underlying white frozenbuffer zone except in the copper-colored depressions. Third,electron microscopy of freeze-etched pink areas revealed a

reticulum of buffer eutectic obscuring underlying cells. Cellsurfaces or partially exposed A fracture faces were observedonly among a few cells at the perimeters of pink areas.

Unsplit pink areas were measured by photographic en-

largement plus planimetry after freeze-fracture and "fixa-tion" by rapid dehydration during lipid extraction (Fig. 4).Accuracy depended on the selection of the area to be pla-nimetered. As well as split membrane, transparency couldindicate regions that were originally unsplit but lost duringfracturing, thawing, or lipid extraction. Loss during fractur-ing, however, is small. When complementary copper andglass sides were fractured, freeze-dried, and matched, thesum of their areas equaled the unfractured equivalent (with-in 1%). Similarly, loss due to thawing and lipid extraction issmall when measured by comparing extracted samples tofreeze-fractured and freeze-dried unextracted samples. Forfreeze-fractured and extracted samples the fraction of un-

split membrane ranged from 3.6 to 20.3% with a mean iL SDof 11.5 + 4.9% (n = 18). For freeze-dried unextracted con-

trols, the fraction of unsplit membrane ranged from 9.0 to15.2% with mean I SD of 12.1 ± 4.4% (n = 3).

Electron microscopy confirmed that the transparent, col-orless areas of 12 cm2 samples actually represented "half"-membrane bound to glass (Fig. 5a). Because the membraneswere flattened against a smooth substrate whose shadowangle was known and low (20 degrees), additional detail wasrevealed in areas between the larger particles; i.e., qualita-tively, there are more smaller particles than conventionallyseen (Fig. Sb). Although the bulk of the membrane was

structurally similar to unbound freeze-etched controls, iso-lated or reticulated holes were observed in some regions(Fig. 5a, inset). Contiguous to these holes were pieces ofhalf-membrane that appeared to have folded over. In an

.-F

erythrocyte "half' membranes on transparent portion of glassafter "freeze-fracture" of 12 cm2 preparations. (a) Erythrocyte Bfaces separated by smooth glass surface, x17,000; inset shows holewith adjacent piece of torn membrane, X37,000; (b) detail of B faceto show particle distribution and general rugosity, X48,000.

earlier study (19), nonrandom fracture was shown for sam-ples of smaller mass, fractured at 2 X 10-6 Torr.

Lipid Quantitation. Although the quantities of lipid ex-tracted were in the nanogram to low microgram range, care-ful spotting and charring, coupled with scanning micro-densitometry, provided both the resolution and sensitivitynecessary to analyze the split samples. Qualitative confirma-tion and quantitative analysis could be made on the sameplate. For cholesterol the sensitivity was better than 10 ngand was linear to about 700 ng, although the range of 50-400 ng routinely was used (Fig. 6). Numerical variation dueto plate preparation, spotting, spraying, charring, and spotfading was reduced by including at least four standards onevery plate.A synopsis of the extraction data is given in Table 1. Two

experiments were designed to verify that the numericalvalue of combined copper and glass extracts would equal thetotal amount of cholesterol extracted from bound but unsplitcells, or an equivalent number of unbound and unsplit cells,and that extraction was complete. In these experiments(Table 1, experiments 6 and 7) the total of copper plus glassequaled 101-104% (n = 6) of the intact, bound erythrocytesand ranged from 88% to 96% of the total equivalent amount

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Proc. Nat. Acad. Sci. USA 73 (1976)

of unbound cells or of calculated values derived from pre-

viously reviewed data (13).Distribution Analysis. To calculate membrane lipid si-

dedness, four quantities were required: the mass of totalmembranes or lipid, MA; the mass of lipid in split mem-branes (copper, MB, or glass side, MC); the fraction of totalmembranes (area) split, fa; and the fraction of membranesplit per cell, jo.. The fraction of outer membrane surfacesplit per cell was previously shown to be 33% (19). The otherquantities were determined for each experiment. Sidednesswas then calculated using two equations in two unknowns(Fig. 7). The ratios, mO/m1, are given in Table 1.The ratio of cholesterol present on the outer portion of the

membrane to that on the inner ranged from 1.02 to 3.34with a mean 4 SD of 2.07 L 0.81. It is of interest that exper-

imental data fell in two classes, representing erythrocytepreparation in phosphate-buffered or in Tris-buffered saline(Table 1). In both classes, however, more cholesterol was

present in the outer half than the inner. For the experimentswith phosphate buffer the mean ratio was 1.34 4 0.31, andfor the experiments with Tris, 2.62 :1 0.57.

DISCUSSIONThe present report describes a quantitative examination ofthe transmembrane distribution of cholesterol. The ap-

proach is based on a recently described freeze-fracturemethod for half-membrane enrichment (19). Presented hereare modifications of the method necessary for bulk analysis,the quantitative requirements for sidedness determination,an analytical approach for calculating outside to inside ra-

tios, and the application of the method to the analysis ofcholesterol.

For chemical analyses, quantities of membrane were re-

quired in amounts larger than produced in the previousstudy (19). Given fresh, thoroughly washed erythrocytes andpolylysine-treated coverglasses, monolayers covering 12 cm2could be formed and frozen within 90 sec. The maximumcalculated yield of total lipid from a monolayer of 2 X 107erythrocytes bound to a single 24 X 50 mm covefglass, at 5X l0-13 g of lipid/erythrocyte (13), is 10 gg. Since cholester-ol accounts for about 24% of the total membrane lipid (13),the amount of cholesterol available for analysis was about

E

28120

0

Z

0 100 200 300 400

CHOLESTEROL (ng)FIG. 6. Quantitation of cholesterol by thin-layer chromatogra-

phy.*Curve shows the sensitivity, linearity, and reproducibility ofcharring coupled with microdensitometry. Points represent themean, bars the SD, of pooled data (six to nine plates per point) ofcholesterol standards prepared over several months.

2.4 jig per intact erythrocyte monolayer. Fracturing reducedthe amount to several hundred nanograms. Nevertheless,pooling several coverglasses produces an adequate samplesize for many microanalytical methods, and even single cov-erglasses may be repeatedly analyzed by the charring meth-od described.

In contrast to other methods for examining membranelipid asymmetry (4-8), the splitting method allows a directchemical analysis of essentially unmodified membrane fromintact cells that are washed, electrostatically bound, and fro-zen. Present evidence that membranes are minimally per-

turbed is based on microscopic observations. By phase con-

trast light microscopy, adsorbed cells do not lyse prior tofreezing even when bound for several hours, if kept fully hy-drated, buffered, and at 0-5'. By electron microscopy, the Bfaces after freeze-fracture appear normal, although some re-

positioning of half-membrane is observed. However, thedata also suggest partitioning of cholesterol in response to to-nicity or type of buffer used to wash and bind the cells.Moreover, the range of data is large and even includes thesymmetric distribution of cholesterol previously noted (16).

Table 1. Half-membrane enrichment (% transparent), cholesterol recovery, and outside/inside ratio

Cholesterol (ng)C Ratio dTransparent (%)b

Total Outside/InsideExp.a ([a) Cu side (MB) Glass side (MC) (MA) (mo/mi)PBS 1 88 ± 3 (4) 358 ± 24 (7) 130 ± 25 (7) 488 1.02

2 88 ± 5 (4) 333 ± 60 (3) 143 ± 21 (3) 476 1.643 91 ± 8(4) 350 (1) 125 (1) 475 1.36

TBS 4 87 ± 6 (3) 246 ± 43 (7) 133 ± 13 (7) 379 3.345 85± 7 (3) 240 ± 37 (8) 128± 27(7) 368 2.396 91 (1) 210 ± 32(4) 86 ± 11 (4) 296 2.017 90 (1) 206 ± 8 (4) 96 ± 5 (4) 302 2.75

a Exps. 1-3, erythrocytes in 310 im osM phosphate-buffered saline (PBS); Exps. 4-7, in 340 im osM Tris-buffered saline (TBS).b Area determined by planimetry after lipid extraction, drying, and photographic enlargement; mean + SD. Numbers in parentheses: cover-

glasses extracted and planimetered.c Data not vertically comparable: final solvent volume varied among different experiments. Numbers in parentheses: thin-layer chromatogi-raphy spots examined. Total indicates the arithmetic sum of copper plus glass. In Exps. 6 and 7, control data of bound but unfractured[293 i 54 (3) and 288 4 28 (3), respectively] and of unbound and unfractured [335 : 37 (4) and 313 4 24 (4)] were obtained.

d Ratio calculated by applying the analysis given in Fig. 7 using tabular data from fa, MA, MB or Mc, plus fos = 0.33 (determined in earlier ex-

periments); mi and mO of cholesterol computed for each experiment and their ratio, mo/mi, given here. Mean ratio for PBS experiments =1.34 4 0.31; for TBS experiments = 2.62 i 0.57; and for both (overall) = 2.07 4 0.81.

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Proc. Nat. Acad. Sci. USA 73 (1976) 177

A*:.~~~i~i. ~:c~. lJ.'

.........| MA

J0

fOs Mm

I I

I I

Mc

MA=m+mo+I]thus:M= MA-mO [2]

MB=faE (I-fOs)mO+ m] (3)thus:MO=IMB- (f0)ml] / (fa)(I- f0o) (4)substituting [4] in [2] and rearranging:mE= EMB-(fa)(I-fos) MAI /(fa)(fos) [5]and:me MA-mi (63

Mc (fO)(fos)mo+(I-fa)mo+ (-fa)ml E7)

m0e[Mc- (1-fa)miI /E(fa)(fos) + (-foa)J I8]substituting [8] in [2] and rearranging:m = {(fa)(fos)+(I-fa)J MA-MC}/(fa)(fos) t9]and:mO=MA- MI [lo]

MA= MB +.MC [II]

FIG. 7. Diagram of analytical approach. Three extractable sources: A, free or bound, unfractured; B, bound, fractured, copper side; C,bound, fractured, glass side. Basic equations for distribution analysis of membrane constituents: of intact membrane [11; split membrane,copper side [3]; or glass side [7]. Sidedness calculations use equations [5], [6] and [9], [10]. Equation [11] provides a control. M = mass ex-tracted from indicated preparation, e.g., from A = MA. mi = mass of the inside portion of the membrane. m0 = mass of the outside portion.f4,o = fraction of the outside portion split by freeze-fracture. f. = fraction of total area split.

Nevertheless, statistically, cholesterol is asymmetrically dis-tributed across the plane of the membrane of phosphate-buffered saline- or Tris-buffered saline-washed, polylysine-bound erythrocytes, and such asymmetry may reflect a con-

dition in vivo. Thus, for a single erythrocyte, a two-to-onedistribution of cholesterol represents about 8 X 10-13 g inthe half-membrane on the extracellular side and 4 X 10-13 g

in the membrane half closest to the cytoplasm.I have emphasized lipid analysis because fracturing of la-

mellar lipids has been studied in model systems (23) andconfirmed for the erythrocyte membrane (24). Moreover, si-dedness analyses are simplified because all erythrocyte lipidis membrane associated. Nevertheless, analysis of polypep-tides or residual enzymatic activity after splitting shouldprovide information about the types of bonds cleaved duringfreezing and fracturing, thus giving information about thevery method that it exploits.The method should be of value to studies of any cell sur-

face that can be homogeneously bound to an optically flatsubstrate. For quantitative sidedness analyses, measurementof the fraction of total membrane split is essential. For theerythrocyte, hemoglobin provides a quantitative, visualmarker of splitting. Although "visual" quantitation is conve-nient, nondestructive, and reasonably sensitive, other meth-ods, e.g., those using spectrophotometry or radioisotopic or

fluorescent markers, could be used. For qualitative sidednessanalyses, fluorescent membrane markers coupled with lightmicroscopy or radioisotopic markers coupled with freeze-fracture autoradiography (25) will allow not only transmem-brane detection of the label but its localization within theplane of the half-membrane.

I gratefully acknowledge numerous helpful discussions with Dr.Wayne Hubbell and with Dr. Walther Stoeckenius in whose labora-tory this research was undertaken. I thank Drs. Hubbell, Stoecken-ius, and Pedro Pinto da Silva and Ms. E. Crump for comments on

the manuscript. This work was supported by grants from the U.S.Public Health Service (HL 06285 and HL 14237). While in press

some of these findings were presented at the 15th Annual Meetingof the American Societv for Cell Biologv in Puerto Rico (26).

1. Stoeckenius, W. & Engelman, D. M. (1969) J. Cell Biol. 42,613-646.

2. Branton, D. & Deamer, D. W. (1972) in Protoplasmatologia(Springer-Verlag, New York-Vienna), Vol. II.E.1, pp. 1-70.

3. Singer, S. J. & Nicolson, G. L. (1972) Science 175,720-731.4. Bretscher, M. S. (1973) Science 181, 622-629.5. Zwaal, R. F. A., Roelofsen, B. & Colley, C. M. (1973) Biochim.

Biophys. Acta 300,159-182.6. Verkleij, A. J., Zwaal, R. F. A., Roelofsen, B., Comfurius, P.,

Kastelijn, D. & van Deenen, L. L. M. (1973) Biochim. Bio-phys. Acta 323,178-193.

7. Gordesky, S. E., Marinetti, G. V. & Love, R. (1975) J. Membr.Biol. 20, 111-132.

8. Whiteley, N. M. & Berg, H. C. (1974) J. Mol. Biol. 87, 541-561.

9. Steck, T. L. (1974) J. Cell Biol. 62, 1-19.10. Bender, W. W., Garan, H. & Berg, H. C. (1971) J. Mol. Biol.

58,783-797.11. Cook, G. M. (1968) Biol. Rev. Cambridge Philos. Soc. 43,

363-391.12. Schmidt-Ullrich, R., Knfifermann, H. & Wallach, D. F. H.

(1973) Biochim. Biophys. Acta 307,353-365.13. van Deenen, L. L. M. & de Gier, J. (1964) in The Red Blood

Cell, eds. Bishop, C. & Surgenor, D. M. (Academic Press, NewYork), pp. 243-7.

'14. Higgins, J. A., Florendo, N. T. & Barrnett, R. J. (1973) J. Ul-trastruct. Res. 42,66-81.

15. Branton, D. (1966) Proc. Nat. Acad. Sci. USA 55,1048-1056.16. Fisher, K. A. (1974) J. Cell Biol. 63, 100a.17. Jacobson, B. S., Welt, M. & Branton, D. (1974) J. Cell Biol. 63,

15Sa.18. Park, R. B. & Pfeifhofer, A. (1974) in Membrane Proteins in

Transport and Phosphorylation, eds. Azzone, G. F., Klingen-berg, M. E., Quagliariello, E. & Siliprandi, N. (North-Holland,Amsterdam, The Netherlands), pp. 97-102.

19. Fisher, K. A. (1975) Science, 190,983-984.20. Fisher, K. & Branton, D. (1974) in Methods in Enzymology,

eds. Fleischer, S. & Packer, L. (Academic Press, New York),Vol. 32, pp. 35-44.

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